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1
Schiffman, Schiffman D., Jonathan Downie, Bradley Demarest, et al. "Novel Use of Molecular Inversion Probes to Interrogate Formalin-Fixed Paraffin-Embedded (FFPE) Samples of Childhood Leukemia." Blood 114, no. 22 (2009): 1589. http://dx.doi.org/10.1182/blood.v114.22.1589.1589.
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Abstract Abstract 1589 Poster Board I-615 Genome-wide, high-resolution analyses of copy number alterations (CNAs) now play an increasingly important role in identifying new genomic loci associated with leukemia biology and prognosis. The most powerful of these studies include large numbers of patients with associated clinical features and outcome data. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution and can detect both gene copy number and allelic imbalance in clinical samples, and have been demonstrated to work on archived formalin-fixed paraffin-embedded (FFPE) samples as old as 20 years. In this pilot study, we report for the first time the successful interrogation of high-resolution CNAs in archived FFPE samples in childhood leukemia. We first extracted genomic DNA from FFPE bone marrow aspirate clots from 18 pediatric patients diagnosed with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) diagnosed between 2006-2008 at Primary Children's Medical Center at University of Utah. DNA from paired remission samples was also extracted for each patient, again using archived FFPE bone marrow aspirate clots. Blast percentages on pre-B ALL marrow clots ranged from 39-99% (Mean 88%, Median 94%). Genomic DNA was isolated using RecoverAll” Total Nucleic Acid Isolation Kit for FFPE Tissues (Ambion®, Applied Biosystems). Clinical features and outcome data were readily available and abstracted from the medical record. The 18 patients in the FFPE cohort included: ages 2-21 years old (Median 5.5 years old), 7 females, presenting WBC 1-75 × 103/uL (Median 3.9), CNS negative disease (n=18), no reported cytogenetic abnormalities (n=8), t(12;21) [n=6], t(9;22) [n=1], and MLL rearrangement (n=1). 7 patients were designated “High Risk” by NCI-Rome Criteria and 1 patient relapsed. The MIP assay was run using the customized 330K Cancer Panel (Affymetrix®, Santa Clara, CA), which includes both cancer-specific SNPs and genome-wide coverage with a median probe distance of 4,207 basepairs (bp). Copy number was calculated by comparing leukemia samples to pooled normal control signal intensity for each probe. CNA calls were based on 5 consecutive probes with >90% call rate, standard deviation < 20%, and copy number ' 1.2 or ≥ 2.8. MIPs revealed remarkably high-quality CNA data for each of the 18 FFPE samples, including the cytogenetically “normal” patients. Both known and novel recurring CNA loci were identified in this cohort. Deletions included: 14q11.2 (n=11 [61%], 51569 bp, no known genes), 22q11.2 (n=10 [56%], 185944 bp, VPREB1), 14q32.33 (n=10 [56%], 631377 bp, no known genes), 7q34 (n=9 [50%], 292586 bp, PRSS1, TRY6, PRSS2), and 12p13.2 (n=6 [33%], 292586 bp, ETV6). Gains included: 10p15.2 (n=10 [56%], 26481 bp, PFKP), 10q26.3 (n=10 [56%], 69691 bp, MGMT), 10p11.21 (n=8 [44%], 922257 bp, FZD8, CCNY, GJD4), and 8p23.3 (n=7 [39%], 90307 bp, ARHGEF10). Interestingly, of 52 amplified segments recurring in 35% or greater of samples, all but one were located in chromosome 10, 14, 17, 18, or 21 suggesting genetic amplification hotspots. Additionally, 100% of IKZF1-deleted samples (n=2/2) compared to 37.5% of IKZF1-normal samples (n=6/16) had M3 marrows (>25% blasts) at Day 7. These same two IKZF1-deleted FFPE samples belonged to the patients with the two highest WBC values at presentation (75.3, 22.9 × 103/uL), in addition to containing two of the six highest bone marrow blast percentages at diagnosis. This is consistent with known findings that IKZF1 deletions are associated with high-risk ALL. In this pilot study of 18 patients, we have shown that archived FFPE bone marrow aspirate specimens (standard of care for all bone marrow procedures) can be used successfully for known and novel CNA analysis in childhood leukemia. We believe this is the first time that high-resolution, genome-wide CNA data from FFPE samples in any type of leukemia have been reported. This is an important development in CNA studies of hematological disease because now it is possible to investigate an unlimited number of archived FFPE childhood leukemia samples from around the world, or to explore rarer and more difficult to find specimens such as relapse or concordant identical twins. Disclosures No relevant conflicts of interest to declare.
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Reading, N. Scott, Archana M. Agarwal, Ronald Hoffman, Josef T. Prchal, and Mohamed E. Salama. "Transcriptional Characterization of Myelofibrotic Bone Marrow Microenvironment Reveals Distinct Tumor Microenvironment in JAK2+ and Calr+ PMF Marrows." Blood 128, no. 22 (2016): 1954. http://dx.doi.org/10.1182/blood.v128.22.1954.1954.
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Abstract Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status. Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84). Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation. Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis. Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. Disclosures No relevant conflicts of interest to declare.
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Oetjen, Karolyn A., Diane E. Bender, Marianna B. Ruzinova, Stephen T. Oh, and Daniel C. Link. "Interrogating the Spatial Architecture of Human Bone Marrow Via Imaging Mass Cytometry." Blood 134, Supplement_1 (2019): 3728. http://dx.doi.org/10.1182/blood-2019-127460.
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Hematopoietic stem cell (HSC) proliferation, self-renewal, differentiation, and trafficking are dependent, in part, upon signals generated by stromal cells in the bone marrow. Stromal cells are organized into niches that support specific subsets of hematopoietic progenitors. Intimate interactions between HSCs and neighboring stromal cells coordinate hematopoietic responses during periods of physiologic stress, while also maintaining the lifelong integrity of the hematopoietic stem cell pool. Hematopoietic niches are comprised of a heterogeneous population of stromal and hematopoietic cells. The identity and function of the known stromal cell subsets have primarily been gleaned through genetic manipulation of mouse models. In humans, the spatial organization of these stromal cells in bone marrow and the signals they generate to regulate hematopoiesis are poorly understood. Current methods to characterize bone marrow mesenchymal stromal cells in humans include: 1) immunostaining of bone sections; 2) analysis of flow sorted stromal cells; and 3) analysis of ex vivoexpanded mesenchymal stem/progenitors. Major limitations to all of these approaches exist that relate to the heterogeneity of bone marrow stromal cells, the lack of markers that reliably distinguish different stromal cell populations, and inherent technical limitations of the assays, such as the number of markers that can be analyzed at one time. Here we report our efforts to use imaging mass cytometry imaging (IMC) to interrogate the complex cellular architecture of human bone marrow. IMC allows for the simultaneous detection of up to 40 markers through the use of antibodies conjugated to elemental metal tags acquired by time-of-flight mass spectrometry. We first used standard immunostaining techniques to develop a panel of antibodies compatible with archived formalin-fixed paraffin-embedded (FFPE) human bone marrow specimens using a heat-induced epitope retrieval method. Hematopoietic lineages have been successfully identified using CD11b, CD68, CD15, CD14, CD16, CD11c, CD20, CD3, CD4, CD8a, CD38, CD45RA, CD45RO, CD235a, CD71, CD34, CD31; stromal cells and structures with CXCL12, alpha-smooth muscle actin, collagen I, vimentin; and nuclear staining using Ki67, Histone H3, and DNA intercalator. We used panels consisting of up to 18 of these antibodies each to test IMC on FFPE human bone marrow specimens. Imaging was performed using the Hyperion Imaging System (Fluidigm), which consists of a UV laser scanning module to capture regions of more than 10,000 cells with 1-micron resolution coupled with a Helios Mass Cytometer. Image analysis was performed by creation of a cell segmentation mask using CellProfiler software and high dimensional analysis in histoCAT. Representative images and corresponding dimensional reduction with t-SNE are shown in Figure 1A-H, demonstrating successful discrimination of distinct hematopoietic lineages. Gating on subpopulations within the t-SNE clusters can be projected on to the original image to illustrate spatial distribution and marker co-expression, as an example CD3+ and CD8+ cells are shown in Figure 1I. These data indicate that IMC allows for highly multiplexed analysis of bone marrow cell populations. Developing imaging techniques for analysis of tissue-banked FFPE bone marrow samples would have broad applications for translational research on hematologic diseases. In particular, this technology has tremendous potential to advance understanding of the spatial architecture of human bone marrow and to investigate alterations in the bone marrow environment in malignant hematopoiesis. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
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Sadeghian, Mohammad Hadi, Maryam Mohammadnia Avval, Hossein Ayatollahi, et al. "Is There Any Relationship between Human Herpesvirus-8 and Multiple Myeloma?" Lymphoma 2013 (February 26, 2013): 1–5. http://dx.doi.org/10.1155/2013/123297.
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Background. Human herpesvirus-8 (HHV-8) is associated with some human diseases including Kaposi’s sarcoma and also some B-cell lymphoproliferative disorders. Few studies have highlighted the potential role of HHV-8 in the development of multiple myeloma (MM) which is known as a malignant proliferation of plasma cells derived from a single clone. Aims. The aim of this study was to find a relationship between HHV-8 and MM using polymerase chain reaction (PCR) method. Materials and Methods. This study was conducted on 30 formalin-fixed, paraffin-embedded (FFPE) bone marrow biopsies of multiple myeloma and 30 normal FFPE bone marrow biopsies. After the sample preparation, Deoxyribonucleic acid (DNA) was extracted by nonheating procedure. PCR for HHV-8 virus was carried out with commercial kit and the PCR products were visualized by gel electrophoresis. Finally, the statistical analysis was performed. Results. HHV-8 virus was not detected by PCR from FFPE blocks of multiple myeloma samples, while only one of the controls showed DNA band of the corrected molecular weights. Fisher’s exact test showed that no statistical differences were found between the two groups (P=0.999). Conclusion. Our report adds to the body of evidence that there is no association between HHV- 8 and MM against a major role of HHV-8 infection in the pathogenesis of clonal plasma cell proliferation.
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Neat, Michael J., Mufaddal T. Moonim, Robert G. Dunn, Helen Geoghegan, and Nicola J. Foot. "Fluorescence in situ hybridisation analysis of bone marrow trephine biopsy specimens; an additional tool in the diagnostic armoury." Journal of Clinical Pathology 66, no. 1 (2012): 54–57. http://dx.doi.org/10.1136/jclinpath-2012-201131.
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Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with formalin-fixed paraffin-embedded (FFPE) sections from numerous tissue types, FISH analysis of FFPE tissue sections from trephine biopsy specimens has been less widely reported, possibly due to technical limitations relating to the decalcification protocols employed. During the last 4 years FISH analysis has been carried out successfully in 42 out of 55 (76%) consecutive trephine biopsy specimens received as part of the standard diagnostic service at our institution. Samples decalcified using EDTA-based protocols were analysed successfully in 31/31 cases (100%), whereas only 11/24 samples (46%) decalcified using formic acid-based protocols were successful. In our experience, FISH analysis of trephine biopsy specimens is a highly reproducible technique and a very useful adjunctive tool in the diagnostic armoury; however, its use in a standard diagnostic setting relies on the use of EDTA-based decalcification protocols.
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Oetjen, Karolyn A., Diane E. Bender, Marianna B. Ruzinova, Daniel A. C. Fisher, Stephen T. Oh, and Daniel C. Link. "Imaging Mass Cytometry Reveals the Spatial Architecture of Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemias." Blood 136, Supplement 1 (2020): 44–45. http://dx.doi.org/10.1182/blood-2020-142238.
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Histologic review of bone marrow trephine biopsies is a central component of the diagnostic and treatment response evaluation of hematologic malignancies. Well-validated antibody reagents are routinely used for immunohistochemistry of these samples to provide additional insight into abnormal antigen expression. However, current immunohistochemistry staining protocols are typically limited to only one or two markers simultaneously. Dysplastic changes in cellular morphology and dyssynchronous expression of lineage markers are common features of myelodysplastic syndromes, myeloproliferative neoplasms and secondary acute leukemias. We have integrated the use of multiple diagnostic validated antibody clones with additional antibodies for hematologic lineages and structural proteins to create a 30-marker panel for imaging mass cytometry (IMC). Antibodies included in this panel identify myeloid, lymphoid, erythroid, macrophage, vascular, megakaryocyte and stromal markers as well as markers of cellular proliferation and apoptosis. Through conjugation to elemental metal tags, the entire panel is stained simultaneously on the tissue sample, then acquired by time-of-flight mass spectrometry on a Hyperion instrument (Fluidigm). Antibody staining concentrations and antigen retrieval conditions were optimized for formalin-fixed paraffin-embedded (FFPE) bone marrow to obtain consistent staining for all markers on the panel. Redundant markers for cell populations were selected to provide further internal validation of the observed staining patterns. After data acquisition, cell segmentation algorithms using CellProfiler and ilastik were applied to quantify marker expression in single cells and Phenograph in HistoCAT was used for cell population clustering. Cluster identities for all cells are associated with the original image location in order to plot the spatial arrangement of populations. Using this highly multiplexed panel, we have imaged sets of bone marrow specimens from patients with normal bone marrow morphology and those with myeloid malignancies. We initially confirmed the staining patterns expected for each antibody patterns of co-expression of lineage markers in normal bone marrow samples. We then extended this panel to examine biopsies from patients with myelodysplastic syndrome, myelofibrosis, and secondary acute myeloid leukemia. We found a clear population of CD71+ CD235a+ erythroid cells with strong expression of the proliferative marker Ki67 located within erythroid islands in normal bone marrow samples and MDS. Cell markers of apoptosis and DNA damage are scattered at low frequency throughout the bone marrow in samples with normal bone marrow morphology, but increased clusters of the DNA damage marker phospho-H2AX are observed in selected cases of myelodysplastic syndromes. Overall, this IMC imaging approach is able to extend the current clinical immunostaining for myeloid malignancies by identifying all major bone marrow cell populations. Through highly multiplexed analysis of bone marrow cell populations, the spatial architecture of cell populations and stromal structures can be elucidated, including erythroid islands, lymphoid aggregates and changes in vascular structures with increasing severity of myelofibrosis. In ongoing studies, the development of these imaging techniques for analysis of archived FFPE bone marrow samples is being applied to translational research on hematologic diseases. Disclosures Oh: Kartos Therapeutics: Consultancy; Disc Medicine: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Blueprint Medicines: Consultancy; Novartis: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy.
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Crassini, Kyle R., Yandong Shen, William S. Stevenson, Stephen P. Mulligan, Oliver Giles Best, and Richard Christopherson. "mRNA Profiling of CLL Cells Derived from the Blood, Bone Marrow and Lymph Node." Blood 132, Supplement 1 (2018): 1850. http://dx.doi.org/10.1182/blood-2018-99-118264.
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Abstract Background Chemo-immunotherapy remains the backbone of therapy for patients with chronic lymphocytic leukaemia (CLL), with evidence pointing towards long term remission and even cure in patients with mutated IGHV status receiving this therapy frontline (1). However, relapse is common especially in those harbouring abnormalities in TP53 or ATM, unmutated IGHV status or a complex karyotype (2). It has become increasingly apparent that the bone marrow (BM) and lymph node (LN) play important roles in promoting the survival and proliferation of CLL cells. Signalling pathways triggered by interactions within these niches, such as the B cell receptor (BCR) pathway, and intracellular proteins such as Bcl-2 are vitally important in the biology of CLL. Novel therapeutic agents, such as ibrutinib, which target components of the BCR pathway, and the Bcl-2 inhibitor venetoclax, have demonstrated the potential of targeted therapies in CLL (3, 4). Novel therapeutic approaches must target the proliferative, drug-resistant compartments of disease within these microenvironments. The NanoString® nCounter platform enables mRNA profiling of archival samples, including formalin-fixed, paraffin-embedded tissue (FFPE). We have previously demonstrated the utility of this technology by comparing the mRNA expression profile of CLL cells derived from the peripheral blood (PB), archival BM and LN tissue as well as PB-derived CLL cells following in vitro co-culture with a human stromal cell line under either normoxic or hypoxic conditions. Here we present an update on our previous work with increased sample numbers in each of the tissues or culture conditions. Methods RNA was extracted from FFPE BM trephines (n = 5) and LN sections (n = 5), using the QIAGEN RNeasy FFPE Kit. All biopsies analysed were comprised of > 80 % lymphocytes, as determined by microscopic review. RNA from PBMC fractions (n = 5) was isolated either immediately or following co-culture with HS5 stromal cells for 24 h under normoxic (n = 5) or hypoxic (n = 5) conditions using the QIAGEN RNeasy Mini Kit. RNA from all preparations was quantified using a NanoDrop™ spectrophotometer. A total of 200ng of FFPE-derived RNA and 100ng of PBMC-derived RNA was analysed per sample on the NanoString® platform using a 260 gene panel. Three-fold changes in mRNA expression were considered significant. Results Of the 260 genes profiled, 89 were upregulated in the BM samples and 52 in the LN samples compared to expression in PB-derived CLL cells. Changes were seen in genes encoding for proteins involved in chemotaxis (CXCL9), the regulation of apoptosis (BCL2L1), surface receptors (FLT3) and genes associated with intracellular signalling, metabolism and cell division. 35 genes were downregulated in the LN samples and 31 in the BM samples. These changes were seen in genes coding for surface receptors (ROR1 and CXCR4), genes coding for intracellular signalling proteins (RAF1) and genes coding for transcription factors (JUN and FOS). Co-culture of PBMCs with HS5 cells induced similar changes to those observed in our comparison of the PBMCs and BM samples; genes coding for 61.5% and 50.0% of the mRNA expression changes observed in the LN were observed in PBMCs cultured under normoxic and hypoxic conditions respectively. A similar comparison of the BM samples identified concordant changes in expression of 46.5% and 39.2% of genes under normoxic and hypoxic conditions respectively. Importantly, changes observed in genes coding for the anti-apoptotic protein MCL1, the surface receptors CXCR4 and ROR1 and the transcription factors ATF, FOS and JUN were consistent across samples from LN, BM and the in vitro model. In summary, we have utilised the NanoString® nCounter platform to profile PB, BM and LN-derived CLL cells and have identified panels of genes that are either up or down-regulated in cells derived from these microenvironments. Furthermore, the high concordance between RNA changes in the in vitro model and the primary tissue suggest the HS5 co-culture system mimics aspects of the tumour microenvironment. These data provide a better understanding of how CLL cells populate and proliferate in the tumour microenvironment and may lead to novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.
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Shergill, Ardaman, Santosh L. Saraf, Sujata Gaitonde, Damiano Rondelli, and Irum Khan. "CCN2 - Exploring a New Biomarker in Myelofibrosis." Blood 126, no. 23 (2015): 4063. http://dx.doi.org/10.1182/blood.v126.23.4063.4063.
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Abstract BACKGROUND: Bone marrow fibrosis in myelofibrosis (MF) is the result of a complex and poorly understood interaction between megakaryocytes, fibroblasts, endothelial cells, cytokines and marrow stroma. Preclinical studies support a pathobiological role of TGF-β. It is overexpressed by megakaryocytes in MF and the TGF-β signature is upregulated and has been targeted in MF animal models. However TGF-β is a pleiotropic cytokine implicated in many cellular processes. CCN proteins are a group of 6 matricellular proteins important in fibrotic diseases and injury repair. CCN1 (CYR61) and CCN2 (CTGF) are transcriptionally activated by mitogenic growth factors such as PDGF, FGF2 and TGF-β. Recombinant CCN2 induces differentiation of human bone marrow mesenchymal stem cells into fibroblasts (Lee et al. JCI 2010). Levels of CCN2 in biologic fluids correlate with severity of fibrosis in scleroderma, liver cirrhosis and nephropathy. Given the proven role of CCN2 as a measurable serum biomarker in pro-fibrotic diseases and a downstream effector of TGF-β, in this retrospective study we examined CCN2 expression in myelofibrosis. We studied its correlation with clinical response following allogeneic stem cell transplant (ASCT), the only therapeutic modality to date which can consistently reverse fibrosis. METHODS: Patients diagnosed with MF at our institution from 1998 to 2015 were identified by diagnostic code (IRB#2013-0896). Bone marrow (BM) specimens at diagnosis and 1 year following ASCT were retrieved. Staging bone marrows from lymphoma patients, read as normal, were used as negative controls. CCN2 localization and expression was assessed by IHC (Abcam 5097). The slides were scored from 0-100% for megakaryocyte cytoplasmic staining by a blinded hematopathologist. RNA was extracted and reverse transcribed from frozen fixed paraffin embedded (FFPE) BM samples using the Qiagen RNeasy FFPE RNA Purification Kit. Assays for CCN1, CCN2 and CCN3 (NOV) and endogenous controls RPLPO and GAPDH were performed using Taqman quantitative PCR assays. mRNA data were analyzed using the software package DataAssist (v3.01; Life Technologies). RESULTS: CCN2 expression is upregulated in myelofibrosis compared to healthy bone marrow. mRNA analysis showed a 27 fold increase (p=0.19) in CCN2 mRNA expression in patients with MF when compared to healthy controls. IHC data also showed increased expression in megakaryocytes of MF patients compared to controls (63% vs 40%, p=0.28). Exploratory analysis showed a 36 fold increase in CCN2 mRNA expression in JAK2 negative (n=3) MF patients compared to JAK2 positive (n=7) patients (p=0.06), suggesting expression may depend on the molecular profile. CCN2 expression in the bone marrow of myelofibrosis patients is significantly downregulated at 1 year after allogeneic stem cell transplant. 13 pre-treatment cases of MF were compared with 6 post-transplant cases. The mean percentage of megakaryocyte with cytoplasmic expression of CCN2 by IHC in MF at diagnosis was 63%, compared to 22% in post-transplant specimens, (p= 0.01). mRNA extracted from the post-transplant marrows showed 0.01 fold expression of CCN2 compared to pre-transplant (p= 0.18). This decrease in CCN2 correlated with clinical and pathologic resolution of disease. The average DIPSS score pre-transplant was 2 (range 0-5) which improved to 0 for all patients post-transplant. The mean BM cellularity pre-transplant was 82% (range 50%-95%) and post-transplant was 48% (range 35%-65%). The average decrease in cellularity after transplant was 29%. Reticulin fibrosis ranged from grade 2-3 in the pre-transplant MF bone marrow samples which improved to grade 0-1 post-transplant. CONCLUSIONS: CCN2 is a downstream effector of TGF-β and a measurable biomarker of fibrosis in fibroproliferative diseases. We show that CCN2 expression in myelofibrosis decreases significantly after ASCT, suggesting its role as a biomarker of fibrosis in this disease. This paralleled clinical and pathologic resolution of disease. This will need to be validated in a larger number of paired patient samples where in addition to bone marrow expression, serum CCN2 levels will be measured by ELISA. Disclosures No relevant conflicts of interest to declare.
