Academic literature on the topic 'Two-level transplant evolution'

Abstract CML relapse after allogeneic SCT is a relatively frequent situation and is clearly correlated to disease status, time from diagnosis to transplant and T cell depletion. Defining other risk factors could help modulate post transplant immunosuppression. We evaluated the value of early minimal residual disease (MRD) quantification to predict relapse of CML patients receiving standard allogeneic SCT. MRD was analysed by RQ-PCR at Day 100 in 38 patients with a follow up after transplant > 1 year and was expressed as BCR-ABL/ABL, qualified by objective evaluation of RNA amplifiability relative to local normal values for MRD samples (the Quality Index or QI). This QI allows objective evaluation of the degree of correction for positive results and modification of the limits of detection for negative results. Thirty six of 38 patients received conventional conditioning regimen with either BU/CY or TBI/CY and classical prevention of GVHD based on cyclosporin and methotrexate. The median time from diagnosis to transplant was 21 months and median follow up of the cohort was 76.8 months. Patients were allocated to two groups according to their Day 100 RQ PCR level. We compared the characteristics and evolution of the 14 patients with a high MRD level (Day 100 RQ PCR >10−4) to that of the 24 patients with a low MRD level (Day 100 RQ PCR <10−4). There were no significant differences in terms of disease status at transplant, median age at transplant, time from diagnosis to transplant, type of conditioning regimen, source of stem cells, sex mismatch, grade of acute GVHD or incidence of chronic GVHD between the two groups. We show that Day 100 BCR-ABL transcript levels >10−4 by RQ-PCR represents an independent risk factor of relapse after conventional non T cell depleted SCT. These data should favour risk-adapted post transplant immunosuppression based on a single time point early evaluation of MRD.

Abstract The level of minimal residual disease (MRD) prior to allogeneic haematopoietic stem cell transplantation (HSCT) was shown to be an independent prognostic factor for the outcome of paediatric patients with high-risk acute lymphoblastic leukemia (ALL). Retrospective studies which used (semi-)quantitation of clone-specific immunoglobulin/T-cell receptor (Ig/TCR) rearrangements documented feasibility and practicality of such an approach. Recently, this approach was disputed by Imashuku et al (BMT 2003) due to great occurrence of the clonal evolution and generally high MRD levels prior HSCT in their cohort. In our prospective study, MRD before and after HSCT was monitored in a cohort of 36 children with ALL consecutively transplanted in our centre between VIII/2000 and VII/2004. We used a quantitative real-time PCR approach introduced and standardised by European Study Group on MRD in ALL. In 25 of 36 patients MRD level prior HSCT was assessed (9 patients lacked adequately sensitive Ig/TCR target; two lacked analysable DNA prior HSCT). Seventeen patients were MRD-negative prior HSCT (including two with MRD level below the quantitative range 10(−4)) and 8 were MRD-positive up to 9x10(−2). In the MRD-positive subgroup, 7 events (6 relapses) occurred post-transplant in striking contrast to only one relapse in MRD-negative subgroup (EFS log-rank p<0.0001). MRD proved to be the only significant prognostic factor in a multivariate analysis (p<0.0001). Adoptive immunotherapy including donor lymphocyte infusions in patients with adverse dynamics of MRD after HSCT had only limited and/or temporary effect. Clonal evolution did not present a problem precluding MRD monitoring in any of patients suffering a post-transplant relapse. We show that MRD quantitation using clonal Ig/TCR rearrangements represents a feasible approach for the risk assessment in paediatric ALL patients undergoing allogeneic HSCT. However, our ability to respond to detectable MRD levels after HSCT and to avert an impending relapse is very limited. The change of the approach to MRD-positive patients prior HSCT is necessary because of very questionable benefit of HSCT in these children. Supported by grants MSM0021620813, FNM 9735 and GAUK 62/2004.

