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Journal articles on the topic 'Multiplex immunohistochemistry'

Author: Grafiati
Published: 4 June 2021
Last updated: 14 March 2023

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1

Wistuba, I., E. Parra, and A. Francisco Cruz. "MS17.04 Multiplex Immunohistochemistry." Journal of Thoracic Oncology 14, no. 10 (October 2019): S191. http://dx.doi.org/10.1016/j.jtho.2019.08.380.

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Sheng, Wenjie, Chaoyu Zhang, T. M. Mohiuddin, Marwah Al-Rawe, Felix Zeppernick, Franco H. Falcone, Ivo Meinhold-Heerlein, and Ahmad Fawzi Hussain. "Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy." International Journal of Molecular Sciences 24, no. 4 (February 4, 2023): 3086. http://dx.doi.org/10.3390/ijms24043086.

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Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.
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Gannot, Gallya, Michael A. Tangrea, Heidi S. Erickson, Peter A. Pinto, Stephen M. Hewitt, Rodrigo F. Chuaqui, John W. Gillespie, and Michael R. Emmert-Buck. "Layered Peptide Array for Multiplex Immunohistochemistry." Journal of Molecular Diagnostics 9, no. 3 (July 2007): 297–304. http://dx.doi.org/10.2353/jmoldx.2007.060143.

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Morrison, Larry E., Mark R. Lefever, Lauren J. Behman, Torsten Leibold, Esteban A. Roberts, Uwe B. Horchner, and Daniel R. Bauer. "Brightfield multiplex immunohistochemistry with multispectral imaging." Laboratory Investigation 100, no. 8 (April 27, 2020): 1124–36. http://dx.doi.org/10.1038/s41374-020-0429-0.

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Forsberg, Peter A., Andrew Hammes, Diana Abbott, Daniel W. Sherbenou, Adriana Rossi, David Jayabalan, Ruben Niesvizky, Tomer M. Mark, and Scott Ely. "Cellular proliferation by multiplex immunohistochemistry identifies aggressive disease behavior in relapsed multiple myeloma." Leukemia & Lymphoma 60, no. 8 (January 11, 2019): 2085–87. http://dx.doi.org/10.1080/10428194.2018.1551537.

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Barrow, Emma, D. Gareth Evans, Ray McMahon, James Hill, and Richard Byers. "A comparative study of quantitative immunohistochemistry and quantum dot immunohistochemistry for mutation carrier identification in Lynch syndrome." Journal of Clinical Pathology 64, no. 3 (December 22, 2010): 208–14. http://dx.doi.org/10.1136/jcp.2010.084418.

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AimsLynch Syndrome is caused by mutations in DNA mismatch repair (MMR) genes. Mutation carrier identification is facilitated by immunohistochemical detection of the MMR proteins MHL1 and MSH2 in tumour tissue and is desirable as colonoscopic screening reduces mortality. However, protein detection by conventional immunohistochemistry (IHC) is subjective, and quantitative techniques are required. Quantum dots (QDs) are novel fluorescent labels that enable quantitative multiplex staining. This study compared their use with quantitative 3,3′-diaminobenzidine (DAB) IHC for the diagnosis of Lynch Syndrome.MethodsTumour sections from 36 mutation carriers and six controls were obtained. These were stained with DAB on an automated platform using antibodies against MLH1 and MSH2. Multiplex QD immunofluorescent staining of the sections was performed using antibodies against MLH1, MSH2 and smooth muscle actin (SMA). Multispectral analysis of the slides was performed. The staining intensity of DAB and QDs was measured in multiple colonic crypts, and the mean intensity scores calculated. Receiver operating characteristic (ROC) curves of staining performance for the identification of mutation carriers were evaluated.ResultsFor quantitative DAB IHC, the area under the MLH1 ROC curve was 0.872 (95% CI 0.763 to 0.981), and the area under the MSH2 ROC curve was 0.832 (95% CI 0.704 to 0.960). For quantitative QD IHC, the area under the MLH1 ROC curve was 0.812 (95% CI 0.681 to 0.943), and the area under the MSH2 ROC curve was 0.598 (95% CI 0.418 to 0.777).ConclusionsDespite the advantage of QD staining to enable several markers to be measured simultaneously, it is of lower utility than DAB IHC for the identification of MMR mutation carriers. Automated DAB IHC staining and quantitative slide analysis may enable high-throughput IHC.
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Harmsen, Marissa J., Arda Arduç, Maaike C. G. Bleeker, Lynda J. M. Juffermans, Arjan W. Griffioen, Ekaterina S. Jordanova, and Judith A. F. Huirne. "Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry." International Journal of Molecular Sciences 23, no. 15 (July 29, 2022): 8434. http://dx.doi.org/10.3390/ijms23158434.