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Martinez, Nerea, Carmen Almaraz, Manuela Mollejo, et al. "Mutational Status of Splenic Diffuse Red Pulp Small B-Cell Lymphoma Revealed By Whole Exome Sequencing." Blood 126, no. 23 (2015): 1448. http://dx.doi.org/10.1182/blood.v126.23.1448.1448.
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Abstract Background: Splenic diffuse red pulp lymphoma (SDRPL) is a rare small B cell neoplasm provisionally included in a category of unclassifiable splenic B-cell lymphoma/leukemias in the 2008 WHO classification. SDRPL is characterized by a diffuse pattern of involvement of the splenic red pulp by small monomorphous B lymphocytes. Patients are normally diagnosed at stage IV when spleen, bone marrow and peripheral blood are involved. This indolent but incurable disease is more common in aged males and it shows with splenomegaly and moderate lymphocytosis. The differential diagnosis with other splenic lymphomas such as marginal zone lymphoma, hairy cell lymphoma and its variant is not always easy, due to the similar clinical presentation and the absence of specific molecular markers. Here we studied the mutational status of 15 SDRPL patients using Whole Exome Next Generation Sequencing. Methods: Genomic DNA was extracted from FFPE/FF splenic tumor or bone marrow samples. When available, DNA from oral mucosa was obtained as the corresponding non-tumor control. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). Validation of variants was performed by PCR based targeted resequencing using a MiSeq instrument (Illumina Inc., San Diego, CA). We performed paired-end-76pb whole exome sequencing on DNA from 15 SDRPL patients. The corresponding normal counterpart from 3 of the patients was sequenced. From one patient FFPE and bone marrow DNA was available for comparison. In total 9 FFPE tissue samples, 3 FF tissue samples, and 4 bone marrow samples were sequenced. Almost 95% of the selected variants were validated by PCR based resequencing in 9 of the patients, while from 6 of the patients no tissue was available for validation. Results: 290 substitutions and 26 indels were obtained after filtering. Whole exome sequencing permitted us to identify variations in several genes of relevant pathways in lymphomas, such as NFkB pathway (IkBKB, TRAF, TANK, SYK), Apoptosis (BAD, DCPS, BCLAF1), MAPK (CXCR4, TCF3, NF1, MAP3K5), Cell cycle (CCND3, POLD3, BUB1), Chromatin (CREBBP, ARID1A, ARID1B, ARID3A, MLL3), MYC regulators (AKAP10, CTCF, EP400) or WNT signaling (SALL1, WNT5B, GPC6). Moreover, CCND3 and MLL3 were recurrently mutated in 2 different patients. Genes specifically found mutated in other splenic malignancies, such as NOTCH2, BRAF, MAP2K1, and KLF2 were not found mutated in this series of SDRPL patients. Conclusions: SDRPL samples contain somatic mutations involving genes regulating relevant pathways for cell survival, such as NFkB, apoptosis, cell cycle, chromatin, or WNT. The mutational signature of the series studied here may indicate that SDRPL is a distinct entity with specific molecular features different to other lymphoid splenic malignancies. Disclosures No relevant conflicts of interest to declare.
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Locke, Darren, Steven Bernstein, Frank Lynch, et al. "An IHC Screen For EphA3 Positive FFPE Tumors." Blood 122, no. 21 (2013): 4965. http://dx.doi.org/10.1182/blood.v122.21.4965.4965.
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Abstract Eph receptors are the largest subgroup of the receptor tyrosine kinases (RTK). Unlike other RTK, these function principally during development. Quiescent in postembryonic tissues, Eph expression by adult tissues is abnormal and implicated in tumor initiation, invasion and metastasis. Aberrant EphA3 expression is seen in solid and hematologic tumors, particularly advanced stage lymphoproliferative and myeloproliferative diseases. In this regard, targeting EphA3 may constitute a novel treatment for hematological malignancy. With clinical utility in mind, i.e., patient selection for anti-EphA3 therapy, a panel of commercial and proprietary (KaloBios) antibodies was screened by Western Blot for reactivity to recombinant Eph receptors (EphA3, EphA4, EphA5, EphA7, EphB2, EphB3). Those with EphA3 binding selectivity were further screened (QualTek) by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) normal and diseased human bone marrow (NLBM, AML) as well the LK63 pre-B-ALL cell line from which EphA3 was originally isolated. Different tissue pretreatments were used for antigen/epitope retrieval of EphA3, including steam-heating in citrate-based or Tris & chelator-based buffers, subsequent/or protease digestion, or neither. Following each antigen/epitope retrieval procedure, antibody reactivity for EphA3 was assessed by light microscopy using enzymatic biotin, tyramide and polymer-based detection techniques. In each instance, the location of the EphA3/antibody complex was visualized with 3,3-diaminobenzidine that precipitates a discrete insoluble reaction product in presence of enzyme (HRP). Nuclei were counterstained with hematoxylin to assess cell/tissue morphology. From this screen, one antibody (with an epitope in the cytoplasmic CT-domain of EphA3) was chosen for further assay optimization based on its selective reactivity towards LK63 and AML and low selective reactivity towards NLBM. Assay optimization included, amongst other aspects, evaluation of EphA3 expression in a panel of NLBM and hematopoietic tumors (200+ specimens inclusive of acute & chronic leukemia, peripheral T-cell & B-cell neoplasm, Hodgkin & non-Hodgkin lymphoma, multiple myeloma, myelodysplasic syndrome, myeloproliferative neoplasm) by a board-certified hematopathologist. In NLBM, the frequency of EphA3+ immature-blast cells (CD34+ or CD117+) was insignificant. Less than 10% CD34+/CD117+ cells were EphA3+. Elevated EphA3 expression (percentage EphA3+ nucleated cells) was observed in most hematopoietic tumors. For example, in multiple myeloma, tumor cells were typically EphA3+/CD138+ plasma cells. For AML, leukemic CD34+ or CD117+ blasts/initiating cells were typically EphA3+. Some CD138- plasma cells or leukemic CD34-/CD117- cells were also EphA3+. Correlation was made between EphA3 expression and specific tumor maturation stages, differentiation status and/or tumor aggressiveness. Tumors in blast crisis presented elevated EphA3 expression, e.g., CML in accelerated or blastic crisis but not chronic phase. Elevated EphA3 expression was noted in pre-B-ALL & pro-B-ALL (early pre-B-ALL) rather than mature B-cell ALL. EphA3 expression for some peripheral B-cell neoplasms correlated well with tumor grade: high grade, poorly differentiated (typically aggressive) B-cell lymphomas or follicular lymphomas were EphA3+. A similar relationship was noted for non-Hodgkin lymphoma (Hodgkin lymphoma was EphA3-). Preclinical screening also provided evidence for EphA3 expression by stroma/fibroblast (mostly lymphoma) and vasculature/endothelium, further rationale for development of reliable tools for profiling EphA3 in hematologic tumors and other malignancies. Using well-characterized normal and diseased FFPE bone marrow biopsies, this IHC assay for EphA3 has subsequently been validated (QualTek) to provide data that is not directly available from routine histopathology review and that supports use of the assay for profiling EphA3 in specific hematologic tumors and for patient selection in early Phase clinical trial/s of an anti-EphA3 monoclonal antibody (KB004, KaloBios). Beyond this, EphA3 targeted therapy with KB004 is anticipated for treatment of solid tumors. Recent genome-wide surveys identify EphA3 amongst the most frequently overexpressed genes in pancreatic, colon and lung carcinoma, melanoma and glioblastoma. Disclosures: Locke: QualTek Molecular Labs: Employment. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity’s Board of Directors or advisory committees. Yonker:KaloBios: Employment, Equity Ownership. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.
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Nath, Shriram Vaidia, Michelle Tamblyn, Susan Telfer, et al. "Ethylene Diamine Tetra Acetic Acid (EDTA) Decalcification of Paediatric Bone Marrow Trephines In a Diagnostic Laboratory." Blood 116, no. 21 (2010): 2566. http://dx.doi.org/10.1182/blood.v116.21.2566.2566.
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Abstract Abstract 2566 Background: In paediatric patients with haematological disorders such as acute lymphoblastic leukaemia (ALL), bone marrow aspiration is sometimes difficult to obtain and bone marrow trephine biopsy (BMTB) is a valuable source of material. In a diagnostic laboratory, the turnaround time is critical for a bone marrow trephine to be decalcified, processed and embedded. In our laboratory, 48 hours was routinely required from the time the bone marrow was performed until the sections were ready for reporting. A hydrochloric acid-EDTA (Ethylene-Diamine-Tetra-Acetic acid) decalcifying solution was used for 4 hours but rendered the trephines unsuitable for special studies such as Fluorescence in Situ Hybridisation (FISH). Aim: To evaluate an alternative decalcification method which preserved the ability to perform FISH on formalin-fixed paraffin-embedded (FFPE) tissue without compromising the turnaround time as a laboratory quality improvement measure. Method (EDTA decalcification): Following overnight formalin fixation, the BMTB was decalcified in a solution containing 20% EDTA with continuous stirring for 7.5 hours. The 20% EDTA, pH 7.1 stock solution was prepared by adding Ammonium Hydroxide (25%, concentrated ammonia) (Merck) to distilled water. EDTA disodium salt (372.24; Ajax Finechem) was added and the pH adjusted to pH 7.1 using concentrated ammonia. BM trephines were then processed routinely, embedded in paraffin and 4μm sections were mounted on Super Frost Plus slides. Haematoxylin-Eosin (H&E) staining, Silver Nitrate staining for Reticulin was performed on all slides and Immunocytochemistry, Immunofluorescence and FISH on selected slides. Patient Characteristics: 20 trephines from 15 patients underwent 7.5 hour EDTA decalcification. The diagnosis in 9 patients was Precursor-B ALL while one each had T-Cell ALL, Acute Myeloid Leukaemia, Hodgkin's disease, Refractory Anaemia, Drug Induced Anaemia and Idiopathic Thrombocytopenic Purpura. The mean age was 9.3 years (range 1.9–16.7years) and the mean trephine length was 12.8mm (range 6–21mm). Results: 100% decalcification was achieved in 18 trephines while in 2 trephines 95% decalcification was achieved on morphological examination. The turnaround time was 48 hours. The quality of H&E, reticulin stain, immunohistochemistry and immunofluorescence was maintained and FISH was successful on these FFPE BMTB tissues. This has lead to incorporation of this method for routine use in our laboratory. Conclusion: 20% EDTA decalcification of paediatric BMTB specimens is feasible without affecting the quality of histological preparations or turnaround time. The main advantage of the EDTA decalcification process is that the tissue is amenable to FISH analysis should it be required. Disclosures: No relevant conflicts of interest to declare.
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Horna, Pedro, Kathryn E. Pearce, Rhett P. Ketterling, and Jess Peterson. "Recurrent Chromosomal Abnormalities in Tumoral Lesions of Small Lymphocytic Lymphoma/Chronic Lymphocytic Leukemia: A Large-Scale Fluorescent in-Situ Hybridization Study on Tissue Biopsy Sections." Blood 134, Supplement_1 (2019): 4282. http://dx.doi.org/10.1182/blood-2019-129116.
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Background: Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) is a lymphoproliferative disorder of small mature B-cells, most commonly presenting with peripheral blood and bone marrow involvement. Tumoral lesions are variably encountered, resulting from either lymph node involvement or, less commonly, infiltration into virtually any extramedullary site. The prognostic assessment of patients with SLL/CLL relies strongly on the identification of recurrent chromosomal abnormalities, which are routinely tested by fluorescence in situ hybridization (FISH) on peripheral blood or bone marrow specimens. However, the incidence of such recurrent chromosomal abnormalities on tumoral lesions has only been studied on small series, and its concordance with peripheral blood and/or bone marrow testing remains unknown. We hereby report a large series of chromosomal abnormalities detected on tumoral SLL/CLL lesions, based on a validated FISH panel for formalin-fixed/paraffin-embedded (FFPE) biopsy sections. Methods: FISH was performed at Mayo Clinic on FFPE tissue sections from biopsies obtained from May 2014 to November 2018, as clinically indicated. Pathology reports and H&E-stained sections were reviewed and were consistent with a diagnosis of SLL/CLL in all cases. Probes were validated to detect previously described chromosomal abnormalities in SLL/CLL, including trisomy 12 (+12) and deletions of 6q23 (6q-), 11q22.3 (11q-), 13q14.3 (13q-) and 17p13.1 (17p-). Available FISH results performed on peripheral blood or bone marrow samples from these patients were also reviewed for comparison. Results: Tissue biopsies involved by SLL/CLL from 346 patients were evaluated by FISH. The majority of specimens were either lymph nodes (65%) or soft tissue masses (29%). The median age was 66.9 years (range: 36 to 91), and the male to female ratio was 2.1:1. FISH abnormalities were identified in 60% of evaluated tissue sections. The most frequently detected aberration was +12 (35%), followed by 13q- (24%), 11q- (15%), 17p- (6%) and 6q- (2%) (Figure A). In particular, the incidence of +12 was significantly higher, and the incidence of 13q- significantly lower compared to frequencies previously reported on peripheral blood or bone marrow specimens (p<0.01 for both abnormalities, in comparison to a recently published cohort of 1585 patients: Br J Haematol 2016;173:105). Most cases had 1 abnormality (55%), with fewer patients having 2 abnormalities (13%), and only 1 case showing 3 abnormalities. Of 47 patients with 2 or more chromosomal aberrations, most (72%) had 13q- in combination with either 11q- (7 cases), +12 (10 cases) or 17p- (7 cases) (Figure 1B). Of 29 patients with positive blood or bone marrow FISH results within 12 months of tissue biopsy, 7 patients (24%) had discrepant results, all of which were limited to discordant +12 and/or 13q- detection. In 14 additional patients with positive blood or bone marrow FISH results more than 12 months before or after tissue biopsy, discordant results were found in 3 cases (21%), all of which indicated gains of 17p- (2 cases) or 11q- (1 case) on the follow-up analysis. Conclusions: We hereby report the largest documented series (to the best of our knowledge) of FISH results performed on tumoral lesions of SLL/CLL. Trisomy 12 was overrepresented in tumoral lesions, as compared to the reported predominance of 13q- in leukemic involvement. Discrepant results between concurrent tumoral and leukemic FISH testing were limited to differences in +12 and 13q- in our series. We also document the acquisition of aberrancies of unfavorable prognosis (17p- and 11q-) on follow up FISH analysis in a small subset of patients, which should be taken into account when considering repeat FISH studies. Figure Disclosures Horna: MorphoSys AG: Research Funding.
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Kobayashi, Nobuhiko, Tsukasa Oda, Makiko Takizawa та ін. "Integrin α7 and Extracellular Matrix Laminin 211 Interaction Promotes Proliferation of Acute Myeloid Leukemia Cells and Is Associated with Granulocytic Sarcoma". Cancers 12, № 2 (2020): 363. http://dx.doi.org/10.3390/cancers12020363.
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Acute myeloid leukemia (AML) with granulocytic sarcoma (GS) is characterized by poor prognosis; however, its underlying mechanism is unclear. Bone marrow samples from 64 AML patients (9 with GS and 55 without GS) together with AML cell lines PL21, THP1, HL60, Kasumi-1, and KG-1 were used to elucidate the pathology of AML with GS. RNA-Seq analyses were performed on samples from seven AML patients with or without GS. Gene set enrichment analyses revealed significantly upregulated candidates on the cell surface of the GS group. Expression of the adhesion integrin α7 (ITGA7) was significantly higher in the GS group, as seen by RT-qPCR (p = 0.00188) and immunohistochemistry of bone marrow formalin-fixed, paraffin-embedded (FFPE) specimens. Flow cytometry revealed enhanced proliferation of PL21 and THP1 cells containing surface ITGA7 in the presence of laminin 211 and stimulated ERK phosphorylation; this effect was abrogated following ITGA7 knockdown or ERK inhibition. Overall, high ITGA7 expression was associated with poor patient survival (p = 0.0477). In summary, ITGA7 is highly expressed in AML with GS, and its ligand (laminin 211) stimulates cell proliferation through ERK signaling. This is the first study demonstrating the role of integrin α7 and extracellular matrix interactions in AML cell proliferation and extramedullary disease development.
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Flodr, Patrik, Pavla Latalova, Petra Pusciznova, et al. "Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease." Blood 126, no. 23 (2015): 5318. http://dx.doi.org/10.1182/blood.v126.23.5318.5318.
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Abstract Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens. Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2. Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%). Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A). Supported by the grant NT 14393. Disclosures No relevant conflicts of interest to declare.
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Lee, Sze Hwei, Tai-Chung Huang, Yuan Chang-Tsu, et al. "Discrepant Mutational Composition between Myeloid Sarcoma and Bone Marrow Leukemia Revealed through Targeted Next Generation Sequencing." Blood 132, Supplement 1 (2018): 1395. http://dx.doi.org/10.1182/blood-2018-99-116637.
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Abstract Introduction: Myeloid sarcoma (MS) is the involvement of acute myeloid leukemia (AML) cells in extramedullary tissues, whose mechanism remains to be further elucidated. How MS affects AML prognosis at a molecular level is also an open question. This unexplained tropism of leukemic blasts to extramedullary tissues is likely caused by the combination of genetic and epigenetic aberrations. Our study investigated the paired samples of myeloid sarcoma tissue and bone marrow (BM) at diagnosis in perspective of elucidating specific genetic abnormalities that underpin the ectopic homing of MS cells. Methods: A 62-patient cohort of pathologically-proven MS diagnosed between 2005-2018 was systemically reviewed. Histomorphology and immunophenotypes of all cases were reviewed by the hematopathologist. Cytogenetic karyotyping in BM cells was carried out with conventional G-banding method. Twenty-five cases of extramedullary presentation at initial diagnosis were further interrogated with next generation sequencing. DNA was extracted from formalin-fix-paraffin-embedded (FFPE) tissue using an error-avoiding reagent against formaldehyde-caused artificial single nucleotide changes (QIAGEN GeneRead). Mutations in 54 genes associated with myeloid disorders were analyzed in Illumina MiSeq sequencer along with the TruSight Myeloid Sequencing Panel. FLT3-ITD in bone marrow was detected by Sanger sequencing. Of these, 16 patients' paired samples (16 FFPE tissue and 16 BM tissue) were analyzed in comparison. Results: MS occurred concomitantly with leukemic marrow in 21 patients, without bone marrow disease in 2 patients, and resulted from transformation of MPN or MDS/MPN in 2 patients. The median age of diagnosis is 45 years (range 17-72 years), and the male to female ratio is 1.28. Twenty patients received standard treatment with systemic chemotherapy, and nine patients underwent subsequent allogeneic transplantation. The median overall survival was 31.6months ± 20.83 months with a median follow up of 28 months. Cytogenetic patterns included 9 patients with normal karyotypes, 5 with complex changes and 4 with a core-binding factor aberration. The median of mutation numbers in MS was three (range 0- 5) per case. In agreement with prior reports, the most common molecular abnormality in MS was NPM1 (11/25, 44%), followed by FLT3-ITD (7/25, 28%) and DNMT3A (6/25, 24%). The coexisting genetic alterations in the BM from patients without evident AML in morphology or immunophenotypic analyses, suggesting a role for clonal hematopoiesis in the BM. All of the cases with RUNX1-RUNX1T1 translocation harbored KIT mutation. Nineteen out of 25 cases (76%) had a mutation in the genes of receptor tyrosine kinase (RTK)- RAS pathway. In MS-BM paired samples, additional driver mutations in the MS samples included DNMT3A, IDH1, ASXL1, KRAS, CBL, NPM1, KIT and U2AF1. Conversely, one patient's BM PTPN11 mutation was lost in his MS lesion. For more details, the cytogenetic changes, mutational profile and the variant allele frequency were summarized in Figure 1. Conclusion: Interestingly, additional driver mutations were identified in the MS samples compared to the BM counterparts, suggesting that a comprehensive characterization of AML heterogeneity might need the mutational profiling of the coexisting MS. AML patients with MS infers a negative prognosis and were treated with frontline allogeneic stem cell transplantation whenever possible in this study. Consistent with prior report, this study showed a higher incidence of genetic alteration of NPM1, FLT3-ITD and DNMT3A in MS along with an enrichment of RTK-RAS signaling pathway mutations. Functional study is needed to delineate the biology underlying the ectopic homing of these commonly mutated genes in AML. (Figure legend) Molecular signature of 25 patients with myeloid sarcoma. Highlight in red represents pathologic mutation, highlight in blue represents mutation of unknown significance. Disclosures No relevant conflicts of interest to declare.