OBJECTIVE: The aim of the present study was to analyze the long-term evolution of patients submitted to endolymphatic irradiation as a pre-transplant preparation. SETTING: Referral center of university hospital. DESIGN: Case-control study. MAIN OUTCOMES MEASURES: The study was designed to evaluate the incidence of rejection, kidney loss, leukopenia, infection, and graft survival in the group treated (group 1) prior to surgery, compared to a control group (group 2) composed of patients under identical clinical conditions (sex, age, type of donor, immunosuppressive therapy and time of transplant) that did not undergo treatment preparation. PATIENTS: Patients were selected from amongst transplantation candidates on a long-term waiting list, some with a high level of antibodies against panel. The control group was chosen from amongst recently transplanted patients. Patients in the treated group received lipoiodine containing 131I with specific activity ranging between 4 and 6 mCu/ml. RESULTS: A significant difference between the two groups was found with regard to the incidence of rejection crises (21.0% in group 1 and 73.6% in group 2; P= 0.003), and the maintenance dose of azathioprine (smaller in group 1; P< 0.01). As to kidney graft loss due to rejection, a tendency to significance could be identified (10.5% in group 1 and 42.1% in group 2; P= 0.063); however, the difference was not significant between the two groups in terms of reversibility of rejection episodes during the first 60 post-transplant days. CONCLUSIONS: The authors concluded that this method, besides being relatively innocuous (there was no compromising of either the thyroid gland or of gonad function and there was no increase in tumor incidence), has an extended immunosuppressive effect, and can be indicated for cadaveric renal allograft recipients, especially those showing high panel reactivity.

Abstract Introduction: Leukemic escape from the graft versus leukemia (GVL) effect of allogeneic hematopoietic cell transplantation (HCT) is poorly understood. During prolonged periods of post-transplant remission, immunologic selection pressure can favor relapse mediated by acquired resistance to GVL-mediated clearance, such as loss of mismatched HLA in haploidentical transplants. We hypothesized that genetic mechanisms of immune evasion can cause late relapses after matched unrelated donor transplants for myeloid malignancies. Methods: We evaluated 580 adult patients with myeloid malignancies who underwent allogeneic HCT at our institution between 2001 and 2014 and experienced subsequent disease relapse. In 19% of these patients (n=112) relapse occurred at least one year after transplantation. The 25 patients included in the study had specimens banked at each of three timepoints: 1) prior to transplantation, with AML or MDS involvement, 2) approximately 100 days after transplantation, during the period of remission 3) at the time of disease relapse. The indications for transplantation were AML (n = 20) and MDS (n = 5). Transplants were from matched unrelated (n=14), matched related (n=9), and mismatched unrelated (n=2) donors. 12 patients received myeloablative conditioning and 13 received reduced-intensity conditioning regimens. Targeted sequencing of 187 genes, selected based on pathogenic involvement in myeloid malignancies or suspected involvement in immune evasion, was performed on all three timepoints from each patient. Genome-wide microarray-based copy number assessment was performed onthe pre-transplant specimen and post-transplant relapse specimens. Results: We identified three recipients with relapse-specific HLA loss via 1-8 Mb deletions or chromosome 6p arm-level copy neutral uniparental disomy (UPD). One of the HLA alterations was a deletion spanning HLA-B and HLA-C in a donor/recipient pair mismatched at HLA-C. However, in two other cases, relapse-specific HLA losses were identified in donor/recipient pairs that were fully matched at A, B, C, and DRB1. These findings suggest that HLA loss may allow leukemic cells to escape allogeneic immune recognition of minor HLA discrepancies and/or the presentation of minor histocompatibility antigens in the context of matched MHC presentation. These three HLA losses were identified among 14 recipients of matched unrelated donor HCT. HLA losses were not identified among recipients of matched related (n = 9) or mismatched unrelated (n = 2) allogeneic HCT. To evaluate potential interactions between canonical myeloid driver mutations and immunologic alterations, we defined the genetic characteristics of paired pre-transplant MDS/AML samples and post-transplant relapsed samples. In 22 out of 25 cases, at least one driver mutation that was present in the pre-transplant sample was also detected in the relapse sample. Clonal genetic evolution was common at the time of relapse and predominantly involved the acquisition of new subclonal mutations affecting mitogenic signaling (n = 9) and myeloid transcription