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Zhang, Wei, Min Xiao, Jianfeng Zhou, and Ken H. Young. "Targeted Next-Generation Sequencing of Cell-Free DNA in Diffuse Large B-Cell Lymphoma." Blood 132, Supplement 1 (2018): 4212. http://dx.doi.org/10.1182/blood-2018-99-116847.
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Abstract Background: Minimal residual disease (MRD) has an emerging diagnostic and prognostic role in Diffuse Large B-cell Lymphoma (DLBCL). However, repetitious bone marrow aspirations or lymph node biopsies would bring great pains to patients. At present, deep next-generation sequencing (NGS) based on circulating tumor DNA (ctDNA), allows quantitative mutational analysis with high sensitivity simultaneously, thereby providing us a promising non-invasive and radiation-free approach to monitor MRD and track sophisticated dynamic evolution of tumor clones. Methods: A total of 21 patients with denovo DLBCL and 6 patients with transformed DLBCL treated at our center were enrolled in this study. Formalin-fixed paraffin-embedded (FFPE) tissues at diagnosis were collected. Peripheral blood samples were obtained at the time of DLBCL diagnosed, remission, relapse, or other specific points in follow-up duration. Genomic DNA (gDNA) and cell-free DNA (cfDNA) were extracted from FFPE specimens and plasma samples, respectively. Targeted sequencing based on Illumina HiSeq 2000 were performed in all samples, which covering the whole coding sequences or hotspot regions in 61 genes known to be significantly associated with DLBCL. Library was prepared using unique molecular identifiers (UMI), which enabled the reduction of most synthesis-based errors. Sequencing depth was performed up to 2000× for FFPE specimens and 25000× for plasma samples, respectively. All mutations detected by cfDNA were validated by droplet digital PCR using Bio-Rad QX200TM. Concordance of filtered variants was determined between FFPE specimens and plasma samples at diagnosis. Correlation between clinical conditions and variant allele frequency (VAF) was also analyzed. This study was approved by Institutional Review Board of Tongji Hospital. Results: The median sequencing depth of FFPE specimens and cfDNA samples was 2312× and 28211×, respectively. Compared sequencing results of FFPE specimens with paired plasma samples at diagnosis, the number of cases with complete concordance, partial concordance, and complete discordance were 7, 12, and 8. For ultimate filtered variants detected in FFPE specimens and plasma at diagnosis, the consistency was 70% in transformed DLBCL and 55% in de novo DLBCL. Taking International Prognostic Index (IPI) score into consideration, the consistency of detection of denovo DLBCL patients with IPI score ≤ 2 was significant lower than that of patients with IPI score ≥ 3 (37% vs. 74%, P < 0.001). Besides, the presence of mutations in cfDNA also predicted MRD-positivity even though the patients achieved complete remission (CR), and the dynamic change of variant allele frequency is also associated with the evolution of tumor clones in most cases. Finally, long-last negativity of variants in cfDNA after treatment was significantly associated with superior progression-free survival. Conclusions: Our study indicated that targeted NGS of cfDNA in DLBCL is a promising test that facilitate disease monitoring and individualized therapy. A high level of concordance between FFPE specimens and plasma samples is associated with progression or evolvement that underlies clinical conditions, and ctDNA analysis may be a practical testing method to assess the prognosis of patients, although further investigations based on an extended sequencing panel and study cohort are warranted. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Duncan, Ian, Natalie Danziger, Daniel Duncan, et al. "Acid-Based Decalcification Methods Compromise Genomic Profiling from DNA and RNA." Blood 134, Supplement_1 (2019): 4659. http://dx.doi.org/10.1182/blood-2019-131362.
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BACKGROUND: Comprehensive genomic profiling (CGP) performed by next-generation sequencing of DNA detects genomic alterations including point mutations, insertions/deletions, copy number variations, and select gene rearrangements. When RNA sequencing is included in CGP, it allows for expanded detection of gene fusions, which are common in hematologic malignancies and sarcomas. When such tumors involve bone, a decalcification step is frequently employed to soften tissues prior to processing and sectioning. While commonly used acid-based decalcification methods work quickly, the resulting nucleic acid damage can be profound. In this study, we examine the effects of decalcification on DNA and RNA sequencing in the clinical setting. DESIGN: 1711 consecutive formalin-fixed paraffin embedded samples were evaluated by CGP during routine clinical care via DNA and RNA sequencing, using a hybrid-capture next-generation sequencing assay (FoundationOne®Heme). Specimen site [e.g. bone/ bone marrow or soft tissue] and decalcification status were extracted from pathology reports and H&E review. Samples were considered decalcified if reported as such in the pathology report or if visible decalcified bone was present on the H&E. Samples documented to be processed with fixatives other than formalin were excluded. Sequencing failures were defined as samples that failed DNA extraction (DNAx), RNA extraction (RNAx), or library construction (LC) due to insufficient nucleic acid to advance into sequencing. Samples were only evaluated for RNA if DNAx was successful (1594 cases). RESULTS: Specimen site was a strong predictor of sequencing failure, with a significant increase in failure rate from bone/bone marrow samples (n=619) compared to samples from soft tissue sites (n=1092) for both DNA (13.4% vs 4.6%, p=4.7E-9) and RNA (42.5% vs 13.5%, p<2.2E-16). Of the bone/bone marrow samples, 237 of 619 samples were decalcified. Decalcification was associated with significantly higher failure rates than non-decalcified samples for both DNA (29.1% vs 3.7%) and RNA (67.4% vs 30.8%) (Table 2). One method of avoiding decalcification for bone marrow samples is utilization of clot preparations, where aspirates are processed as an FFPE block. Clot preparations fail sequencing significantly less often than decalcified core biopsies (DNA: 3.3% vs 18.8%, p=9.2E-06; RNA: 39.2% vs 70.4%, p=2.5E-03) (Table 3). CONCLUSIONS: CGP of samples acquired from bone and bone marrow sites is challenging, with a lower success rate for DNA and RNA sequencing than soft tissue sites. The higher overall failure rate correlates with use of decalcification agents leading to degradation of nucleic acids and impacts RNA sequencing significantly more than DNA (67.4% vs 30.8% failed). Clot preparations of bone marrow samples performed better than core biopsies for both DNA and RNA. The higher overall RNA sequencing failure rates still observed in in non-decalcified bone/bone marrow are predominantly due to RNA failure of non-decalcified clot preparations. These samples likely have increased failure rates secondary the use of non-standard fixatives (e.g. B+, Bouin's, AZF, etc.) not documented in the pathology report and the frequency of hypocellular clot preparations in conjunction with higher requirements for RNA yield compared to DNA yield. To increase CGP success rates, decalcification should be avoided when possible. Peripheral blood and bone marrow aspirate samples rarely fail sequencing (<1%, data not shown) and are preferable to decalcified samples if adequate tumor is present. Bone marrow clot preparations perform better than bone marrow core biopsies and clot preparations should be fixed with 10% neutral buffered formalin. If decalcification is required for processing, EDTA based decalcification methods and/or minimizing decalcification times is recommended. Disclosures Duncan: Foundation Medicine, Inc.: Employment. Danziger:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Duncan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Hemmerich:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Edgerly:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Huang:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Vergilio:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Elvin:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. He:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Britt:Foundation Medicine, Inc: Employment. Reddy:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Sathyan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Alexander:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ross:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Brown:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ramkissoon:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Severson:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment.
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Dhakal, Binod, Shruti Sharma, Svetlana Shchegrova, et al. "Personalized, ctDNA analysis to detect minimal residual disease and identify patients at high risk of relapse with multiple myeloma." Journal of Clinical Oncology 39, no. 15_suppl (2021): 8029. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.8029.
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8029 Background: Despite treatment with high-dose chemotherapy followed by autologous stem cell transplantation (AHCT), MM patients invariably relapse. MRD-negativity post-AHCT has emerged as the most important prognostic marker. Currently, MRD in MM is monitored via bone marrow aspirate sampling. Marrow MRD assays are limited by the spatial heterogeneity of marrow MM localization; extramedullary disease and sampling variability of marrow aspiration. Sensitive, non-invasive blood-based MRD assay is an unmet need. ctDNA as a noninvasive biomarker can be utilized to predict relapse in MM. Here we attempt to evaluate MRD using ctDNA in AHCT recipients with MM. Methods: In this retrospective, single-center study, we analyzed ctDNA MRD in blood samples collected from 28 patients with MM after upfront AHCT. A total of 80 plasma timepoints were available pre and post AHCT with a median follow-up of 92.4 months. Multiparameter flow cytometry (MFC) at 10-4 level was used to assess the MRD from the BM biopsy. Individual bone marrow aspirates or FFPE slides from the time of MM diagnosis and matched normal blood were whole-exome sequenced, and somatic mutations were identified. MRD assessment at 3 months post-AHCT was performed by ctDNA analysis using a personalized, tumor-informed (SignateraTM, bespoke mPCR NGS assay). The prognostic value of ctDNA was evaluated by correlating MRD status with clinical outcomes. Results: Table provides the baseline disease characteristics. Median age was 67 [41-75] years and 16 [57.1%] were males. ctDNA was detectable in 70.8% (17/24) of pre-AHCT, 53.6% (15/28) of ̃3 months post-AHCT, and 39.2% (11/28) of patients during the surveillance phase post-AHCT. Of the 15 ctDNA MRD positive patients, 93.3% (n=14) experienced relapse on follow-up (hazard ratio: 5.64; 95% CI: 1.8-17; p=0.0003). Patients negative for ctDNA at 3 months post-AHCT had significantly superior progression-free survival (PFS) compared to positive (median PFS, 84 months vs. 31 months; p=0.003) The positive predictive value (PPV) for relapse among patients positive for ctDNA at 3 months post-AHCT was 93.3%, and significantly higher than marrow MFC of 68.4%. Conclusions: Our study shows the feasibility that a tumor-informed assay on archival blood samples is predictive of relapse post-AHCT. Future prospective studies with real-time marrow NGS and ctDNA samples are needed to define the role of ctDNA in MM and its prognostic significance.[Table: see text]
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Snyder, Matthew J., William D. Bradford, Priya S. Kishnani, and Laura P. Hale. "Idiopathic Hyperammonemia following an Unrelated Cord Blood Transplant for Mucopolysaccharidosis I." Pediatric and Developmental Pathology 6, no. 1 (2003): 78–83. http://dx.doi.org/10.1007/s10024-001-0271-3.
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Bone marrow transplantation (BMT) has been shown to reverse or stabilize some manifestations of mucopolysaccharidosis I (Hurler syndrome). Idiopathic hyperammonemia (IHA) is a rare complication of solid organ and BMT that is characterized by elevated serum ammonia, normal liver enzymes, and abrupt onset of neurologic deterioration. We present the case of a 14-month-old male patient with Hurler syndrome who developed fatal IHA (ammonia = 2297 μmol/L) 31 days after a cord blood transplant. A complete autopsy was performed, with examination of both frozen and formalin-fixed paraffin-embedded (FFPE) tissues using a variety of special stains and electron microscopy. Hyperammonemia was documented by analysis of antemortem serum and postmortem cerebrospinal and vitreous fluid. Other causes of hyperammonemia, including Reye syndrome, were excluded. Histologic changes included centrilobular microvesicular steatosis of the liver and storage product present in multiple organs. The highly water-soluble mucopolysaccharide (MPS) storage product was best identified by colloidal iron staining of FFPE and unfixed air-dried fresh frozen liver sections. Alcian blue stains failed to convincingly demonstrate MPS in any of the liver sections. This is the first published report, to our knowledge, of IHA in a posttransplant patient younger than 18 years old or following transplantation for Hurler syndrome. Demonstration of the hepatic centrilobular microvesicular steatosis characteristic of IHA was complicated by the diffuse storage of MPS within the liver. MPS storage can be best detected in the liver using colloidal iron staining. Oil-red-O staining may be useful to document microvesicular steatosis in cases with a clinical history of hyperammonemia following solid organ or BMT. Determining if certain subsets of children are at increased risk for IHA requires further study.
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Reading, N. Scott, Josef T. Prchal, Ronald Hoffman, and Mohamed E. Salama. "Digital Immune Expression Profiling Coupled with Immunohistochemistry for Interrogation of Microenvironment in Formalin Fixed Paraffin Embedded Specimens of Marrow and Spleen from PMF Patients." Blood 126, no. 23 (2015): 2832. http://dx.doi.org/10.1182/blood.v126.23.2832.2832.
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Abstract Background: Gene expression profiling studies have demonstrated aberrant expression of inflammatory response genes in myeloproliferative neoplasm (MPN) granulocytes and/or CD34+ cells. Our understanding of the immune response to primary myelofibrosis (PMF) hematopoietic stem cells and tissue-specific microenvironments is not complete due to a limited availability of bone marrow (BM) aspirates and fresh spleen samples from PMF patients. In order to overcome this obstacle, we utilized a novel approach with mRNA enrichment analysis which utilizes formalin fixed, paraffin embedded (FFPE) specimens of BM and spleen from PMF patients to identify immune and other microenvironment cell types and to construct pathway activation patterns. Methods: We applied enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) consisting of 770 standard gene panel and 20 custom gene panel for identification of immune cells and assessing immunological milieus in the microenvironement of matched, archival FFPE spleen and BM samples from MPN patients.. Up to 500ng of RNA (at 100ng/ul) isolated from FFPE BM and/or spleen specimens from PMF patients was used for digital expression profiling in accordance with the manufacturer's protocol. The panel included 109 genes that define 24 immune cell types and populations, and 40 housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 2.5 and the PanCancer Immune Profiling Advanced Analysis Module (v.1.0.22). Findings identified from the digital expression profiles on cells types were confirmed via immunohistochemical evaluation. Results: Twenty-six archival FFPE tissue samples (13 BM and 13 spleen) obtained from PMF patients who had undergone therapeutic splenectomy and BM biopsy at the same time, and normal tissue controls, were analyzed as described previously (Liew et al 2015). Following data normalization, genes were selected based on P < 0.05 (unpaired t -test) and fold change > 2.0 differentially expressed mRNA levels in the BM (n=208) and spleen (n=108). These genes were distributed across several functional categories including: TNF superfamily (e.g. TNFRS13C, CD70, LTB), signal ligands (cytokine, chemokine) (e.g. JAK3, IFI16, SPP1), B and T cell functions (TIGIT, CXCR5, CXCL14), and cell adhesion (e.g. ITGB3). In supervised clustering of the significantly expressed genes, the first bifurcation of the dendrogram separated controls from PMF samples in both BM and spleen. Twenty-seven genes were significantly differentially expressed by both PMF BM and spleen, compared to control specimens. Interestingly, the PMF BM samples were further separated in a second bifurcation of the dendrogram into 3 subgroups, indicating immune transcriptional diversity within PMF samples (Figure 1). Further characterization of these subgroups and potential clinical relevance are being studied in a larger number of specimens in order to achieve statistical power. Cell type analysis indicated a significant (P =<0.05) difference in activated CD4 T-cells, T helper-1 cells, CD8 T-cells, and B-cells across all BM and spleen samples. Macrophages (P =<0.001) were increased in the spleen, and neutrophils (P =0.01) were increased only in BM samples. A decrease in CD8-positive T-cells in PMF samples (p =0.009) was confirmed using immunohistochemistry with computer assisted image analysis. Heterogeneity of Tregs in PMF spleen samples (n=10) was further confirmed by immunohistochemistry (n-3). Conclusions: Digital immune expression profiling coupled with immunohistochemistry is a novel approach for characterization of tumor microenvironment in fibrotic PMF marrow and spleen. Our preliminary findings indicate a consistent decrease in cytotoxic CD8 T-cells but varying expression of Tregs. In addition, we identified several genes in various immune functional categories within PMF patients that could potentially serve as therapy targets. Disclosures Hoffman: All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salama:Promedior: Consultancy.
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Greene, John Patrick, Orla Casey, Karen O'Leary, et al. "The Irish Programme for Stratified Prostate Cancer Therapy (iPROSPECT)." Journal of Clinical Oncology 34, no. 2_suppl (2016): 335. http://dx.doi.org/10.1200/jco.2016.34.2_suppl.335.
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335 Background: iPROSPECT is a prospective longitudinal translational study, funded through the Irish Cancer Society in partnership with Movember that is examining blood and tissue markers that might correlate with treatment response and survival and aid future stratification of metastatic prostate cancer (mPCa) patients with regard to optimal personalised treatment regimens. Methods: Patients with metastatic prostate cancer who are beginning or changing treatment are eligible for inclusion. Nine hospital sites supported by four research laboratories are open to recruitment. Cohort 1 (n = 15) includes patients with newly diagnosed mPCa who are beginning treatment with androgen deprivation therapy (ADT). Cohort 2 (n = 45) includes patients with CRPC who are beginning or changing treatment. All enrolled patients have blood drawn for plasma, serum and CTC analysis at baseline and thereafter every 4 months. All eligible patients with metastatic bone disease are offered an optional substudy of bone marrow biopsy performed prior to starting treatment. Any patient with visceral or nodal disease is offered a targeted CT guided biopsy. All patients are offered the PCa Research Consortium questionnaire, FACT-P and the Brief Pain inventory short form. Results: Between March and August 2015, 15 patients within cohort 1 (100%) have been enrolled and 22 patients in cohort 2 (48.8%). In cohort 1, the median age of patients was 66, median PSA at entry was 9.2 with an average Gleason score of 8. In cohort 2, the median age of patients was 68, median PSA at entry was 236.4 with an average Gleason score of 8. Eight patients underwent bone marrow biopsy, and 2 patients underwent CT guided biopsies. All patients had archival FFPE tissue collected. Questionnaires were collected from all eligible patients. Conclusions: The recruitment rate to date has indicated successful implementation of this study. Results will identify how clinical and biological patterns change over time and relate to each individual’s treatment response and progression of disease. [Table: see text]
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Locke, Darren, Frank Lynch, Steven Bernstein, Koushan Siami-Namini, and Geoff T. Yarranton. "An IHC Screen for EphA3 Positive Myelofibrosis (MF) and Other Myeloproliferative Neoplasms (MPNs)." Blood 124, no. 21 (2014): 5588. http://dx.doi.org/10.1182/blood.v124.21.5588.5588.
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Abstract MF is a neoplastic stem cell disorder in which a multipotent hematopoietic stem cell acquires a clonal proliferative advantage, and its progeny inappropriately releases fibrogenic factors into the bone marrow microenvironment, leading to secondary bone marrow fibrosis. The cytokines implicated in the pathogenesis of MF include transforming growth factor β (TGF-β), basic fibroblastic growth factor (b-FGF), and platelet-derived growth factor (PDGF). Some of these cytokines, such as b-FGF and PDGF can act as angiogenic factors. Patients with MF have higher concentrations of circulating vascular endothelial growth factor (VEGF) and b-FGF than do control subjects. In addition, a higher degree of bone marrow angiogenesis has been reported in patients with MF. Eph receptors form the largest subgroup of receptor tyrosine kinases (RTK). The Ephrins are the ligands of the Ephs and stimulate bi-directional signaling allowing cell movement and shape change. EphA3 is a member of the Eph family and is expressed mainly during fetal development. Aberrant expression of EphA3 is detected in some solid and hematologic tumors. Recently, the expression of EphA3 has been detected in bone samples from subjects with MF. Antibody targeting of EphA3 may therefore constitute a novel approach to treating MF and other hematologic malignancies. Using well-characterized normal and diseased FFPE bone marrow biopsies, an IHC assay for EphA3 expression, which was developed and validated previously for use in AML and MDS patient screening, was evaluated further for use on MPNs, such as MF, polycythemia vera (PV) and essential thrombocythemia (ET). This IHC assay for EphA3 expression was validated (intra-assay variability/precision and inter-assay variability/reproducibility established by variance of SHS for serial tissue sections) using MPNs with negative, low, medium and high intensity and/or percent positive expression of EphA3. A numerical scoring scheme (semi-quantitative simplified H-Score [SHS]) was used by a-board certified anatomic & clinical pathologist to capture tumor & non-tumor (specifically, fibroblast) reactivity of EphA3-specific monoclonal antibody. A survey of ten (10) normal bone marrow (NLBM), thirty five (35) cases of primary MF, five (5) post PV-MF cases, six (6) PV cases and four (4) cases with ET was made with the validated IHC assay. For NLBM, there was low level reactivity in a subset of immature-blast cells that represent a small fraction (1-5% or less) of the total population of nucleated cells. There was no reactivity in fibroblasts, if present. Low EphA3 immunoreactivity of NLBM samples per se is consistent with published RT-PCR data. For MPNs, significant EphA3 expression in both tumor and tumor-associated stromal fibroblasts was noted (not solely in areas of fibrosis, although not all MPN samples studied were in the fibrotic phase). Interpretation of EphA3 status in these NLBM and MPNs was used to refine the semi-quantitative SHS scheme in anticipation of setting patient sample cut-off. Between 60-70% of MF samples were deemed EphA3+ using this IHC assay for EphA3 expression. Targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating MF and other MPNs. The Phase 2 component of a clinical study is ongoing in which the activity of KB004, a non-fucosylated anti-EphA3 Humaneered® antibody, will be characterized in disease specific cohorts including AML, MDS, MF and other MPNs. Disclosures Locke: QualTek Molecular Labs: Employment. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership.
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Maciocia, Paul, Margarida Neves, Shimobi Onuoha, et al. "Analysis of T-Cell Receptor Beta-Constant Region Expression for Rapid Assessment of T-Cell Clonality." Blood 132, Supplement 1 (2018): 2867. http://dx.doi.org/10.1182/blood-2018-99-112569.
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Abstract Introduction T-cell malignancies are heterogenous disorders, representing approximately 10-15% of cases of non-Hodgkin's lymphoma and 15% of acute lymphoblastic leukaemia. Diagnosis may be complicated both by the fact that non-malignant oligoclonal T-cell proliferations often mimic T-cell cancers, and due to the lack of consistent markers of malignancy which discriminate healthy and malignant T-cells. B-cell lymphoproliferations, by contrast, can be reliably identified as clonal by confirmation of restricted kappa or lambda light chain expression. We have recently described an analogous method for evaluation of T-cell clonality, by evaluating expression of one of two virtually identical and mutually exclusive alleles at the T-cell receptor beta constant region: TRBC1 or TRBC2. We demonstrated that approximately 35% of normal T-cells and T-cell malignancies expressed TRBC1, using a TRBC1-specific antibody suitable for use in fresh/ frozen tissues. We have now developed a novel antibody with TRBC2 specificity, which recognises an intracellular, non-surface exposed epitope and can be used in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. Here we demonstrate TRBC1/2 assessment can be used to identify T-cell clonality using immunohistochemistry on FFPE tissues, and confirm our results using reference methods including Sanger and massively parallel sequencing. Methods/ Results To generate anti-TRBC2 antibody, we immunised New Zealand rabbits with a peptide comprising the intracellular portion of the TCR beta-2 chain (VKRKDSRG). Protein A-purified immunoglobulin from the sera of immunised rabbits was tested for reactivity to TRBC1 (VKRKDF) and TRBC2 peptides by ELISA. Strong TRBC2-specificity with limited TRBC1 binding was seen. Immunoglobulin was then depleted of residual TRBC1 reactivity by depletion on TRBC1 peptide-coated agarose beads. Following this, no reactivity to TRBC1 peptide could be detected by ELISA. We then stained fresh frozen and FFPE samples of pelleted TRBC1 and TRBC2 cell lines with the novel, polyclonal anti-TRBC2 antibody. We confirmed that anti-TRBC2 antibody stained only TRBC2 and not TRBC1 cell lines, and that the antibody performed equivalently in fresh or FFPE preparations. We then showed that in a range of normal tissues, including tonsil, spleen and others, a staining pattern restricted to only a proportion of T-cells was seen. In bone marrow sections, some staining of megakaryocytes was also seen, as has been previously demonstrated for other T-cell markers such as linker for activation of T-cells (LAT). No other non-T-cell staining was noted. We obtained paired FFPE and frozen samples of several TCR+ T-cell malignancies. We performed staining for TRBC1 and TRBC2 on both frozen and FFPE samples: TRBC1 staining was performed only on frozen samples. In addition, we extracted DNA from these tissues and performed PCR for TCR beta VDJ rearrangement using standard BIOMED protocols. Clonal proliferations were identified by heteroduplex gel analysis and capillary electrophoresis (Sanger) sequencing was performed on dominant bands. In addition, pooled PCR products were subjected to massively parallel sequencing to quantify clone frequency and to confirm VDJ identity. We demonstrated mutual exclusivity between samples staining positive for TRBC1 and TRBC2, and concordance between TRBC1/2 status identified by Sanger sequencing, NGS and antibody-based methods. Further, in a number of TCR+ malignancies, non-T-cell haematological tumours and reactive lymphoproliferations, where only FFPE material was available, we used anti-TRBC2 antibody to evaluate TRBC2 expression. We demonstrated that in non-T cell tumours, a limited pattern of staining was seen, restricted to a proportion of infiltrating normal T-cells only. T-cell tumours, by contrast, were uniformly TRBC2-negative or positive, across a range of entities. 18/29 (62%) of T-cell neoplasms were TRBC2-positive. Conclusions We have generated a new TRBC2-specific polyclonal reagent suitable for use in FFPE tissues, which displays minimal cross-reactivity against other cell types. A monoclonal reagent is in preparation. We suggest that TRBC1/2 assessment can thus be used to rapidly identify clonal T-cell lymphoproliferations and may be a helpful addition to the diagnostic haematopathology toolkit. Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: UCLB. Neves:Autolus Ltd: Employment. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.
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Georg, Greiner, Michael Gurbisz, Franz Ratzinger, et al. "Molecular Quantification of Tissue Mast Cell Burden in Systemic Mastocytosis: A New Approach for Diagnostics and Prognostication." Blood 132, Supplement 1 (2018): 3043. http://dx.doi.org/10.1182/blood-2018-99-116492.
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Abstract Background: The somatic KIT D816V mutation leads to an activation of the receptor tyrosine kinase KIT and represents a diagnostic criterion for systemic mastocytosis (SM). In the majority of patients, only a very few KIT D816V+ mast cells (MC) or MC precursors, if any, are found in peripheral blood (PB) and bone marrow (BM) aspirates. In contrast, the MC count in BM biopsies is typically much higher. Melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) is widely used for detecting KIT mutations in biopsies.1 We have previously proposed dPCR as a new standard method of KIT D816V testing in SM that can also reliably quantify the variant allele fraction (VAF) in formalin-fixed paraffin-embedded (FFPE) material.2 While multilineage involvement of KIT D816V indicated by a high VAF in PB was associated with an aggressive clinical course,3-6 the mutant allele burden in tissue sections has not been studied in a large series of SM patients. Methods: The VAF of KIT D816V in the tissue was assessed by dPCR (PrimePCR ddPCR, Biorad) on DNA isolated from 211 FFPE BM sections from 116 patients with SM (median age 53 years; 58 females, 58 males) and 57 controls (lymphoma patients undergoing staging biopsy). A total of 91 SM patients were diagnosed with indolent SM (ISM) and 25 with advanced SM (aggressive SM, SM with associated hematologic neoplasm, and mast cell leukemia according to WHO criteria). Results of diagnostic samples were compared to qualitative analysis of KIT D816V clamp-PCR. BM MC infiltration was quantified in a representative area of the tryptase stained section and expressed as % of nucleated cells. Total tryptase levels in serum were determined by a fluoroenzyme-immunoassay. Results: The KIT D816V mutation burden in FFPE BM sections of SM patients showed marked differences between patients ranging from 0.027% to 60% VAF (median: 1.9%). The VAF in BM sections was largely independent from that in liquid specimen (PB and BM aspirate). In particular, a number of ISM patients showed substantially higher VAF in FFPE tissue than in liquid specimen (Figure 1A). In line with this, a higher correlation of KIT D816V mutation burden to BM MC infiltration and serum tryptase levels was observed for FFPE tissue (Spearman's coefficient of correlation r = 0.68 and r = 0.68 respectively; Figure 1B-C) than for liquid specimen (r = 0.48 and r = 0.58 respectively). When analyzing subgroups of SM, FFPE BM sections of patients with advanced SM had a significantly higher KIT D816V allele burden (median 23.40%) compared to patients with ISM (median 1.65%; p<0.001 Figure 1D). Finally, we assessed the sensitivity and specificity of dPCR for KIT D816V analysis in FFPE BM section compared to clamp PCR. While a specificity of 100% was observed for both assays, dPCR showed a significantly higher sensitivity to detect the mutation in BM tissue of ISM patients compared to clamp PCR (97% vs. 89%; p<0.05). Conclusion: dPCR is a sensitive method to detect KIT D816V and reliably quantify the mutant allele burden in FFPE BM sections of SM patients. The mutational burden in the tissue represents a new molecular marker of the disease burden in SM since it reflects both the multilineage involvement of KIT D816V and the MC burden while the mutation burden in PB primarily reflects the KIT D816V multilineage involvement. Although the tissue mutational burden in patients with advanced SM is higher compared to ISM, the difference is not as substantial as described for PB. Thus, the prognostic value of KIT D816V allele burden measurement in SM needs to be redefined for BM sections. Potential future applications of KIT D816V quantification in FFPE BM sections include treatment response monitoring and definition of high disease burden in smoldering SM (SSM). We propose to include dPCR-based KIT D816V mutant allele burden measurement in BM tissue as a new biomarker for disease burden in SM in future clinical trials. References: 1. Sotlar K, et al. Am J Pathol. 2003;162(3):737-746. 2. Greiner G, et al. Clin Chem. 2018;64(3):547-555. 3. Erben P, et al. Ann Hematol. 2014;93(1):81-88. 4. Hoermann G, et al. Allergy. 2014;69(6):810-813. 5. Jara-Acevedo M, et al. Mod Pathol. 2015;28(8):1138-1149. 6. Broesby-Olsen S, et al. J Allergy Clin Immunol. 2013;132(3):723-728. Figure 1 Figure 1. Disclosures Sperr: Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Hoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria.
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Wen, Yu-Ye, Erica Fang, Yanchun Li, Condie Edwin Carmack, and Marilyn M. Li. "The efficacy of targeted next-generation sequencing for detection of clinically actionable mutations in cancer." Journal of Clinical Oncology 30, no. 15_suppl (2012): 10598. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.10598.
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10598 Background: The emergence of next-generation sequencing (NGS) technologies has significantly accelerated the identification of cancer-causing mutations and the development of personalized cancer care. However, the clinical application of these technologies to detect cancer gene mutations has been extremely limited due to the long turn around time, the high cost, and large amount of input DNA required by existing NGS-based tests. Methods: We have assessed the performance of a novel NGS technology that merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life Technologies, Inc.) in our clinical diagnostic laboratory. The test interrogates 739 common mutations in 46 cancer genes including many clinically actionable mutations concurrently. First, we studied 12 tumor samples including 4 archived FFPE, 4 blood/bone marrow, and 4 cell line samples with known mutations to evaluate the sensitivity and specificity of the test. We then studied 34 de-identified, archived FFPE tumor samples of unknown genotype to further evaluate the efficacy of the test. Results: We successfully identified all known mutations previously detected by Pyposequencing or Sanger sequencing technologies. Multiple serial dilution studies showed that the test could detect mutations at frequencies as low as 5% with 99% confidence. For the samples of unknown genotype, we detected 23 COSMIC mutations in 16 samples including HRAS, BRAF, MET, TP53 mutations in lung cancer, KRAS, PIK3CA, TP53, APC, BRAF, ERBB2 mutations in colon cancer, TP53 and KRAS mutations in breast cancer, and KIT and PDGFRA mutations in GIST. Analysis of the variant call data showed that a minimum of 100X coverage is required in order to detect mutations at 10% frequency or above; a minimum 300K final library reads are necessary in order to minimize/eliminate amplicon dropout. Conclusions: The targeted NGS test can effectively detect cancer gene mutation with input DNA as low as a few nanograms, turn around time can be as short as two days, and can significantly lower cost compared to traditional Sanger sequencing. Our experience demonstrates that this technology holds great potential for clinical use, including diagnostic and therapeutic applications.
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Kuo, Szu-Yin, Hongxiang Liu, Yung-Liang Liao, et al. "A parallel comparison of T-cell clonality assessment between an in-house PCR assay and the BIOMED-2 assay leading to an efficient and cost-effective strategy." Journal of Clinical Pathology 64, no. 6 (2011): 536–42. http://dx.doi.org/10.1136/jcp.2010.086637.
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AimsDiagnosis of T-cell lymphoproliferation is sometimes challenging, and in certain instances pathologists rely heavily on the clonality assessment results of T-cell receptor (TCR) gene rearrangement (TCR-GR). Many investigators have designed various in-house primer sets for PCR-based study targeting different loci of TCR genes. In recent years, the commercial BIOMED-2 protocols have become available. The in-house primers are very cheap while the BIOMED-2 primers are expensive. This parallel study aimed to compare the sensitivity of the in-house TCRG primers (two reactions) and the BIOMED-2 TCR primers (six reactions) in an attempt to develop a sensitive and cost-effective strategy for TCR-GR assessment.MethodsPCR-based analysis was performed on 69 samples of T-lineage neoplasms including 60 formalin-fixed paraffin-embedded (FFPE) tissues, 5 samples from peripheral blood (PB) and 4 samples from bone marrow (BM) aspirate.ResultsForty-seven (78%) FFPE and all PB or BM aspirate samples yielded control DNA products suitable for clonality assessment including 4 precursor and 50 mature T-cell neoplasms. The detection rates of clonal TCR-GR were 63% (34/54) by the two in-house TCRG primers, 85% (46/54) by all six BIOMED-2 reactions, 91% (49/54) by combining the in-house and BIOMED-2 TCRG reactions and 94% (51/54) by combining the in-house and all BIOMED-2 reactions. By using the in-house and BIOMED-2 TCRG reactions with a total of four tubes, clonal TCR-GR was detected in 91% of the cases. The reagent cost for this combination was one-third of that for the six BIOMED-2 reactions and the detection rate was also higher than the latter alone (91% vs 85%).ConclusionsAs the in-house primers were custom made and are much cheaper than the commercial kits, the authors concluded that this four-tube strategy was cost-effective and efficient for TCR-GR clonality assessment.
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Daniel, Sugganth, Erica Gornstein, Garrett Michael Frampton, et al. "BRCA1/2 reversion mutations in prostate cancer identified from clinical tissue and liquid biopsy samples." Journal of Clinical Oncology 35, no. 15_suppl (2017): 5024. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.5024.
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5024 Background: Prostate tumors with genomic alterations (GA) in BRCA1 or BRCA2 ( BRCA) may be sensitive to treatment with PARP inhibitors (PARPi). However, secondary reversion mutations (revGA) can arise that may restore BRCA function and underlie reduced sensitivity to PARPi or platinum (Pt)-based therapy. Comprehensive genomic profiling (CGP), using either tissue or liquid biopsies, can detect the variety of clinically relevant revGA that can arise. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for 1911 patients with predominantly relapsed, refractory or metastatic prostate carcinoma was analyzed by hybrid-capture, next-generation sequencing for all classes of GA: base substitutions, indels, rearrangements, and copy number changes. RevGA were any GA that could restore the reading frame if in cis with a nonsense or frameshift (fs) GA. Results: 216/1911 (11.3% ± 1.4%) tumors had ≥1 deleterious BRCA GA. Of these, 7 samples harbored potential revGA in BRCA1 (1) or BRCA2 (6): prostate acinar adenocarcinoma (5), neuroendocrine carcinoma (1), or undifferentiated carcinoma (1). Of these, 2 samples were liquid biopsies (blood), 4 were FFPE tissue samples (liver), and 1 sample was a bone marrow core biopsy. All samples with revGA were metastases. Potential revGA were of 3 types: overlapping indel (4), compensatory fs (2), and missense (1). One case harbored 2 revGA, an overlapping indel and a compensatory fs. Alteration frequencies for TMPRSS2 (fusions), PTEN, and AR were similar with or without BRCA mutations; revGA-positive samples had a modest increase in PTEN alterations (42.9% vs 33.8%, NS). The frequency of CDK12 alterations was significantly reduced in BRCA-mutated tumors (0.9% vs 6.9%, p = 0.00002). Clinical histories for patients with reversion mutations will be presented. Conclusions: CGP of 1911 prostate carcinomas reveals ≥1 deleterious BRCA1/2 in 11.3% of samples. From these, a series of 7 cases, all metastases, with co-occurring potential revGA were identified. Although rare, revGA can be acquired during treatment and may underlie resistance to PARPi or Pt-based therapy. BRCA revGA can be detected from both tissue and liquid biopsies.
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Shirane, Shuichi, Marito Araki, Soji Morishita, et al. "Dynamic Increase in JAK2V617F Allele Burden Is a Predictive Parameter for the Transformation into Myelofibrosis from Polycythemia Vera and Essential Thrombocytosis." Blood 124, no. 21 (2014): 1832. http://dx.doi.org/10.1182/blood.v124.21.1832.1832.
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Abstract Patients diagnosed with polycythemia vera (PV) or essential thrombocythemia (ET), a subtype of myeloproliferative neoplasms (MPN), sometimes suffer disease transformation into myelofibrosis (MF) associated with poorer prognosis. Thus, predicting which patients have a risk of MF transformation is an important task. Following the identification of a driver mutation JAK2V617F in a majority of MPN patients, several studies was performed to investigate the potential of JAK2V617F allele burden as a diagnostic marker for MF-transformation. However, the results differ between cohorts presumably due to a lack of accurate JAK2V617F allele burden measurement. Since we have previously developed alternative-binding probe competitive polymerase chain reaction (ABC-PCR) that accurately determines the JAK2V617F allele burden, we assessed the predictive value of JAK2V617F allele burden in MF-transformation in Japanese MPN cohort. In a retrospective study, we compared JAK2V617F allele burdens between formalin-fixed paraffin embedded-bone marrow (FFPE-BM) from initial diagnosis and peripheral blood (PB) from follow-up visits. We first examined whether the allele burdens in FFPE-BM and PB were comparable when they are collected at the same time. Determining the allele burdens in a set of FFPE-BM and PB taken from same patient within a 3-month period, we observed that allele burdens from these specimens are significantly correlated (n=26, R²=0.97), which is consistent with previous report with a larger cohort (Blood 122; 3784-6). Thus, in subsequent analyses, we set a base line of the mutant allele burden determined from FFPE-BM, which is then compared with allele burdens from PB during the disease duration. We examined 14 PV and 20 ET patients (mean disease duration 69.2 months) defined by WHO 2008 MPN criteria. From first diagnosis to the time when MF-transformation was first recognized, JAK2V617F allele burden was significantly increased (mean increase 19.5±17.3%, p=0.044) in patients with MF-transformation (n=11). While patients with no MF-transformation (n=23) presented limited changes (mean increase 3.9±16.1%) over a similar duration period. When subclassifying patients into three groups based on the change or the base line value of JAK2V617F allele burden, MF-transformation was more frequently (p=0.034) observed in patients whose JAK2V617F allele burden was either increased by more than 10% during the follow-up (group A) or higher at first diagnosis than the mean values for each disease (PV; 71.7%, ET; 35.5%) (group B). MF-transformation was 2 out of 4 (50%) in the group A, and 9 out of 22 (41%) in the group B. In contrast, MF transformation in the rest of the patients (group C) was 0 out of 8 (0%). Hydoxyurea-treated (n=16, 6 PV and 10 ET) and –untreated (n=18, 8 PV and 10 ET) patients do not show significant difference in frequencies of MF-transformation, confirming that Hydoxyurea has no preventative effect against MF-transformation. In conclusion, our study showed that higher JAK2V617F allele burden at first diagnosis or a dynamic increase in allele burden during the follow-up period is a predictive factor for MF-transformation. Thus, a routine measurement of the JAK2V617F allele burden by an accurate assay system is recommended to predict MF-transformation. Disclosures No relevant conflicts of interest to declare.