factors (n = 11). TP53 alterations, including point mutations and 17p deletions were identified in 6 out of 25 patients, including two that remained stable before and after transplantation, and four that were newly detected at the time of relapse. Conclusions: We identified recurrent HLA loss via 6p UPD and segmental deletions in 3 out of 14 patients with late relapse after matched unrelated HCT. Although HLA loss has been observed at relapse after haploidentical HCT, the role of HLA loss as a mechanism of relapse after non-haploidentical HCT has remained unclear. HLA loss in cases with late relapse indicates a prolonged period of immunologic equilibrium, where effective GVL serves as a selective pressure for clonal genetic mechanisms of alloimmune evasion. In the context of MURD HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention. Disclosures Ho: Jazz Pharmaceuticals: Consultancy. Nikiforow:Kite Pharma: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 5504 Backgroud Allogeneic transplantation (HSCT) is the only potentially curative therapy for myelofibrosis, even in the era of new drugs that only impact on selected manifestations of disease. The time to evolution of myelofibrosis in advanced phases is variable (months to several years). The choice to proceed to allogeneic transplantation is based on several clinical variables; conditioning regimen and time to transplantation may influence the outcome of HSCT. Here we present our experience in a group of patients with myelofibrosis transplanted in Our Center with a myeloablative regimen based on full dose of treosulfan. Methods We retrospectively evaluated 20 patients (15 male) with primary myelofibrosis (14) and post-ET (essential thrombocythemia) myelofibrosis (6) who underwent HSCT at our institute from July 2005 to February 2013. In this cohort we included 4 patients in blast phase, 3 of which received a brief course of chemotherapy prior to transplant; 4 patients who underwent a second allogeneic transplantation for disease relapse (2) or graft failure (1); 1 patient referred at our Institute in graft failure 3 months after first HSCT. Only 1 patient had splenectomy before HSCT. Results The median age at diagnosis was 59 years (range 41-76). JAK2 V617F mutation was positive in 7 and unknown in 3 patients. In post-ET MF patients, the median time to transformation in MF was 134 months (60-144). The median time from diagnosis to transplant was 14 months (4-101 months). The median age at transplant was 62 years. DIPSS score at transplant was low in 1, int-1 in 3, int-2 in 13 and high in 3 patients. We adopted myeloablative regimen based on treosulfan 42 gr/ms and fludarabine 150 mg/ms, plus TBI 4 Gy in 7 patients. GvHD prophylaxis was based on ATG, Rituximab and CSA/MTX (8 pts) or rapamycin (11 pts). The graft was PBSCs (19) and BM (1) from related HLA-identical donor (7 pts), matched unrelated donor (5 pts) and haploidentical donor (8 pts). The incidence of graft failure and poor graft function was 5% and 20% respectively. The median time to neutrophils engraftment was 20 days. The incidence of acute and chronic graft versus host disease was 50% and 40% respectively. Cumulative incidence of TRM (transplant related mortality) was 45 %; day 100 OS (overall survival) was 70% while 2 year OS was 40%. In multivariate analysis there was no statistically significant correlation between individual factors (including WBC, hemoglobin, platelets, ferritin, beta2-microglobulin, IgA, IgG, IgM, LDH, psuedocholinesterase levels, age, Sorror comorbidity score, donor match, bone marrow kariotype) and survival. However, we found that a linear composition of level of peripheral blasts (0%, ≥1%, ≥5%), bone marrow CD34+ cells (<5%, ≥5%, ≥20%), spleen size and number of red blood cells units transfused pre-HSCT is able to predict the outcome (p-value 0.03), even if each factor alone is not strongly correlated with survival. There was no statistical significant correlation between the outcome and DIPSS score, Bacicalupo score and Lille score. Conclusions A myeloablative regimen based on full dose treosulfan provide a 95% rate of primary allogeneic engraftment and 40% overall survival in myelofibrosis at advanced stage, with low extra-hematological toxicity in frail patients. Allogeneic transplantation after myeloablative low-toxicity regimens may be offered in myelofibrosis in early chronic phase of disease and for younger patients, two conditions usually linked with durable engraftment, low toxicities and low rate of relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 974 SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Abstract Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation. To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease. Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region. iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy. In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures: No relevant conflicts of interest to declare.

Abstract Hematopoietic stem and progenitor cell (HSPC) gene therapy relies on stable therapeutic levels of gene-modified HSC engraftment. The variant methylguanine methyltransferase gene, P140K, can increase gene modified cells levels in vivo after administration of O6-benzylguanine (O6BG) and non-myeloablative bis-chloroethylnitrosurea (BCNU). However, the biological consequences of chemoselective pressure on HSPC are unknown. It is known that HSPC transplantation causes leukocyte telomere shortening, likely due to proliferative demand on engrafted blood progenitor cells required for hematopoietic reconstitution following myeloablative conditioning. Additionally, telomere shortening is associated with chromosomal instability preceding malignant evolution in blood cells. We and others demonstrate stable telomere length in pigtail and rhesus macaques following myeloablative transplantation of autologous lentivirus gene modified HSPCs. However, the selective pressure placed on P140K-modified HSC after O6BG/BCNU treatment may contribute to telomere attrition. To test this hypothesis, we performed a longitudinal assessment of telomere length on peripheral blood leukocytes using a quantitative PCR method in five pigtail macaques. Macaques received myeloablative total body irradiation (1020cGy) followed by infusion of autologous HSPC gene-modified with either a gammaretrovirus or a lentivirus encoding the P140K transgene. In all animals, O6BG/BCNU effectively increased the contribution of gene-modified cells in the blood. However, in two animals (J02370 and M02426), chemoselection was associated with a loss in total leukocyte telomere length. We sorted modified and non-modified cells in J02370 based on a fluorescent marker, and found that gene-modified leukocytes in animal J02370 displayed significantly shorter telomeres than the non-modified leukocytes. To determine the relative number of gene-modified clones contributing to this phenomenon, we investigated gene-modified clonal contribution over time in each of the five animals. Telomere shortening in animals J02370 and M02426 correlated temporally with emergence of clonal dominance in vivo, whereas animals displaying stable leukocyte telomere length maintained clonal diversity in vivo. In both J02370 and M02426, shorter total leukocyte telomere length was stably maintained with stable levels of the respective dominant clones for up to 1,700 days. Telomere shortening in these animals could be attributed to either increased cell division during clonal outgrowth, or to clonal selection of a progenitor that originally began with shorter telomeres. Importantly, our data suggest that chemoselective pressure on transduced HSPC does not impact telomere length in the setting of polyclonal hematopoiesis. While clonality may exert a negative effect on telomere length, this level of telomere attrition is not associated with malignant transformation or bone marrow failure in this animal model. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Chronic lymphocytic leukemia (CLL) is typically considered a malignancy of mature B-cells and is thought to originate from the oncogenic transformation of a single pre- or post-germinal B-cell. Activation-induced deaminase (AID), an enzyme that induces somatic hypermutation (SHM) at the heavy and light chain Ig loci, has been shown to be active in CLL cells in vitro (Patten et al., Blood 2012). Previous studies suggest that multiple CLL-specific Ig clonotypes related by SHM may be present in patients (pts) with dominant CLL clones possessing somatically mutated or unmutated Ig loci (Logan et al., PNAS 2011; Campbell et al., PNAS 2008). To our knowledge, evolution of the dominant CLL-specific Ig clonotype over the course of treatment has not been demonstrated. Here we utilized the LymphoSIGHT™ method, a next-generation sequencing-based method for lymphocyte characterization and quantification, to quantify clonal evolution at the Ig heavy and kappa chain (IGH and IGK) loci in 63 pts with CLL. Methods: Samples were collected at Stanford University and the Dana-Farber Cancer Institute. Peripheral blood mononuclear cells were isolated, and genomic DNA was extracted. Using unbiased universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al., Blood 2012). CLL-specific clonotypes were identified for each patient based on their high frequency (>5%) within the B-cell repertoire of a diagnostic (dx) sample. The highest frequency CLL clonotype identified in a dx sample is termed the “index clonotype”. Dx and post-treatment peripheral blood samples were assessed for evidence of evolved CLL clonotypes using LymphoSIGHT. A clonotype was considered “evolved” based on CDR3 sequence homology to the dx “index clonotype.” Results: CLL clonotypes were identified in dx samples from 63 pts (51 unmutated IGHV; 12 mutated), and we assessed post-treatment samples for the presence of CLL clonotype-associated oligoclonality. Two of 63 pts exhibited clonal evolution in post-treatment samples. One patient with unmutated CLL was MRD negative for over 7 years following allogeneic hematopoietic cell transplant (HCT), and subsequently became MRD positive with the evolved clonotype (differing by 1 nucleotide from the index clonotype) leading to clinical relapse 9 months after MRD positivity, while the original index clone