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Au, Qingyan, Arezoo Hanifi, Erinn Parnell, et al. "Phenotypic Characterization of the Immune Landscape in the Bone Marrow of Patients with Acute Myeloid Leukemia (AML) Using MultiOmyxTM Hyperplexed Immunofluorescence Assay." Blood 134, Supplement_1 (2019): 1455. http://dx.doi.org/10.1182/blood-2019-122206.
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Background: Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disorder. Bone marrow (BM) constitutes the home niche for leukemia cells in AML. Emerging data indicates that the BM microenvironment becomes immunosuppressive and plays a crucial role in cancer development and progression. Regulatory T cells (Treg), tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) all contribute to immunologically permissive microenvironment for cancer cells. Based on phenotypical characteristics, MDSC can be further subdivided into granulocytic MDSC (G-MDSC, polymorphonuclear MDSC) and monocytic MDSC (M-MDSC). Although increasing evidence suggests that the immune system impacts the pathogenesis and prognosis in AML patients, only limited data has been published to comprehensively describe the immunological composition of AML BM microenvironment. Methods: In this study, we aimed to perform comprehensive characterization of the immune cells in the BM of patients with AML. Using MultiOmyx hyperplexed immunofluorescence (IF) assay and proprietary imaging analysis, we studied BM tissues of 20 AML patients and 6 normal controls with a total of 13 markers essential in cancer immunology. The normal and AML BM FFPE sections were stained with CD34, Arginase1, CD11b, CD14, CD15, CD33, CD68, CD163, HLA-DR, CD3, CD4, CD8 and FOXP3. Results: Overall, MultiOmyx 13-plex panel staining results revealed an immune suppression-skewed immune profile in AML BM in this study. We observed that both M-MDSC and G-MDSC accumulated within the TME in AML BM samples, with higher frequency of G-MDSCs over M- MDSCs. The data also revealed an abundant M2 macrophages present in the TME of the AML samples. The detection of both MDSCs and M2 macrophages in these samples supports the hypothesis that these cells contribute to the establishment of an immunosuppressive TME. Using the MultiOmyx proprietary algorithm, which takes into account the staining patterns, we quantified the counts and density of different immune cells in both AML patient and normal BM samples. There was a significantly higher frequency of M2 TAM in AML than normal BM. Increased M-MDSC to G-MDSC ratio was also noted in patients with AML. Further, the spatial distance from the different subsets of immunosuppressive cells to CD34+ blasts was measured in AML samples using nearest neighbor analysis. The data indicated that G-MDSC were spatially closer to CD34+ blasts in AML than M-MDSC. Conclusions: The direct assessment of immune phenotypes and their spatial relationship by MultiOmyx IF assay provides essential information in understanding the immune landscape in AML BM. Together, our data suggests that AML blasts may directly recruit immunosuppressive Tregs, MDSC and TAM and this may be one of the escape strategies. The potential for eradicating AML lies in rational combinations of immunotherapies with strategies of the induction of anti-tumor immunity and the elimination or reprogramming of the immunosuppressive TME. Disclosures No relevant conflicts of interest to declare.
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Palmerini, Emanuela, Michela Pasello, Robin Lewis Jones, et al. "Irinotecan and temozolomide upfront and in relapsed Ewing sarcoma: A translational study on MGMT (O6-methylguanine–DNA methyltransferase) and ABCG2 (MGMTLiberati)." Journal of Clinical Oncology 38, no. 15_suppl (2020): e23564-e23564. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e23564.
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e23564 Background: Activity of temozolomide (TEM) and irinotecan (IRI) in recurrent Ewing sarcoma (EWS) was demonstrated. Few data are available on TEMIRI use upfront. Biological predictive factors are lacking. Methods: This multi-institutional retrospective study (NCT03542097) included 59 patients with EWS. 8 patients with very high risk (HR) EWS (multivisceral ± bone marrow) received TEMIRI (TEM 100 mg/m2/day oral, and IRI 40 mg/m2/day intravenous, days 1-5, every 21 days) upfront, 51 patients after relapse (28% in 1st line, 72% ≥ 2nd line). Overall response rate (ORR: CR+PR), SD, and PD, progression-free (PFS) and overall survival (OS) were assessed. The relationship between pre-treatment expression of MGMT and ABCG2 (when FFPE tissue available) with ORR, PFS and OS was evaluated. Results: Median age was 27 years (range 4-62 years): 47 patients (80%) were adults (≥18 years), 35 (61%) had ECOG 0 and 42 (71%) presented with multivisceral disease (+ bone marrow in 5). MGMT was positive in 16/30 (53%), ABCG2 in 4/33 (12%). ORR for upfront TEMIRI (n = 8) was 50% (CR + PR = 1 + 3), with SD 50%, while in recurrent EWS (n = 49, 2 patients no measurable by RECIST) ORR was 31% (CR + PR = 4 + 11), SD 38%, and PD 31%. A better ORR was observed in adult (p = 0.008) & ECOG 0 (p = 0.001) patients; MGMT & ABCG2 expression did not influence ORR. 6-mos PFS was 87% after TEMIRI upfront, 43% at recurrence (p = 0.06), and 65% vs 28% (p = 0.02) for ECOG 0 vs ECOG 1-2, respectively. 6-mos PFS was 62% in MGMT+ vs 33% in MGMT- (p = 0.4) and 75% in ABCG2+ vs 50% in ABCG2- (p = 0.7). Median time to progression (TTP) with upfront TEMIRI was 9 months (range 5-28 months), with 1 patient with ongoing CR at 56 months; median TTP at relapse was 3 months (1-29 months). MGMT and ABCG2 expression did not influence 1-yr OS, whereas ECOG (0 vs 1-2) did (88% vs 31% p < 0.001). Grade 3-4 diarrhea, neutropenia, and thrombocytopenia incidence was < 5%, 1 patient with recurrent kidney EWS died of acute liver failure. Conclusions: TEMIRI showed promising activity in a very unfavorable cohort of EWS patients, with manageable toxicity. Performance status correlated with 6-month PFS & OS whereas MGMT & ABCG2 did not. Clinical trial information: NCT03542097 .
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He, Jie, Jose A. Bufill, Michelle K. Nahas, et al. "Clinical Utility of Comprehensive Profiling of Genomic Alterations in Hematologic Malignancies." Blood 124, no. 21 (2014): 1072. http://dx.doi.org/10.1182/blood.v124.21.1072.1072.
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Abstract Background: The clinical utility of mutation profiling in hematologic malignancies requires robust detection of all classes of genomic alterations in a single analysis across different tumor types. We developed a novel, hybrid capture-based NGS assay (FoundationOne® Heme) designed to comprehensively assess the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events. We have analyzed the genomic alterations including substitutions, indels, copy number alterations and genomic rearrangements of 795 samples with various hematolymphoid malignancies including lymphoma, leukemia and multiple myeloma to demonstrate the diagnostic, therapeutic and prognostic utility of this test in routine clinical practice. Method: Genomic profiles of 746 out of 795 (94%) consecutive samples received in a CLIA-certified laboratory (Foundation Medicine) were successfully characterized by the FoundationOne® Heme test including 527 from bone marrow aspirates, 176 FFPE samples and 92 samples derived from blood. The clinical cohort is composed of 375 multiple myeloma cases, 185 leukemia cases, 150 lymphoma cases, 75 MDS/MPN cases, and 15 unknown hematologic disorder cases (Figure A). Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes and 31 frequently rearranged genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ~7M on-target unique pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, copy number alteration and gene rearrangements. Results: 705/746 (95%) patients had at least one alteration reported as a somatic driver mutation, 68% of cases harbored at least one alteration linked to a targeted therapy or would inform enrollment in a clinical trial consistent with therapeutic relevance. These potentially actionable alterations included mutations in KRAS (14% of cases), NRAS (13%), CDKN2A (8%), DNMT3A (6%), IDH1/2 (5%), BRAF (4%) and FLT3 (3%) (Figure B). In addition, 64% of cases harbored at least one alteration that have been shown to predict outcome and have prognostic relevance, including TP53 (14% of cases), TET2 (7%), ASXL1 (7%), CDKN2B (5%), CREBBP (5%), MLL (5%) and NPM1 (3%)(Figure B). We also detected 344 genomic rearrangements in 280/746 (38%) patients. Of the 344 reported rearrangements, 166 were known fusions in various disease types and 178 were novel rearrangements involving known oncogenes and tumor suppressor genes, including a novel RCSD1-ABL2 fusion in a patient with B-cell ALL who will derive benefit from kinase inhibitor therapy as part of their anti-leukemic regimen. Genomic rearrangements (detected in 38% of cases) can lead to various functional consequences: 260 (76%) rearrangements detected in this cohort are predicted to create activating fusions; 19 (6%) rearrangements are predicted to be activating intragenic rearrangements in MLL, FLT3 and CARD11; and 60 (17%) rearrangements are likely truncating events in tumor suppressors. Conclusions: We demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets or prognostic predictors in a broad spectrum of hematologic malignancies. Moreover, our data suggests that the FoundationOne® Heme comprehensive genomic profiling test can alter therapeutic strategy in routine clinical practice. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures He: Foundation Medicine: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc.: Employment, Equity Ownership. Young:Foundation Medicine: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment. Donahue:Foundation Medicine: Employment. Young:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. van den Brink:Foundation Medicine, Inc: Consultancy. Intlekofer:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.
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Au, Qingyan, Jun Fang, Anna Juncker-Jensen, et al. "Characterization of Myeloid-Derived Suppressor Cells and Tumor Associated Macrophages Using MultiOmyxTM Hyperplexed Immunofluorescence Assay in Hodgkin Lymphoma." Blood 132, Supplement 1 (2018): 4135. http://dx.doi.org/10.1182/blood-2018-99-115434.
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Abstract Tumor microenvironment (TME) consists of heterogeneous subsets of myeloid cells and plays a crucial role in promoting cancer development and metastasis. Tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) all contribute to an immunologically permissive microenvironment for cancer cells. On basis of the expression of surface markers, MDSC can be further subdivided into granulocytic MDSC (G-MDSC, polymorphonuclear MDSC) and monocytic MDSC (M-MDSC). In solid tumors, these different myeloid cell populations are well characterized and extensively studied. However, in hematological malignancies the role of myeloid cell subsets has been less studied. A recent study showed increase in MDSC in the bone marrow (BM) at time of diagnosis in acute myeloid leukemia (AML) patients (Sun H. et al. Int J Hematol. 2015). Significantly higher numbers of G-MDSC and M-MDSC were present at diagnosis in classic Hodgkin lymphoma (cHL) (Romano A. et al. Br J Haematol. 2015). The accumulation of TAMs was also reported to be associated with poor prognosis in cHL (Steidl C. et al. N Engl J Med. 2010). Collectively, these results indicate that the tumor-resident myeloid cells play an important clinical role, thus highlighting the need for monitoring and deeper characterization of various myeloid subsets in hematological malignancies, especially in the tumor FFPE sections. Herein, we report an analysis of MDSCs and 'protumoral' M2 macrophages using MultiOmyx hyperplexed immunofluorescence (IF) assay in 9 clinical samples diagnosed with HL. MultiOmyx is a proprietary multi 'omic' technology that enables detection and visualization of up to 60 biomarkers on a single 4µM FFPE slide (Gerdes MJ. et al. PNAS 2013). The HL FFPE sections were stained with a 13-marker panel including Arginase 1, CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, CD3, CD4, CD8 and FOXP3. We observed that both M-MDSC (Fig 1A, characterized as CD11b+CD14+CD15-CD33+HLA-DR-) and G-MDSC (Fig 1B, identified as CD11b+CD14-CD15+CD33+HLA-DR-) accumulated within the TME in all 9 HL samples, with higher frequency of G-MDSCs over M-MDSCs. Arg1 expression was detected exclusively in G-MDSC population (Fig 1C). The data also revealed an abundant M2 macrophages (Fig 1D, characterized as CD68+CD163+) present in all HL samples. The detection of both MDSCs and M2 macrophages in HL samples supports the hypothesis that these cells contribute to the establishment of an immunosuppressive TME. Using the MultiOmyx proprietary algorithm, which takes into account the staining patterns, we will next quantify the counts and density of different tumor-resident myeloid subsets and measure the spatial distance between each subset of tumor-resident myeloid cells to the neoplastic Reed-Sternberg cells. Correlation study will also be performed to determine if significant correlations exist between MDSCs and TAMs and how these immunosuppressive myeloid cells are related to the Regulatory T cells (CD3+CD4+FOXP3+) in HL samples. In addition to HL samples, the same 13-plexed panel will be utilized to characterize the myeloid cell population from AML patients. TAMs and MDSCs are emerging as potential biomarkers for diagnosis and prognosis of cancer as well as therapeutic targets. The comprehensive myeloid cells phenotyping offered by MultiOmyx 13-plexed panel has the potential to monitor the changes of immunosuppressive myeloid cells in response to immune modulating drugs such as MDSC-targeting drugs (e.g. PDE-5 inhibitors, COX-2 inhibitors), TAM-targeting agents (e.g. anti-CSF1R) and combined therapy in treatment of lymphoma and leukemia. Disclosures No relevant conflicts of interest to declare.
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Sharon, Barrans, Andrew J. Davies, Ming Wang, et al. "Real-Time Molecular Classification of Diffuse Large B-Cell Lymphoma (DLBCL) By Gene Expression Profiling (GEP): Successful Delivery of a Routine Service for Randomization of Patients Onto the Multicenter Remodl-B Trial (ISRCTN 51837425)." Blood 126, no. 23 (2015): 331. http://dx.doi.org/10.1182/blood.v126.23.331.331.
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Abstract Germinal Center (GCB) and activated (ABC) B-cell subtypes of DLBCL can be identified by Gene expression profiling (GEP). These subgroups are biologically distinct, harboring mutations in different pathways. Patients classified as ABC more often have mutations of the NF-kB pathway and an inferior response to standard R-CHOP therapy. The REMoDL-B trial utilised GEP to stratify patients for the addition of bortezomib to R-CHOP, based on the hypothesis that this agent may selectively improve the outcome of the ABC subtype. GEP was performed on RNA extracted from diagnostic formalin-fixed paraffin-embedded (FFPE) biopsies using Illumina WG-DASLTM during the 1st cycle of R-CHOP. Patients were classified as GCB, ABC or Unclassified before cycle 2 using the cross-platform DAC classifier (Care et al, PLOS ONE 2013) and randomised to continue R-CHOP+/-bortezomib. Work is underway to compare data generated on Affymetrix arrays and targeted RNA-seq (Illumina TRex), as well as validation by targeted mutational analysis of 18 genes associated with DLBCL (TNFAIP3, CARD11, CD79A, CD79B, MYD88, TRAF3, TNFRSF11A, PRDM1, TP53, FAS, B2M, CD58, EZH2, MLL2, MEF2B, EP300, CREBBP, KDM2B) using Fluidigm multiplex PCR and Illumina MiSeq on DNA also from the FFPE blocks. The trial closed to recruitment in May 2015 and 1147 samples have been analysed. One hundred and fifty three (13%) biopsies were unsuitable for GEP (for insufficient tumor tissue, inappropriate block sent). The remaining samples were classified as ABC (n=261, 23%), GCB (n=471, 44%) and Unclassified (n=214, 19%), with only 11 samples (1%) failing to yield a GEP result. GEP was successful in a range of sample types, including needle and endoscopic biopsies, bone marrow trephines and formal biopsies, with results obtained from as little as 40ng of total RNA, all from FFPE samples. Mutational data were available in 199 samples, with 73% of these having a mutation detectable in 1 or more genes (range 0-5) at a AAF (alternative allele frequency) cutoff at 10%. MYD88 was most commonly mutated (in 30% of ABC and 7% of GCB). EZH2 mutations were restricted to the GCB category (26%) and MYD88, CD79a/b and PRDM1 were more commonly associated with the ABC group. MYD88/PRDM1 were the most frequently associated events, with MYD88/CD79a/b and MYD88/NF-kB being mutually exclusive. Where MYD88 was seen in GCB cases, coexisting mutations imply an origin from transformed follicular lymphoma. B2M mutations were commonly identified across all subtypes (n=26), but specifically enriched in Type III (unclassified) cases (25%), which supports the hypothesis that mutational immune escape may be a feature of DLBCL, in common with other tumor types. Cross platform validation is highly concordant using Affymetrix arrays from a pilot series (27/27 gave the same classifier output, with correlating confidences also seen between platforms). RNA-seq analysis is ongoing, however initial analysis shows 86% concordance with the DASL output. Comprehensive cross platform comparison data will be available for presentation at the meeting. This study demonstrates the feasibility of GEP classification of DLBCL at diagnosis in a large international trial. The molecular classification can also be replicated using different technologies. Mutational analysis confirmed the association between DLBCL subtype and specific mutational hotspots. Disclosures Sharon: Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425). Davies:Takeda: Honoraria; Seattle Genetics: Research Funding. Jack:Jannsen: Research Funding. Johnson:Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425).
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Willard, Nicholas, Andrew Donson, Timothy Ritzmann, et al. "EPEN-08. THE TREM1 POSITIVE HYPOXIC MYELOID SUBPOPULATION IN POSTERIOR FOSSA EPENDYMOMA." Neuro-Oncology 23, Supplement_1 (2021): i15. http://dx.doi.org/10.1093/neuonc/noab090.058.
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Abstract We have previously shown the importance of immune factors in posterior fossa ependymoma (PF EPN). Recently, we found eight transcriptionally unique subpopulations of myeloid cells infiltrating PF EPN with one population particularly enriched in PFA1 tumors. This subpopulation, denoted as hypoxia myeloid subpopulation, is defined by genes associated with angiogenesis, hypoxia response, wound healing, cell migration, neutrophil activation, and response to oxygen levels. TREM1 (Triggering receptor expressed on myeloid cells 1) was found to be expressed almost exclusively within this hypoxia myeloid subpopulation. TREM1 encodes for a receptor belonging to the immunoglobulin superfamily that is expressed on myeloid cells, and stimulates neutrophil and monocyte inflammatory responses. However, single-cell RNAseq give little data suggesting location of cells within the tumor microenvironment. We performed immunohistochemistry (IHC) on our bank of ~90 FFPE PFA EPN samples using TREM1 to characterize and identify the location of the hypoxia myeloid cells. The TREM1 positive cells have an ambiguous cytomorphology reminiscent of a monocyte with modest cytoplasm and a mono-lobated nucleus. IHC also showed that TREM1+ myeloid cells are largely localized to the interface of necrosis and viable tissue, most frequently in a perivascular and intravascular distribution. The latter finding suggests that the TREM1+ cells are derived from the bone marrow and that they may be associated with the mesenchymal tumor population (MEC), which we have previously described as being enriched in PFA1 tumors and localizing to perinecrotic zones. This is supported by parallel IHC analysis of subpopulation-specific markers in the same cohort of PFA EPN which showed the highest TREM1 correlation was with CAIX, a marker of MEC. In PFA matched primary/recurrent pairs, the proportion of TREM1+ cells were increased at recurrence in the majority of cases, suggesting an evolving interaction between this TREM1+ hypoxia myeloid subpopulation and neoplastic cells over the disease course.
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Pichardo, Janine D., Julie T. Feldstein, Maria Arcila, et al. "A Comprehensive Clinical Next Generation Sequencing-Based Assay Can Impact Hematopathologic Diagnosis in a Significant Subset of Patients with Hematologic Malignancies." Blood 124, no. 21 (2014): 2984. http://dx.doi.org/10.1182/blood.v124.21.2984.2984.