remained undetectable. The patient was treated with ibrutinib upon clinical relapse and continues to have detectable MRD with the same evolved CLL clonotype (Fig 1A). In a second patient with mutated IGHV, we observed several evolved clonotypes in the dx sample. Multiple evolved clonotypes, including 5 that exhibited a significant increase in their frequency relative to the index clonotype, were present in the follow-up sample after treatment with fludarabine and rituximab (Fig 1B). These evolved clonotypes differed from the index clonotype by 1-4 nucleotides, but otherwise shared CDR3 identity, excluding independently arisen B cell clonotypes. Conclusions: We observed evidence of clonal evolution at Ig loci in a small subset (3.2%) of pts with CLL undergoing treatment. The presence of evolution in pts with CLL indicates that either the SHM mechanism, including the AID enzyme, remains active after neoplastic transformation, or the evolved clonotypes arose through a mechanism distinct from SHM. These evolved CLL clonotypes may have a selective advantage, and may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Additional cases are under investigation and updated results will be presented. Figure 1. CLL clonal evolution during therapy. MRD levels of two related Ig clonotypes, expressed as leukemia molecules per million leukocytes in peripheral blood, are shown at multiple time points following allogeneic HCT (A). In another patient undergoing conventional treatment, the level of each individual evolved clonotype as a fraction of the total CLL molecules is plotted at dx and post treatment time points. The index clone, evolved clones with increasing levels post-treatment, and evolved clones with decreasing levels post-treatment are shown in red, blue, and white, respectively (B). Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Moorhead: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

AbstractThe annual course of irradiance was recorded at two vertical and even-aged neighbouring Quercus stems, one rich in L. pulmonaria, one without. Irradiance never exceeded 610 μmol photons m−2 s−1 at the L. pulmonaria site, whereas the L. pulmonaria-deficient site could experience 2 h daily 2000 μmol photons m−2 s−1, and 6 h above 1000 μmol photons m−2 s−1 during a clear day in early spring. Thalli of L. pulmonaria were transplanted to these two stems. During the first 40 days (April–May), transplants at the L. pulmonaria-deficient site developed severe chlorophyll degradation, and a substantial reduction in maximal PS II efficiency (Fv/Fm) even when measured after a 48-h recovery period at low light intensity. Extensive bleaching was formed along light-exposed sides of the tiny ridges on the upper side. Subsequent to this damage, FV/FM gradually rose to nearly normal levels during the following year. This apparent recovery was probably mainly due to irreversible loss of damaged chlorophyll, but also to some level of acclimation. No damage was observed in control transplants on the L. pulmonariarich tree, which were the only transplants gaining sufficient growth for new attachment to the new substratum during the 397-day transplantation period. Nevertheless, a fine-scale, but highly significant seasonal variation in FV/FM of control transplants reflected variations of even low irradiance levels. FV/FM, as measured after a 48-h recovery period at low light intensity, is an efficient meth for recording permanent high light damages at and shortly after damage is formed. However, FV/FM is not a useful estimator of chronic long-term damage.

The dissertation thesis deals with Evolution optimization of control algorithms. The first part of the thesis describes the principles and partial methods of evolution optimization methods especially those used in two-level transplant evolution method. Later the grammatical evolution method is described, which modified algorithm became impulse for creation of transplant evolution method. The transplant evolution method and its two-level modification are new evolutionary algorithms proposed in this work, which were used for optimization of structure and parameters of general controllers control algorithms. The transplant evolution algorithm and its extended two-level modification are described in detail in next chapters. The proper settings of evolutionary algorithms are important for minimization the time of optimization and for finds results approaching the global optimum. For proper setting the parameters of differential evolution was created meta-evolution algorithm that is described in chapter named meta-evolution. The basic concepts of control, chosen methods of system identification and controller parameters settings are described in next part. This part describes algorithms of digital controllers and some specific methods uses in digital control. The demonstrations of control algorithm optimizations of various types of controllers are showed in experimental part. The optimized algorithms of general controllers are compared with various types of PSD controllers which were set by various algebraic methods or differential evolution for various models of systems. In the conclusion of this work is stated a recommendation for further development of evolutionary optimization of controllers are focusing on parallel and distributed computing.