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Abstract High throughput genomic studies have identified novel recurrent somatic alterations with prognostic or therapeutic relevance in hematological malignancies. By contrast, few studies have investigated the impact on pathologic assessment, and clinicopathologic diagnosis. We therefore assessed the impact of a CLIA-certified CAP-accredited comprehensive clinical grade next-generation sequencing-based assay, FoundationOne Heme (FOH), on hematopathologic assessment of patients at our institution. The FOH assay targets 405 cancer-related genes and 31 frequently rearranged genes by DNA-capture and sequencing, and 265 frequently-rearranged genes by RNA-capture and sequencing. We prospectively tested 92 cases as part of routine clinical practice, including malignant lymphoid neoplasms (n=51) and suspected myeloid malignancies (n=41). (Table 1) The samples submitted included 30 blood, 38 bone marrow and 24 FFPE specimens. We were able to obtain genomic profiling data in90 of the 92 submitted cases (98%) including all FFPE specimens. A total of 282 genomic abnormalities (range 0-11, average 3.2 abnormalities/case) including substitutions, insertions/deletions and gene fusions were detected. There was excellent concordance between conventional cytogenetic or molecular genetic assays and FOH assay. Only 10 cases lacked any genomic abnormality. Six of these were morphologically normal bone marrows submitted to rule out myeloid neoplasia. Of the 4 remaining cases without recurrent somatic alterations, 2 cases were derived from myeloma patients where the sample analyzed had less than 20% plasma cells and 2 cases were from patients with myelodysplasia with likely low tumor content. In 84 cases with a diagnosis of hematologic malignancy, we identified genomic abnormalities with diagnostic relevance in 42 cases (50%). Most importantly, in 12 cases (14% of cohort), the presence of specific genomic alterations led to a change or refinement of the diagnosis. (Table 2) This included two cases in which a diagnosis of T-LGL was confirmed based on the presence of STAT3 mutations, three cases of lymphoma/myeloma in which a specific diagnosis was reached based on identification of pathogenic fusions/rearrangements, and 4 cases of MPN/MDS which could be confirmed based on the presence of known MPN/MDS disease alleles. In addition, we identified genomic alterations with prognostic relevance in 54 cases (64%), and with potential therapeutic impact in 64 cases (76%). Our data demonstrate that the FOH assay can be performed with a very high success rate (98%) in a routine clinical setting and that comprehensive genomic profiling can substantively impact pathologic assessment and diagnosis of a wide spectrum of hematologic malignancies. Genomic testing provided critical diagnostic information in half of the cases, in some instances refining or changing the conventional pathological diagnosis. These findings suggest that comprehensive targeted genomic testing has an important role to play not only in identifying prognostic and therapeutic targets but also in hematopathology diagnosis, and should be considered as a first line testing platform in hematologic malignancies. Table 1:Clinical characteristicsTotal number of casesN=92 Median age58 (18-82) SexMale58 (63%)Female34 (37%) Myeloid neoplasms33 (36%)AML14MDS11MPN6CMML1CML1 Lymphoid neoplasms51 (55%)DLBCL17B-ALL6TLPD6MCL5Myeloma5CLL3MZL3FL3BCL-NOS2CHL1 Normal marrow6 (7%) Sample failed2 (2%) Genomic abnormalities in hematologic malignancies80/84 (93%)Diagnostic42/84 (50%)Prognostic54/84 (64%)Potential therapeutic64/84 (76%) Table 2: Genomic changes which led to improved diagnosis and classification of the hematologic malignancy Case Diagnosis/Problem Genomic alteration Final/refined diagnosis 1 Neutropenia, T-LGL? STAT3 (N647I) T-LGL 2 Neutropenia, T-LGL? STAT3 (D661Y) T-LGL 3 T-LPD? JAK3 (M511I) T-PLL 4 T-LPD? TET2 (Q1523*), TP53 (R175G) PTCL, NOS 5 Transformed FL CIITA-DSCAML1 (Fusion) PMBL 6 CHL vs ALCL IGH-BCL2 (Rearrang.) DLBCL 7 BCL-NOS IGL-MYC (Rearrang.) BCL between DLBCL/BL 8 Myeloma IGH-MAF8 (Rearrang.) High risk myeloma 9 MDS? STK11 (F354L) RUNX1-MECOM (Fusion) MDS 10 MDS? MLL (ITD) MDS 11 MDS? KDM6A MDS 12 MPN MPL (R514_W515>KK) ET Disclosures Moskowitz: Seattle Genetics, Inc.: Consultancy, Research Funding; Genentech: Research Funding; Merck: Research Funding. Horwitz:Research: Celgene, Millennium, Infinity, Kiowa-Kirin, Seattle Genetics, SpectrumConsulting: Amgen, Bristol-Myers Squibb, Celgene, Jannsen, Millennium, seattle genetics: Consultancy, Honoraria, Research Funding. Stein:Janssen Pharmaceuticals: Consultancy. He:Foundation Medicine: Employment. Stephens:Foundation Medicine, Inc. : Employment, Equity Ownership. Miller:Foundation Medicine: Employment. Younes:Novartis: Research Funding; J & J: Research Funding; Curis: Research Funding; Bayer; Bristol Meyer Squibb; Celgene; Incyte; Janssen R & D; Sanofi; Seattle Genetics; Takeda Millenium: Honoraria. Dogan:Foundation Medicine: Consultancy.
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Looney, Timothy, Michelle Toro, Geoffrey Lowman, et al. "Optimized IGH Chain Sequencing for Detection of Very Low Frequency B Cell Malignancies from RNA or DNA Input." Blood 134, Supplement_1 (2019): 5777. http://dx.doi.org/10.1182/blood-2019-130525.
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Background High throughput sequencing (HTS) of rearranged TCRB and IGH chains has been demonstrated as a means to detect malignant T and B cells at a frequency as low as 10E-6. However, onerous input requirements (typically 20-30ug gDNA input over multiple library preparations) have impeded widespread adoption of 10E-6 as a threshold for minimal residual disease (MRD) translational research studies. Here we demonstrate an optimized highly multiplex PCR approach for amplifying IGH chains from fresh or FFPE-preserved RNA or DNA input material. Coupled with automated clonotyping and clonal lineage detection, we demonstrate detection of malignant B cell clones at a frequency of 10E-6 from a single library preparation. Methods Rearranged IGH chains were amplified using multiplex framework 3 and joining gene primers targeting all human IGH variable and joining gene alleles in the IMGT database (Oncomine IGH-SR assay). Libraries were generated from 25 or 100ng total RNA or 2ug gDNA derived from (1) peripheral blood leukocyte (PBL) or bone marrow (BM) spiked with Ramos B-cell cell line and (2) PBL or BM spiked with synthesized chronic lymphocytic leukemia (CLL) rearrangements from literature. Sequencing analysis was performed using the Gene Studio S5 and Ion Reporter to identify clonotypes, track clones across samples, and identify B cell clonal lineages. Clonal lineages were defined such that lineage members have a shared variable and joining gene identity, identical CDR3 lengths, and CDR3NT sequences within 85% similarity of other lineage members. Automated rarefaction analysis in Ion Reporter was used to determine optimal sequencing depth. Results Ramos and synthesized spike in controls were detected at a frequency of 10E-5 using 25ng of PBL total RNA and sequencing to 3M reads depth, and 10E-6 using 100ng input and 10M reads depth. gDNA-based libraries required 2ug and 3M reads depth to detect spike-in rearrangements at a frequency of 10E-5, while 10E-6 was achieved by combining the results from four 2ug gDNA libraries, each sequenced to 3M reads depth. Input and sequencing depth requirements were consistent across PBL or bone marrow derived libraries. Rarefaction analysis confirmed that the sequencing depth was appropriate for the targeted limit of detection. Conclusions These results demonstrate routine detection of B cell malignancy IGH chains at a frequency of 10E-6 using a limited amount of RNA or DNA material, comparing favorably to existing HTS-based approaches. We anticipate this approach to become a routine component of rare clone tracking applications including those involving B-ALL and CLL, particularly where sample material is limited or a low limit of detection is of paramount importance. Disclosures Looney: Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.
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Sengar, Manju, Sridhar Epari, Hasmukh Jain, et al. "Prevalence and Clinico-Pathological Attributes of c-MYC Rearranged High-Grade B-Cell Non Hodgkin Lymphomas (NHL): Single Centre Experience." Blood 126, no. 23 (2015): 5031. http://dx.doi.org/10.1182/blood.v126.23.5031.5031.
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Abstract Introduction: MYC rearrangement in high-grade B-cell NHL(non-burkitt), either as single hit or double hit, has significant prognostic and therapeutic implications. There is remarkable paucity of data on frequency and clinico-pathological features of MYC-rearranged large B-cell lymphomas from developing world. Method: This study included de-novo high grade B-cell NHL cases (>15 years of age) registered at Tata Memorial Centre between January 2013- December 2014. Demography details, clinical features (stage at presentation, bone marrow involvement, presence and extent of extranodal involvement, B-symptoms, international prognostic index),original histopathological diagnosis, chemotherapy and radiotherapy details were recorded from electronic medical or paper case records. Response to therapy, relapses or death if any, status at last follow up, dates of relapse and/or death and last follow up were recorded. All cases were reviewed by expert hematopathologists to categorize the high grade B-cell NHL as per the WHO-2008 classification of hematopoietic malignancies. All cases with adequate formalin-fixed paraffin embedded (FFPE) blocks were evaluated by FISH for c-MYC , BCl-2 and BCl-6 using Vysis dual colour break apart probes. Cases which showed >10% cells with split signal were considered to harbor rearrangement. Result: A total of 114 cases of de-novo high-grade B-cell NHL with adequate FFPE blocks were evaluated. Based on WHO 2008 classification, 112 cases were classified as diffuse large B-cell lymphoma (DLBL)) and 1 each as Burkitt's (BL) and B- cell lymphoma unclassifiable -intermediate between DLBL and BL(BCLU). A total of 9/112 (8%) cases of DLBL showed MYC rearrangement. One of these 9 cases had both MYC and BCl-2 rearrangement. BCLU did not demonstrate MYC or BCl-2 rearrangement. The Ki-67 index was variable (40-95%)in MYC rearranged cases. The median age of the c-MYC rearranged cases was 55 year (range-23-79 years). 88% were males. Advanced stage disease, bulky mass and extranodal disease was seen in 67%, 44% and 55% of cases respectively. All but one patient had high LDH, however none of the patients had elevation more than 2XULN. These patients received rituximab based chemoimmunotherapy (RCHOP-4,RCEOP-4, REPOCH-1) and 77% achieved complete response. At median follow up of 5 months, 1 year-overall survival and progression free survivals were 80% and 75% respectively. There were no significant differences in clinical features (except higher proportion of males in the c-MYC rearranged subset), LDH levels, ki-67 index , response to therapy and survival between DLBL with or without c-MYC rearrangement Conclusion: In our study 8% of all DLBL cases showed c-MYC rearrangement. The frequency of double hit lymphoma was less than 1% (0.8%). Disclosures No relevant conflicts of interest to declare.
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Cengiz Seval, Guldane, Isinsu Kuzu, Seher Yuksel, et al. "Next Generation Sequencing May be Helpful in Designing Novel Combinations Among Heavily Pretreated Myeloma Patients, Refractory to All Available Approved Anti-Myeloma Drugs." Blood 134, Supplement_1 (2019): 3197. http://dx.doi.org/10.1182/blood-2019-127541.
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Introduction: Multiple genetic alterations that occur at diagnosis or relapse are not only prognostic characteristics of multiple myeloma (MM) but also provide evidence for clonal evolution. Uncovering and dissecting true driver events in MM might provide rational for new potential targets and therapeutic approaches. However, whereas genetic diagnostics in MM namely FISH and gene expression profiling are well-established prognostic tools, individual mutation profiling has not yet been adopted for this purpose. Herein, we aimed to analyze the Next Generation Sequencing (NGS) platform results investigating mutational profiling of patients with relapsed and refractory MM (RRMM). Also, the clinical results of those who had a targetable mutation and were treated "off label" will be presented. Material and Methods: A total of 14 consecutive patients with MM referred to our center between November 2018 and May 2019 were studied. Plasma cells were isolated from bone marrow samples using Selection Kit microbead specific for EasySepTM Human CD138 marker (StemCell Technologies). DNA extracted form magnetic bead enriched cells, bone marrow aspiration smears for bone marrow involved, from FFPE tissue samples for extramedullary-involved cases. NGS method was performed on llumina Miseq platform (USA) by using QIAseq targeted DNA panel (12)- Human myeloid neoplasm panel, covers all exons and exon-intron junctions of 141 target genes. For the data analysis QCI Analyze Universal 1,5.0 was performed. The PCL analysis was performed on CD138 and Ki67 double immune stained paraffin sections, and the quantification was done by using 3DHistech digital pathology platform. Results: We obtained 16 samples of DNA from 12 heavily pretreated and two newly diagnosed myeloma patients. Female/male: 5/9 with a median age of 57 years (range, 39-87) patients had received a median four lines (range; 1-13) of treatment. Out of a panel of 141 genes, 59 mutations in 26 genes were detected (Figure-1). Among these recurrent genomic abnormalities, concomitant missense protein coding alterations were detected in all patients. The PTEN mutation was the most frequently detected, followed by mutations of RAS/MAPK pathway genes. The hotspots of mutation in KRAS codon 61 and NRAS included codons 61 and 13 as well as codon 600 in BRAF. In addition, we detected novel ie myeloproliferative and myelodysplasia associated mutations previously un-described in myeloma. A diverse range of recurrent gains and losses were detected in our cohort. Two patients at diagnosis also carried mutations of PTEN and KRAS. Based on these results three patients were able to obtain off-label approval for treatment with Everolimus (for PTEN) (Patient-1) or Trametinib (for KRAS) (Patient 5 & 6) in combination with Pomalidomide (EvoPomDex) w/wo Daratumumab or Tra-PomDex. Patient-1 had extensive extramedullary disease (EMD) in the skin, which responded completely to Dara-EvoPomDex combination. Complete disappearance of initial lesions (presented in Figure-2) and VGPR duration was only two months. Subsequent refractoriness and appearance of new lesions lead to death of the patient, one year from the initiation of EMD. Patient-6, also presented with EMD, was treated with TraPomDex as the seventh treatment line. TraPomDex treatment was well tolerated, the most significant adverse event diarrhea, infections and pancytopenia. Her biochemical response was a transient VGPR, which was lost during interruption of treatment due to infection. She also died four months after initiation of TraPomDex. Patient-5, plasma cell leukemia, has been on Tra-PomDex for a month and his response is PR yet. Conclusion: The detection of mutations can improve our ability to treat multiple refractory patients who have ran out of all therapeutic options. Though the responses observed among such very heavily pretreated patients are not durable, they are highly promising. Also, of additional importance is detection of age-related cumulative mutations belonging to background bone marrow precursors. Detection of sub-clonal mutations is very helpful in depth analysis of clonal response to treatment and clonal evolution. In the coming years, the identification of actionable mutations in myeloma opens the way for targeted therapy. Acknowledgement:This study is supported by Ankara University Research Grants (Project: 14A0230003) and Turkish Academy of Sciences. Disclosures Ozcan: Takeda: Honoraria, Other: Travel support, Research Funding; Bayer: Research Funding; AbbVie: Other: Travel support, Research Funding; Sanofi: Other: Travel support; Abdi Ibrahim: Other: Travel support; Celgene Corporation: Other: Travel support, Research Funding; Janssen: Other: Travel support, Research Funding; Archigen: Research Funding; Roche: Other: Travel support, Research Funding; Jazz: Other: Travel support; MSD: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Other: Travel support; BMS: Other: Travel support. Beksac:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: trametinib and everolimus for myeloma patients
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Palmerini, Emanuela, Michela Pasello, Robin Lewis Jones, et al. "Does MGMT (O6-methylguanine–DNA methyltransferase) have a role in metastatic Ewing sarcoma (ES) patients (pts) undergoing temozolomide (TMZ) and irinotecan (IRI)?" Journal of Clinical Oncology 35, no. 15_suppl (2017): 11030. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.11030.
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11030 Background: TMZ+IRI has significant activity in metastatic ES. Epigenetic silencing of the MGMT DNA gene by promoter methylation has been associated with response to TMZ in glioblastoma. Our aim was to assess if MGMT methylation 1) has a role in ES progression and 2) is predictive of response to TMZ. Methods: 1) In 10 ES cell lines presence of MGMT gene (Real-time PCR), methylation of its promoter (methylation-specific PCR) and protein expression (western blot) were assessed. MGMT protein (IHC) and methylation of its promoter was searched in 97 ES pts samples (74 localized; 23 metastatic). 2) In metastatic ES pts treated with TMZ+IRI, with pre-treatment FFPE tissue and measurable disease, the relation between RECIST response, PFS and MGMT expression (IHC) was assessed. Results: 1) The expression of MGMT gene and its protein was detected and concordant (p = 0.02) in all ES cell lines evaluated; methylation was a rare event. In ES tissue samples the methylation of the MGMT gene was found at a low intensity as compared with the unmethylated gene, but the protein expression was relatively low: 36% in localized, 65% in metastatic pts (p 0.03). 2) 24 pts (median age 19 years, range 3-50 years; F/M: 7/17) treated with TMZ + IRI from 2010 to 2015 were identified. Line of treatment: 8 patients were in 1st line; 16 ≥ 2nd line. Median n of cycles was 6 (range: 2-31). Pattern of metastases: 16 multiple sites, 4 lungs, 3 multiple sites + bone marrow, 1 bone. MGMT was positive in 63% of cases. ORR: 16.5% (1 CR , 3 PR); SD: 50% (13 pts); PD: 33.5% (7 pts). According to MGMT expression the ORR was 11% in negative and 20% in MGMT positive patients (p = 0.8). 6-mos PFS rate was 59% (38-80 %IC), no differece according to MGMT expression (pos 61% vs neg 56%, p 0.7). Conclusions: Whereas in cell lines the MGMT gene and its protein expression is a generalized event, in tissue samples MGMT protein is present in a minority of localized pts, and might be associate with tumor progression. Methylation of MGMT gene does not seem responsible for its regulation in ES, and post-transcriptional mechanisms are more likely to be involved. The presence of MGMT protein does not predict the response to TMZ + IRI in this small series.
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Scarpa, Frank J., Madhuri Paul, Rachel Daringer, et al. "TP53/NPM1-mutated acute myeloid leukemia as a molecularly distinct disease entity." Journal of Clinical Oncology 39, no. 15_suppl (2021): 7030. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.7030.
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7030 Background: TP53-mutated acute myeloid leukemia (AML) is a distinct disease entity associated with a dismal prognosis. This disease group is distinguishable by its low frequency of SNVs, unremarkable transcriptional signatures, and lower leukocyte and myeloblast counts compared to TP53 wildtype disease. Response to gold-standard hypomethylating agents is typically transient. NPM1 mutations in this disease subset are rare despite the fact that NPM1c has been shown to negatively regulate the tumor suppressive functions of p53 .Methods: Bone marrow, peripheral blood, or FFPE tissue samples from 10,118 patients with suspected myeloid disease were sequenced using a dual DNA/RNA 297 gene myeloid panel. Results were validated in a separate independent dataset using a 54 gene TruSight myeloid panel (N = 2463). FISH/cytogenetic data was analyzed across myeloid disease. Patients with confirmed AML (n = 460) were included in the NGS portion of this study. Statistics were performed using Fisher’s exact test for categorical variables and two-tailed T-test for continuous variables. Results: All TP53-mutated myeloid disease (n = 1282 / 10,118) was associated with fewer co-mutations except DNMT3A (13.4%; n = 172), and complex cytogenetics (36.4%; n = 134/381). TP53+/NPM1+ status across all myeloid disease was not associated with a complex karyotype (7.6% vs 38.5%; 1/13 vs. 133/368, p = 0.02). Among AML patients, NPM1+/TP53+ patients (n = 18) were more co-mutated with DNMT3A (33.3% vs. 10.3%, P = 0.01), FLT3 (33.33% vs. 2.5%, P < 0.0001), IDH1 (27.8% vs. 4.4%, P = 0.002), IDH2 (22.2% vs. 6.4%, P = 0.03); and PTPN11 (22.2% vs. 2.5%, P = 0.003) when compared to TP53+/NPM1- patients. NPM1+/TP53+ AML had more mutations in recurrently mutated genes (4.5 vs 2.1; P < 0.0001) than TP53+/NPM1- AML. Conclusions: TP53+/NPM1+ AML harbors molecular signatures which clearly distinguish it from ordinary TP53-mutated AML, and is more reflective of de novo and NPM1+ AML. Further clinical outcome studies are needed to determine the therapeutic and prognostic implications of this subset, and whether other TP53-mutated patients can be better risk stratified.[Table: see text]
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Tatetsu, Hiro, Miho Watanabe, Emily F. Mason, et al. "Oncofetal Protein SALL4 Is Highly Expressed in Myelodysplastic Syndrome Alongside with NAT10 and P53." Blood 136, Supplement 1 (2020): 34. http://dx.doi.org/10.1182/blood-2020-134533.
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Introduction: Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and refractory cytopenias as a result of ineffective hematopoiesis. We have reported that oncofetal protein SALL4 can be used as a prognostic biomarker in MDS disease. SALL4 is a transcription factor that is important for development and embryonic stem cell properties. While SALL4 expression is down-regulated or absent in most adult tissues, SALL4 is re-expressed in various cancers. We have previously reported that aberrant expression of SALL4 can be detected in MDS and AML patients. Studies in murine models, as well as in human samples, have demonstrated that SALL4 is essential for leukemic cell survival and that SALL4 transgenic mice develop an MDS-like phenotype prior to transformation to AML, suggesting that SALL4 can be a driver of MDS/AML pathogenesis. However, SALL4 expression status and related pathway in bone marrow (BM) cells and relationship with somatic gene mutations in MDS patients has not been explored. In this study, we evaluated the expression of SALL4 and related factors using single-cell mass cytometry (CyTOF) and NanoString technology in MDS patients alongside with target sequence for MDS-related mutations. Materials and methods: We evaluated the expression of SALL4 using single-cell mass cytometry (CyTOF) utilizing 28 antibodies including surface lineage markers and intracellular proteins, such as p53, ki67, c-myc and pAKT to identify SALL4 expressed cells and related pathway in bone marrow (BM) for 10 MDS patients. SALL4 expression was also analyzed in formalin-fixed paraffin-embedded (FFPE) 14 patient samples utilizing two SALL4 probes designed for NanoString technology alongside with target sequence for MDS-related mutations. One probe only detects expression of SALL4A, which is full length SALL4, while the other probe can detect both SALL4A as well as its splicing variant SALL4B. We examined the expression of a panel of 17 known SALL4 downstream target genes. Results: First, we assessed an expression level of SALL4 using CyTOF in 10 MDS patients as compared with normal BM samples (N=5). We observed that Lin-CD34+CD38+ progenitor cells in 8 out of the 10 MDS patients had higher SALL4 expression levels, while the progenitor cells of normal BM cells did not express SALL4. Similar results were observed in Lin-CD34+CD38- hematopoietic stem cells, CD11b+ mature myeloid cells, CD19+ B cells, CD235+ erythroid cells. Conversely only 3 MDS patient had weak SALL4 expression in CD3+ T cells. Subsequently, we investigated the correlation with p53, ki67, c-myc and pAKT. SALL4 is only correlated with p53 (P=0.005), while others were not. Next, we also assessed an expression level of SALL4A and SALL4A&B in 14 MDS patient bone marrow (BM) samples using Nanostring nCounter. We observed that 12 out of 14 MDS patients had higher SALL4 expression levels as compared with control normal BM samples (N=3). Next, we evaluated whether MDS patients expressed SALL4A, SALL4B, or both. Of the 14 MDS patients, 8 predominantly expressed SALL4A Of these 8 MDS patients, 5 harbored RNA splicing-related mutations, such as SF3B1 (N=4), U2AF1 (N=1). Only 1 out of 7 patients who predominantly expressed SALL4B demonstrated a splicing related mutation. Subsequently, we investigated the correlation between SALL4 and its known downstream targets. An expression of SALL4A is moderately correlated with CBLb (γ=0.53), CDH1 (γ=0.42) and NAT10 (γ=0.41). The expression of SALLA&B is moderately correlated with NAT10 (γ=0.47) and RUNX1 (γ=0.46). NAT10 (N-acetyltransferase 10) has been reported to promote transcription of RNA polymerase I and is a critical regulator of p53 homeostasis. It was identified to be a potential SALL4 downstream target in our early Chip-seq studies. In the isogenic K562 cell lines with SALL4 overexpression, higher levels of NAT10 were observed, suggesting that NAT10 is one of the downstream targets of SALL4. Conclusions: Our study has demonstrated for the first time that 1) SALL4 was expressed in various MDS BM cells confirmed by CyTOF 2) there are SALL4 splicing variants in MDS patients, particularly with SF3B1 mutations 3) a novel SALL4/NAT10/p53 link has been identified in these MDS patients. Future studies on mechanism(s) and biological role(s) of SALL4 splicing variants in MDS are needed. Disclosures No relevant conflicts of interest to declare.
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Batlevi, Connie, Craig H. Moskowitz, Andrew D. Zelenetz, et al. "Identification of Actionable Genomic Alterations Across Different Lymphoma Histologies Using a Comprehensive Next Generation Genomic Sequencing Clinical Assay." Blood 124, no. 21 (2014): 3000. http://dx.doi.org/10.1182/blood.v124.21.3000.3000.
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Abstract Background: Several retrospective series have characterized the genomic landscape in many lymphoma subtypes. However, as these studies used different sequencing methods, depth of coverage, and specimen source, the incidence of alterations in many lymphoma subtypes remain unknown. Even when common genomic alterations were identified within a lymphoma subtype, the incidence and spectrum of the alterations varied. In this study, we prospectively examined the incidence of genomic alterations across different lymphoma subtypes using the Foundation One Heme (FOH) assay. Methods: FOH testing was offered to patients in routine clinical practice. Formalin-fixed paraffin-embedded (FFPE) tissue, bone marrow aspirate or peripheral blood were examined using the FOH targeted sequencing assay. Hybridization capture from 405 cancer related genes and 31 genes commonly rearranged in cancer was applied to ≥ 50ng of DNA extracted from 49 tumor specimens and sequenced to high, uniform coverage. Genomic alterations, which include: base substitutions, small insertions and deletions (indels), rearrangements, and copy number alterations, were determined. Results: Specimens from 48 non-consecutive patients with lymphoma (5 new diagnoses, 42 relapsed) were prospectively examined. Represented subtypes include DLBCL (44%), FL (17%), CLL (10%), MCL (10%), MZL (8%), T cell lymphoma (4%), and Hodgkin lymphoma (2%). Median age of diagnosis was 48.1 years (range 29.5-80.6) with a male predominance (69%). The majority (75%) had advanced stage disease at diagnosis, with a median of 3 lines of therapy (range 1-12, n=44). Forty-nine specimens, (1 patient with 2 specimens) were collected from FFPE (70%), peripheral blood (22%) or bone marrow aspirate (8%). Reports resulted after a median 28 days (range 10-48 days) from the time specimens were shipped to Foundation Medicine. Across 48 patients, 90 distinct alterations were detected with a median of 5 alterations/patient (range 0-11). Genomic alterations were more commonly observed in relapsed aggressive lymphoma compared to relapsed indolent lymphoma (median 6.4 vs 4 alterations/patient, p<0.05) or in newly diagnosed patients. The most common alteration was various IgH translocations (46%) and alterations in cyclin dependent kinases (CDK) genes (CDKN2A, CDKN2B and CDKN2C) (31%). Alterations of p53 (23%) and MLL2 (21%) were also frequently observed. Several rare alterations not previously described in primary lymphomas were identified, including alterations in ATRX, CDH1, MSH6, pyruvate carboxylase, and thrombospondin receptor (CD36). Alterations associated with approved agents or clinical trials were found in 89% of patients and were more frequent in relapsed aggressive B-cell NHL (median 2/patient) versus relapsed indolent B-cell NHL (median 1/patient, p<0.02). Commonly identified actionable alterations included CDK alterations in DLBCL, MCL and CLL, and alterations in the PI3K oncogenic pathway in DLBCL, FL and MZL. Conclusion: Application of a comprehensive next generation genomic sequencing assay provides an opportunity to both describe the spectrum and compare the incidence of genetic alterations across different lymphoma subtypes. Preliminary data suggest the vast majority of patients have one or more genomic alterations linked to approved agents or clinical trials. Data collection is ongoing. While the true incidence of each genomic alteration is not defined, the dataset provides the frequency of genomic alterations in a clinically relevant population. Moreover, these data will facilitate design of clinical trials by providing the opportunity to select patients based on shared genomic alterations rather than lymphoma subtype. Table 1.Clinical characteristics, N=48Age at diagnosis57.1 (29.5-80.6)N (% of patients)Sex Male Female33 (68.7%)16 (33.3%)Subtype DLBCL21 (43.8%)FL8 (16.7%)CLL5 (10.4%)MCL5 (10.4%)MZL4 (8.3%)T cell lymphoma2 (4.2%)Hodgkin lymphoma1 (2.1%)Other3 (6.3%)Stage at Diagnosis I/II III/IV Not recorded10 (20.8%)36 (75.0%)2 (4.2%)Biopsy sent At diagnosis At relapse6 (12.5%)42 (87.5%)Response to First line Therapy Near CR or CR PR SD PD Unknown22 (45.8%)9 (18.8%)5 (10.4%)3 (8.3%)8 (16.7%)Genomic Alterations with clinical value Potentially Prognostic Potentially Actionable Alterations previously not well described37 (77.0%)43 (89.6%)7 (14.6%) Figure 1 Figure 1. Disclosures Moskowitz: Seattle Genetics, Inc.: Consultancy, Research Funding; Genentech: Research Funding; Merck: Research Funding. Horwitz:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Infinity: Research Funding; Kiowa]Kirin: Research Funding; Seattle Genetics: Consultancy, Research Funding; Spectrum: Research Funding; Amgen: Consultancy; Bristol]Myers Squibb,: Consultancy; Jannsen: Consultancy. Hamlin:Gilead, Spectrum, Seattle Genetics, Genentech: Consultancy; Spectrum, GSK, Jansen and Jansen/Pharmacyclics, Portola, Seattle Genetics: Research Funding. Matasar:Genentech: Consultancy; Spectrum: Consultancy. Miller:Foundation Medicine: Employment. Stephens:Foundation Medicine: Employment, Equity Ownership. He:Foundation Medicine: Employment. Younes:Novartis: Research Funding; J & J: Research Funding; Curis: Research Funding; Bayer; Bristol Meyer Squibb; Celgene; Incyte; Janssen R & D; Sanofi; Seattle Genetics; Takeda Millenium: Honoraria.
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Lipson, Doron, Michelle K. Nahas, Geoff A. Otto, et al. "Identification Of Actionable Genomic Alterations In Hematologic Malignancies By a Clinical Next Generation Sequencing-Based Assay." Blood 122, no. 21 (2013): 230. http://dx.doi.org/10.1182/blood.v122.21.230.230.
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Abstract Background Rapid advancements in cancer genomics and in the development of targeted therapies provide expanding opportunities to use genomic profiling to improve patient outcomes. However, most patients do not have access to clinical genomic profiling platforms, and currently available assays capture a small set of known mutations or translocations tailored to specific tumor types. The spectrum of somatic alterations in leukemia, lymphoma, and myeloma includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and gene fusions; no current assay captures the different types of alterations in a single clinical genomic test. We developed a novel, CLIA-certified next-generation sequencing-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of all classes of genomic alterations using archived FFPE, blood and bone marrow aspirate samples with high accuracy in a clinically relevant timeframe. Methods DNA and RNA were successfully extracted from 350/362 (96%) specimens from 319 patients, including 57 FFPE samples, 150 blood samples and 142 bone marrow aspirates. The initial sample cohort included 20 ALL, 83 AML, 53 CLL, 57 DLBCL, 48 MDS, 32 MPN and 57 multiple myeloma samples. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 374 cancer-related genes and 24 frequently rearranged genes by DNA-seq, and 258 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 590x for DNA and >20M total pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, CNAs and gene fusions. Results Sufficient tumor content (≥20%) was present in 317/350 (91%) of the samples (289/319 patients), and a total of 885 alterations were identified (3.1 alterations per sample), including 555 base substitutions, 213 indels, 36 splice mutations, 51 CNAs and 36 fusions/rearrangements. The most frequent alterations across all hematologic malignancies included mutations in TP53 (9%), ASXL1, KRAS, NRAS, IDH2, TET2, SF3B1, JAK2, MLL2, DNMT3A, RUNX1, and SRSF2 (2-5% each); FLT3 ITDs (2%); MLL PTDs (1%); homozygous loss of CDKN2A/B (3%); and focal amplification of REL (1%). Rearrangements in BCL2/6, MYC, MLL, MLL2, NOTCH2, ABL1 and ETV6 were identified using DNA and RNA targeted sequencing, demonstrating the ability of this platform to reliably identify gene fusions with immediate clinical relevance. Overall high accuracy of the assay for substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across alteration types with high specificity (PPV>99%) [Frampton et al, Nat Biotech, in press]. Comparison of detected alterations to previous molecular testing for JAK2, NPM1, IDH2, FLT3 and CEBPA in MPN/AML samples demonstrated 97% sensitivity (33/34) in our ability to identify known mutations in these clinical samples. We identified additional clinically relevant mutations that were not detected using standard clinical assays, including alterations in JAK2, FLT3 and IDH2, which can inform therapeutic decisions. The use of our content rich sequencing platform allowed us to identify clinically actionable mutations in hematologic malignancies, including IDH1/2 mutations in a spectrum of myeloid/lymphoid malignancies, recurrent BRAF mutations in refractory CLL and myeloma, and mutations in the JAK-STAT signaling pathway in diffuse-large B cell lymphoma. These results demonstrate that a targeted sequencing platform which includes a large set of known disease alleles/therapeutic targets can identify mutations with therapeutic relevance in disease contexts where gene-specific assays are not currently performed in the clinical setting. Conclusions We have developed a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used for clinical sequencing of frozen/paraffin samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets in a broad spectrum of hematologic malignancies. Disclosures: Lipson: Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment, Equity Ownership. Otto:Foundation Medicine, Inc: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc: Employment, Equity Ownership. Wang:Foundation Medicine, Inc: Employment, Equity Ownership. He:Foundation Medicine, Inc: Employment, Equity Ownership. Rampal:Foundation Medicine: Consultancy. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Donahue:Foundation Medicine, Inc: Employment, Equity Ownership. Sanford:Foundation Medicine, Inc: Employment, Equity Ownership. Greenbowe:Foundation Medicine, Inc: Employment, Equity Ownership. Frampton:Foundation Medicine, Inc: Employment, Equity Ownership. Fichtenholtz:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc: Employment, Equity Ownership. Parker:Foundation Medicine, Inc: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment, Equity Ownership. Levine:Foundation Medicine, Inc: Consultancy.
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Schiffman, Joshua D., Patrick D. Lorimer, Vladimir Rodic, et al. "High Resolution Genome-Wide Copy Number Analysis of Pediatric Burkitt Lymphoma Identifies Copy Number Alterations In the Majority of Patients." Blood 116, no. 21 (2010): 3123. http://dx.doi.org/10.1182/blood.v116.21.3123.3123.
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Abstract Abstract 3123 Although cure rates are high for pediatric Burkitt lymphoma (BL), a subset of patients relapses and succumbs to the disease. BL is characterized by translocation of the MYC gene with an immunoglobulin gene, but secondary changes including gain of 1q, gain of 13q, loss of 13q, loss of 17p, and others have been described by both conventional cytogenetic and oligo array CGH approaches. Secondary changes may contribute to the clinical heterogeneity of BL as evidenced by the fact that loss of 13q is associated with a worse prognosis in pediatric BL (Poirel et al., Leukemia 23:323, 2009). However, high resolution, genome-wide copy number analysis has not yet been reported in pediatric BL. The objective of this study was to identify copy number alterations (CNAs) in pediatric BL using a genome-wide approach, and to examine the relationship between CNAs and clinical parameters including outcome. After institutional review board approval, we identified 30 pediatric BL patients treated at Primary Children's Medical Center (n=25, Salt Lake City, UT) and Penn State Hershey Medical Center (n=5, Hershey, PA) with available formalin-fixed, paraffin-embedded (FFPE) diagnostic biopsy specimens. Age, site, and gender data were available for all specimens, and 22/24 of the Utah patients were treated according to the COG 5961 protocol with full clinical information and follow-up available. DNA was isolated from FFPE biopsies containing at least 80% tumor. In addition, germline DNA was isolated from negative staging bone marrow clot sections on Utah patients (n=25) to serve as a pooled normal reference and to provide germline copy number variation data on individual patients. Tumor and paired normal DNA was submitted for Molecular Inversion Probe (MIP) assay (330K Cancer Panel, Affymetrix, Santa Clara, CA). The Nexus Copy Number (BioDiscovery, El Segundo, CA) software package was used to analyze the MIP data with the following stringent call criteria: SNPRank segmentation, 5.0E-4 significance threshold, 1000 kb maximum contiguous probe spacing, minimum of 5 probes per segment, gains ≥ 2.7 copy number value, and loss ≤ 1.3 copy number value. Patients included 23 males and 7 females with a mean age of 8.0 years (range 2 – 18). At presentation, lactate dehydrogenase (LDH) levels ranged from 443 – 13851 U/L and uric acid levels ranged from 1.5 – 13 mg/dL. Patients were Murphy stage I (n=1), II (n=9), III (n=13), and IV (n=2). A total of three patients relapsed (one stage II patient and two stage III patients) and three died (the two stage III patients that relapsed and a different stage II patient who did not have a complete clinical response). 27 of the 30 tumor samples and 22/25 paired normals had adequate DNA for MIP analysis. The 3 tumor samples without adequate DNA were clinically similar to the others. We identified a total of 103 CNAs (defined as change seen in the same cytoband in 1 or more patients), which included 63 gains and 40 copy number losses. 23/27 cases (85%) had at least one gain or loss. We identified 21 recurrent CNAs (same cytoband affected in 2 or more patients), which included 14 gains and 7 losses. We found gains of 1q in 10/27 patients (37%), gains of 13q in 4/27 patients (15%) and losses of 17p in 3/27 patients (11%). Despite a relatively small sample size, deletion of 17p13 was significantly associated with relapse (p=0.041). To our knowledge, this is the first report of high-resolution genome-wide copy number analysis of pediatric BL. Furthermore, we show for the first time that FFPE archived materials can be used for high resolution gene copy number analysis in lymphomas. We identified CNAs in 85% of the pediatric BL cases, which include previously described changes of 1q, 13q, and 17p. Although sample size limited statistical power, deletion of the 17p13 locus was associated with relapse. We plan to extend these studies in a larger sample of patients to evaluate the potential prognostic significance of both the 17p13 locus deletion as well as additional recurrent CNAs. Disclosures: No relevant conflicts of interest to declare.
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Liegel, Jessica, Manoj Bhasin, German A. Pihan, et al. "Transcriptome Sequencing Demonstrates Unique Signature Associated with Durable Clinical Response to DC/AML Fusion Vaccine." Blood 134, Supplement_1 (2019): 3832. http://dx.doi.org/10.1182/blood-2019-129451.
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Introduction Our group has pioneered a personalized vaccine in which patient-derived acute myeloid leukemia (AML) cells are fused with autologous dendritic cells (DC/AML fusion), presenting a broad array of leukemia associated antigens with DC mediated costimulation. In a clinical trial of AML patients who were vaccinated after chemotherapy-induced remission, 71% remained free of disease at median follow up of 57 months. We sought to identify factors associated with durable remission after vaccination using genomic analysis of the bone marrow microenvironment including single cell RNA-seq and TCR clonal diversity analysis. Methods Banked bone marrow samples both prior to and 1 month post-vaccination were selected from patients who maintained long disease remission for greater than 5 years and those who had early relapse. FFPE marrow core biopsy samples (N=10) were the source for gene expression analysis. NEBNext ultra II directional library prep kit and Illumina NextSeq 500/550 system were used to generate reliable high quality RNA sequencing data. Differentially expressed genes were identified by p-value (≤0.01) and fold change (≥2) using Linear Models for Microarray (Limma) approach. Ingenuity Pathways IPA 9.0 was then used to define pathways and upstream regulators. Flash frozen samples (N=4) were analyzed by RNAseq at the single cell level using a standard 10X genomics approach with cell cluster annotation performed with Single Cell Wizard software. Banked peripheral blood was used to evaluate TCR diversity with Takara SMART-Seq next-generation sequencing to amplify variable regions of TCR- α/β subunits. Results Heatmaps depict significant differential gene expression in bone marrow biopsies both pre- and post-vaccination in patients who remained in long-term remission (responders) compared to those who relapsed (non-responders). Prior to vaccination there was modest upregulation of immune activation pathways including IL-7, IL-17A as well as inhibition of TGF-b in responders, suggesting a role of the micro-environment in modulating response. Significantly upregulated pathways in responders after vaccination (p value <0.01) were related to immune activation including NO and Reactive Oxygen Species in Macrophages, IL-2, IL-15, IL-6, IL-7,IL17A, and B cell activation. TGF-b was also downregulated in responders post-vaccination. To characterize the cellular components of the immune micro-environment, single cell analysis was assessed in bone marrow aspirates both pre- and post-vaccination (N=2). Increased cellular heterogeneity pre-vaccination, and increases in T and NK populations post-vaccination, were noted in responding patients who had durable remissions. Furthermore, the temporal changes in expression of TCR clonotypes showed an increase in TCR diversity post-vaccination (N=2). Of note, a patient who achieved a durable remission had (i) loss of specific clonal populations present at the time of diagnosis and (ii) the emergence of newly expanded TCR signatures that were further expanded with subsequent vaccinations and remained present during the follow up period. In contrast, the TCR diversity in a non-responder was low and static with no difference in the TCR clonotypes after vaccination. Conclusions In a cohort of AML patients vaccinated after chemotherapy-induced remission, we found distinct gene signatures amongst patients with long term response as compared to those with early relapse. These signatures have potential to serve as predictive and early biomarkers of vaccine response, and will be investigated in a larger cohort from an ongoing trial. The transcriptomes indicate that vaccine response is dependent on a robust immune microenvironment, as characterized by upregulation of cytokines, activation of T cells, B cells and macrophages, and reduction of TGF-b-mediated negative immunoregulation. We also found that vaccine response is associated with durable oligoclonal expansion within the T cell repertoire, which purportedly represent functionally potent anti-AML shared- and neo-antigen specific T cell populations. This provides an especially unique opportunity to identify target antigens by TCR-epitope pairing. Indeed, information regarding AML antigens targeted by the immune system in the induction of durable remissions could further advance the field of AML treatment by integration in combinatorial therapeutic strategies. Figure Disclosures Stone: Roche: Consultancy; Arog: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Stemline: Consultancy; Takeda: Other: DSMB; Agios: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Macrogenics: Consultancy; Argenix: Other: DSMB; Arog: Consultancy, Research Funding; Biolinerx: Consultancy; Astellas: Consultancy; Daiichi-Sankyo: Consultancy; Celgene: Consultancy, Other: DSMB; Jazz: Consultancy; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy; Biosight: Consultancy; Pfizer: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Stemline: Consultancy; Jazz: Consultancy; Actinium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Biolinerx: Consultancy; Biosight: Consultancy; Novartis: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy, Research Funding; Biolinerx: Consultancy; Agios: Consultancy, Research Funding; Roche: Consultancy; Macrogenics: Consultancy; Trovagene: Consultancy; Argenix: Other: DSMB; Argenix: Other: DSMB; Otsuka: Consultancy; Takeda: Other: DSMB. Kufe:Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria; Hillstream BioPharma: Equity Ownership. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Rosenblatt:Partner Tx: Other: Advisory Board; Dava Oncology: Other: Education; Parexel: Consultancy; BMS: Other: Advisory Board ; Merck: Other: Advisory Board; Amgen: Other: Advisory Board; BMS: Research Funding; Celgene: Research Funding; Imaging Endpoint: Consultancy.
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Weitzel, Jeffrey N., Judy Ellen Garber, Danielle Castillo, et al. "Clinical conundrums: Developing a strategy for discerning TP53-associated chip and coherent clinical care." Journal of Clinical Oncology 38, no. 15_suppl (2020): 1514. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.1514.
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1514 Background: Germline TP53 mutations are associated with Li-Fraumeni syndrome (LFS). However, approximately 20% of commercial laboratory multigene panel test (MGPT)-detected pathogenic TP53 variants represent aberrant clonal expansion (ACE), rather than a germline finding, and are often detected in individuals that lack classic features of LFS. Clonal hematopoiesis (CH) is a form of ACE, and in the absence of an abnormal hemogram is termed Clonal hematopoiesis of indeterminate potential (CHIP). CHIP is often associated with a pathogenic variant (PV) in hematopoietic pathway gene(s) at a variant allele frequency (VAF) less than expected for a heterozygous germline finding. The prevalence increases with age and exposure to chemotherapy. The presence of a skewed VAF is usually noted in a comment on a genetic test result, however, clinicians without genetic training often lack understanding of the comment and need strategies to discern the difference between germline findings, CHIP, and post-zygotic mosaicism. Our studies illuminate possible strategies for discernment for clinicians. Methods: Among 113 cases with MGPT-detected TP53 PVs, enrolled in the Clinical Cancer Genomics Community Research Network registry, we obtained additional tissues, family history and complete blood count (CBC) reports on 42 cases. DNA extracted from formalin fixed paraffin embedded (FFPE) tumor/normal tissues, blood, saliva, eyebrow plucks, was analyzed using a previously validated custom myeloid and CH gene (n = 79) amplicon-based QIAseq panel. PVs with VAF > 2% were included in analyses. Results: Germline status was confirmed for 6 cases (one with a CH PV), post-zygotic mosaicism was supported for 5 cases and 2 were indeterminant. 12 had results supporting ACE/CH, with additional CH-associated PV(s) identified in 5/12 (41%); n = 2 of each TET2, ATM, TP53; and increasing VAF over time for the driver TP53 PV was noted in 2. Of these 2 one was identified to have a hematopoietic malignancy identified through analysis of the CBCs and bone marrow biopsy in parallel with the increasing VAF. Additional results are pending for 7 cases. Conclusions: With the use of our multi-tissue NGS strategy, serial sampling of suspected ACE/CH cases, family history and CBC analyses we were able to discern the status of most TP53 genetic findings. This work has direct translational impact, refining risk estimation and improving the clinical care of patients with TP53 PVs, while avoiding unnecessary LFS-related care and enabling appropriate care for those with ACE.
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Scarpa, Frank J., Madhuri Paul, Wendy A. Wolfson, Lawrence M. Weiss, Vincent Anthony Funari, and Forrest Blocker. "STAG2-mutated AML patients: ASXL1 cohesin binding motif status and mutation landscape." Journal of Clinical Oncology 38, no. 15_suppl (2020): 7538. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.7538.
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7538 Background: ASXL1 and the cohesin complex ( STAG2, RAD21, SMC1A, and SMC3) are commonly mutated chromatin regulators with significant clinical implications in AML. The ASXL1-cohesin interactome regulates gene expression through chromatin accessibility via ASXL1’s cohesin binding motif (CBM). ASXL1 variants are most commonly located in the ASXM1 domain and onwards, and characteristically lead to loss of the PHD domain. Gain-of-functions in truncated ASXL1 are suggested to increase catalytic activity of BAP1, which binds the ASXH domain at AA 351, and to gain an interaction with BRD4, which binds somewhere between the ASXN and ASXH domains, to drive H3K4Me3 and H2AK119Ub. Methods: 2463 suspected AML patient bone marrow, peripheral blood, or FFPE tissue samples were evaluated using an all exon amplicon-based 27 gene NGS panel. Patients with a VAF <10% in ASXL1 were excluded to avoid reporting artifacts, particularly in variant c. 1934dup. Statistics were performed using Fisher’s exact test. Results: Mutations in STAG2-mutated patients were enriched for sAML, as evidenced by the higher number of mutations in ASXL1, SRSF2, and BCOR (associated with sAML) compared to NPM1, DNMT3A, and PTPN11 (pAML). STAG2 mutations were found in 173 samples representing 93.5% of cohesin mutations. Of all ASXL1 mutations (VAF 10.1– 54.5%; median 32.2%) 4.0% occurred in the CBM. While 23.5% of samples with mutations outside ASXL1 CBM had concomitant mutations in STAG2, none of the 18 samples with CBM mutations (VAF 11.3 – 51.7%; median 42.5%) had any cohesin gene mutation (P = 0.0174). The proportion of BCOR (27.8% vs 9.2%; P = 0.024) and CEBPA (27.8% vs 8.2%; p = 0.016) mutated patients in the CBM+ group was significantly higher than the CBM- group. JAK2 (16.7% vs 5.4%), KRAS (22.2% vs 13.6%), EZH2 (22.2% vs 13.6%), and RUNX1 (38.9% vs 27.7%) mutations were also higher though not significantly in this group. Mutations throughout all of ASXL1, the 13 amino acids after the CBM, and hotspot variants all had STAG2 mutations at a frequency of 20.9-44.4%, further suggesting mutual exclusivity. Conclusions: STAG2 mutations and mutations in the CBM were mutually exclusive events and harbored different co-mutation frequencies. In compromised ASXL1 CBM cases, BCOR and CEBPA transcriptional regulators are significantly more mutated, but in cases of ASXL1 mutation outside the CBM, cohesin mutations are preferred, suggesting alternative chromatin accessibility mechanisms driving leukemogenesis. This observation has not been previously reported in the literature to our knowledge.
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Maura, Francesco, Niccolò Bolli, Daniel Leongamornlert, et al. "Whole Genome Sequencing Reveals Recurrent Structural Driver Events in Peripheral T-Cell Lymphomas Not Otherwise Specified." Blood 132, Supplement 1 (2018): 4115. http://dx.doi.org/10.1182/blood-2018-99-112094.
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Abstract Historically, the differential diagnosis between different nodal peripheral T-cell lymphoma (PTCL) subtypes based on morphological and phenotypic grounds has posed great challenges. In the last few years, our knowledge of the molecular bases of different PTCLs has significantly expanded. However, peripheral T-cell lymphomas not otherwise specified (PTCL-NOSs) are still regarded to as a heterogeneous category encompassing PTCL cases not fitting other, more homogeneous, subtypes. In fact, PTCL-NOS is one of the few lymphoma subtypes where no recurrent driver mutations have been reported so far. In order to better characterized the PTCL-NOS genomic landscape, we decided to investigate 11 PTCL-NOS patients by a whole genome sequencing (WGS) approach (median coverage 27X). Ten out of eleven samples were collected from FFPE blocks and 2 were removed from analysis: one due to low cancer cell fraction (CCF) and the other based on cluster generation issues during sequencing likely caused by a hyper-fragmented DNA. Among the remaining 9 cases, we extracted 59,617 somatic base substitutions (range 2,471-10,756, median 6,358 per patient) and 20,531 small insertion-deletions (indels) (range 84-6,397, median 1,580). We were able to characterize the spectrum of FFPE-induced artefacts, mostly composed of point mutations and indels within LINE-1 (L1) elements, predominantly of the L1PA family. This is a crucial quality control step that could be applied to similar future studies from archive samples. Four samples were heavily involved by FFPE-related artefacts and were excluded for this reason. Using a non-negative matrix factorization (NNMF) algorithm we investigated for the first time the PTCL-NOS mutational signature landscape. We did not find novel processes in this entity, but rather known processes operative in other lymphoid malignancies. Among those: signatures 1 and 5, deriving from the age-related process of spontaneous deamination of methylated cytosines; signatures 2 and 13 deriving from aberrant activity of the APOBEC family of DNA deaminases; signatures 17 and 8, pertaining to two yet poorly characterized processes. The contribution of different processes to the mutational spectrum of each case was profoundly heterogeneous. Combining our data set with 64 previously published whole exome sequencing cases (23 ALCL, 15 AITLs, 9 PTCL-NOSs and 16 EATL-II), we confirmed the lack of recurrent driver mutations among PTCL-NOS. Taking advantage of WGS data, we therefore focused on structural variants (SVs: inversions, translocations, internal tandem duplications and deletions) and copy number alterations (CNAs). We found 372 SVs, with a stunning median of 73 per sample (range 56-86). Even more interesting, at least one complex event was observed in all but one patients, including one whole genome duplication (WGD) and five chromothripsis events in three patients, suggesting a critical role of SVs in shaping the PTCL-NOS genome. We found that known onco-drivers were recurrently disrupted by such events: the most frequent target was CDKN2A, deleted in 4 out of 5 patients, 2 of which carried homozygous deletions. Interestingly, PTEN loss was observed in 2 out of 4 CDKN2A-deleted patients. Given the high prevalence of these deletions, we extended our observation to an independent validation set of ALCLs (n=56), AITL (n=22) and PTCL-NOS (n=59) investigated by FISH (n=36), next generation sequencing (n=25) or SNP6 array series (n=76). Overall, CDKN2A was deleted in 22/59 (37%) PTCL-NOSs cases, and in 17/22 (77%) both alleles were lost. PTEN was deleted in 12/59 (20%) PTCL-NOS cases, all of which also carried a CDKN2A loss. Strikingly, the co-occurrence of CDKN2A and PTEN was found only among PTCL-NOS, and in none of the other entities. With the limitations of the small sample size, the presence of CDKN2A bi-allelic deletions was associated with inferior survival (25% [95% CI: 9-66%] 5-y OS for deleted cases vs 52% [95% CI: 28-96%] for wt/hemizygous cases, p=0.042) among patients treated with an autologous bone marrow transplant front line program for advance stage and high-risk disease (n=19). Our observations point at SVs as a main driver of PTCL-NOS, often involving known cancer genes and their downstream pathways. Furthermore, our data highlighted recurrent gene deletions that may be relevant for differential diagnosis within this category of lymphomas. Disclosures Bolli: Celgene: Honoraria. Chiappella:Roche: Other: lecture fees; Amgen: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Nanostring: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Teva: Other: lecture fees. Corradini:Celgene: Honoraria, Other: Advisory Board & Lecturer; Novartis: Honoraria, Other: Advisory Board & Lecturer; Roche: Honoraria, Other: Advisory Board & Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Sandoz: Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Amgen: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer.
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Kharfan-Dabaja, Mohamed A., Rami S. Komrokji, Qing Zhang, et al. "TP53 and IDH2 Somatic Mutations Are Associated with Poor Outcomes Following Allogeneic Hematopoietic Cell Transplantation for Myelodysplastic Syndrome." Blood 126, no. 23 (2015): 4382. http://dx.doi.org/10.1182/blood.v126.23.4382.4382.
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Abstract Background: Despite growing understanding of clinical and genetic basis of myelodysplastic syndromes (MDS) and increased use of allo-HCT, the disease remains incurable in approx. half of cases. Availability of next generation gene sequencing is an important tool that helps further prognosticate outcomes. Pts, materials and methods: We performed targeted amplicon-based next generation sequencing (NGS) of the 26 most frequently mutated genes in MDS. DNA was extracted from paraffin embedded bone marrow (FFPE) samples obtained during pre-transplant work-up at a median of 30 (9-53) days prior to allo-HCT. Sequencing was performed on the Illumina platform with an average read depth of 1500x. The Qiagen GeneRead software suite was used for alignment and variant calling. Frameshift, nonsense, and missense variants that were not present in germline databases at >1% frequency and those that were predicted to be functionally significant by SIFT and Polyphen were deemed true mutations. Cutoff for variant allele frequency was set at 20%. Kaplan-Meier estimates were used for overall survival (OS) and Cox regression for multivariable analysis. Results: We identified 139 pts who received an allo-HCT between 01/2005 and 06/2012 for MDS (89%), AML (4%) or CMML (7%). However, 38 samples were excluded for inability to isolate DNA due to poor sample quality. Patient, disease, and transplant characteristics are summarized (Table 1). Median age of pts was 58 (22-74) yrs and the majority (77%) had received prior azacitidine. Somatic mutations (≥1) at time of allo-HCT were identified in 39% of cases. The most common mutations in decreasing frequency were: ASXL1 (11%), DNMT3A (6%), IDH2 (5%), KRAS (4%), RUNX1 (4%), TP53 (4%), TET2 (3%), SRSF2 (3%), SF3B1 (2%), EZH2 (1%), BCL (1%), MLL (1%), and WT1 (1%). FLU-BU was the most common preparative regimen (92%). Median F/U for all surviving pts was 36 months. Median OS for all pts was 29 (95% CI=10-48) mos. There was no difference in OS between pts harboring ≥1 mutation vs. those with none (34 (95%CI=10-57) mos vs. 29 (95%CI=16-42) mos, p=0.7), or in the presence of >5% vs. ≤ 5% BM myeloblasts at time of allo-HCT (20 (95%CI=10-31) mos vs. 34 (95%CI=13-55) mos, p=0.5), or if pts were ≥ 60 yrs of age vs. <60 (34 (95%CI=15-34) mos vs. 29 (7-52) mos, p=0.7). Pts with R-IPSS (poor and very poor risk) at time of allo-HCT had over 2-fold lower OS vs. R-IPSS (very low, low, and int) (15 vs. 41 mos, p=0.06). When analyzing OS based on specific mutations, presence of mutated TP53 (6 (95%CI=0-17) mos vs. 34 (95%CI=14-53) mos, p=0.02) or IDH2 (11 (95%CI=6-16) mos vs. 34 (95%CI=12-56) mos, p=0.006) predicted for significantly worse OS after allo-HCT. No difference in OS was observed when mutated ASXL-1, TET-2, DNMT3A, or RUNX-1, were present. Multivariable analysis (adjusted for %BM blasts and karyotype) identified TP53 (HR=3.3 (95% CI 1.1-10), p 0.03) and IDH2 (HR=3.9 (95% CI 1.5-11), p 0.006) as independent predictors of poor outcomes after allo-HCT. Conclusion: Our study confirms the adverse prognostic significance of TP53 mutation in MDS pts undergoing allo-HCT and identifies mutated IDH2 as an independent predictor of inferior OS in allo-HCT. Identifying these extremely high-risk populations (TP53 and IDH 2 mutations) is useful as standard allo-HCT seems ineffective (OS< 12 mos); and there is an unmet need to improve outcomes by integrating novel agents as post-transplant consolidation or maintenance. The lower frequency of somatic mutations in our study sample compared to previously reported might be explained by our more stringent criteria to screen out FFPE artifacts, among others. Given the relatively small sample size, our findings merit validation in a larger data set.Table 1.VariablesN=101Recipient median age (range), yrs58 (22-74)Recipient sexM=54%WHO at DxRA=2% RARS=3% RCMD=16% RAEB-I=31% RAEB-II=26% CMML=8% MDS-unclas=5% MDS/MPN=1% MDS-RS=5% AML=4%Azacitidine before allo-HCTNo=22% Yes=77% Unk=1%Best response to Azacitidine pre-HCTCR=8% CR marrow=4% PR=12% HI=5% SD=43% PD=7% Missing=22%Donor sourceMRD=35% MUD=46% MMUD=17% Cord=3%Cell sourcePBSC=96% BM=1% Cord=3%Preparative regimenFLU-BU=92% FLU-MEL=3% Other=5%GVHD prophylaxisMTX=66% SIR=19% MMF=15%R-IPSS at allo-HCTVery low=15% Low=24% Int=18% High=20% Very high=20% Missing=4%Cytogenetic scoring2=56% 3=16% 4=22% 5= 2% Missing= 4%Mutation presentNo=61% Yes=39% Disclosures Komrokji: Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Pharmacylics: Speakers Bureau; Incyte: Consultancy. Field:PDL Biopharma: Research Funding. Perkins:PDL Biopharma: Research Funding. Lancet:Seattle Genetics: Consultancy; Pfizer: Research Funding; Boehringer-Ingelheim: Consultancy; Kalo-Bios: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.
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Yu, Yongxin, Francys Alarcon, Yanglong Mou, et al. "A Novel Multimodal Next Generation Sequencing Assay with Total Nucleic Acid Input to Provide Comprehensive Genomic Profiling for Hematologic Malignancies." Blood 136, Supplement 1 (2020): 29–30. http://dx.doi.org/10.1182/blood-2020-140460.
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Background: A cost effective and comprehensive genomic profiling (CGP) approach for diagnosis, risk stratification and therapy would be useful for the evaluation of oncologic specimens. Available approaches involving additive testing for DNA and RNA abnormalities through traditional methods (e.g. Sanger, FISH, cytogenetics, qRT-PCR) are not comprehensive, require multiple different workflows and are sample consuming, often resulting in incomplete testing. While there are next generation sequencing (NGS) assays designed for detecting DNA and RNA abnormalities, they have separate workflows that require twice the amount of sample and effort. To address this, we developed a novel total nucleic acid (TNA) extraction method and single tube workflow utilizing TNA and a custom multimodal chemistry designed for hematologic malignancies. This consolidated workflow enables an efficient discovery based approach for both DNA/RNA abnormalities including single nucleotide variants (SNVs), InDels, copy number variants (CNVs), large structural changes from DNA and gene fusions and gene expression levels from RNA. This method maximizes data derived from valuable samples while delivering a comprehensive profile of the patient's tumor which can help guide therapeutic and clinical decisions. Methods: Total nucleic acid (TNA) was extracted from bone marrow and peripheral blood of 95 patients (CML, CMML, CLL, AML and myeloid disorders). 297 genes that have DNA mutations specific to hematological cancers were targeted, along with 213 genes that were targeted for clinically significant RNA abnormalities. Enriched genomic and transcriptomic regions of interest from 85 patients were successfully sequenced with unique dual indices on an Illumina NovaSeq 6000. DNA variant detection as well as fusion detection from RNA were compared to traditional orthogonal NGS assays that use DNA input or compared to qRT-PCR and Sanger sequencing assays that use RNA as input. Results: In this study, we developed an efficient and high-quality TNA extraction method that can purify enough total nucleic acid from bone marrow, peripheral blood, cytogenetic pellets, flow suspension, and FFPE samples for the downstream NGS assay. The average OD 260/280 value was 1.9 and the OD 260/230 was 2.18. After sequencing, 256/262 (97.7% accuracy) SNV and Indel variants that were candidate pathogenic mutations were concordant from 38 patients. Meanwhile, 100% (7/7) of all BCR/ABL1 gene fusions which had an international scale (IS) value above 6.4% were concordant. In addition, 69 fusion positive samples containing 20 unique gene fusions which had been previously reported by an independent ArcherDX assay designed specifically for gene fusions were also evaluated with this chemistry. Analysis revealed a 92.5% (64/69) concordance. More importantly, the QIAseq multimodal TNA NGS assay detected both DNA and RNA abnormalities in a single tube. For example, in one myeloid leukemia patient, we not only identified pathogenic variants of ASXL1 and JAK2 which had been previously detected by a DNA NGS assay, but also detected a concurrent BCR-FGFR1 fusion which had been previously reported by a FISH assay. Moreover, we were able to provide more comprehensive genomic profiling by investigating many DNA and RNA abnormalities simultaneously. In our study, for 5 patients that previously been tested for BCR-ABL1 fusion only, we are able to assess BCR-ABL1 fusion status from RNA as well as identify pathogenic DNA variants at the same time, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71, which provides valuable information to assist diagnosis and treatment in a cost effective and efficient way. Conclusions: We developed a single tube TNA based workflow with a custom multimodal chemistry that simultaneously detects many DNA and RNA abnormalities in a cost effective and efficient way while reducing sample requirements. This unique TNA NGS assay provides comprehensive genomic profiling for hematologic malignancies and improves the diagnostic testing options for precise patient care. Disclosures Yu: NeoGenomics: Current Employment. Alarcon:NeoGenomics: Current Employment. Mou:NeoGenomics: Current Employment. Jung:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Thomas:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Shinbrot:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Jiang:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Ye:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.