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There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.
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Oscar, Brück, Sami Blom, Riku Turkki, Panu E. Kovanen, Antonio Ribeiro, Nina Linder, Johan Lundin, Olli Kallioniemi, Teijo Pellinen, and Satu Mustjoki. "Immune Cell Profiling in CML Bone Marrow By Multiplex Immunohistochemistry." Blood 128, no. 22 (December 2, 2016): 1897. http://dx.doi.org/10.1182/blood.v128.22.1897.1897.

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Abstract Background In most solid tumors, CD8+ cytotoxic T-cells and type 1 T-helper cells are associated with a positive prognosis, but a strong immunosuppressive microenvironment may hamper their effectiveness. This notion has contributed to the development of new immune-activating therapies, such as immune checkpoint inhibitors. Although having demonstrated long-term remissions in many different solid tumor types, immune checkpoint inhibitors have not been evaluated comprehensively in hematological malignancies. In this study, we aimed to characterize the cellular and molecular immunological profiles of chronic myeloid leukemia (CML) patients' bone marrow (BM) samples. Methods BM biopsies were taken at the time of diagnosis from chronic phase CML patients (n=57) treated in the Helsinki University Hospital during years 2005-2015. We used non-leukemic (NL) BM biopsies (n=10) as controls. Using hematopathologic expertise, we constructed tissue microarray (TMA) blocks from duplicate BM spots characterized with high leukemic cell infiltration. We stained TMA slides using multiplexed immunohistochemistry (IHC) combining fluorescent and chromogenic staining allowing detection of up to six markers and nuclei simultaneously. Marker panels included T and B-lymphoid (CD3, CD4, CD8, CD20), myeloid dendritic (CD11c, BDCA-1, BDCA-3), macrophage (CD68, pSTAT1, c-MAF), natural killer cell (CD3 and CD56) and leukemia cell (CD34) markers. In addition, we examined immune checkpoint molecules (PD1, CTLA4, OX40, LAG3, TIM3) and their ligands in leukemic cells (HLA-G, PD-L1, PD-L2, HLA-ABC), as well as activation markers (CD25, CD27, CD57, Granzyme B and CD45RO). We analyzed leukemia patients' immune checkpoint expression profiles quantitatively using the image analysis software Cell Profiler and cell analysis software FlowJo and compared results with NL BMs' immune cell profiles. Results The proportion of CD3+ T cells of all cells was significantly higher in CML BM vs. NL BM (median 6.0% [interquartile range (IQR) 3.6-10.7] vs. 2.1% [IQR 1.5-4.5], p=0.001). There was no significant difference in CD8+ cytotoxic T cell levels, but CD4+ helper T cells were 8-fold more abundant in CML as compared to non-leukemic BM (p<0.0001). The proportion of both memory CD45RO+CD8+ T cells (62.2% [IQR 47.4-69.8] vs. 47.3% [IQR 27.9-56.2] of CD8+ T cells, p=0.03) and memory CD45RO+CD4+ T cells (61.8% [IQR 51.8-68.5] vs. 40.0% [IQR 25.6-57.9] of CD4+ T cells, p=0.004) were significantly higher in leukemic patients. Although the proportion of PD1+CD8+ T cells did not differ between CML and NL BM, there was a significantly lower proportion of PD1+CD4+ T cells in CML BM vs. NL BM (25.1% [IQR 17.0-38.7] vs. 69.5% [IQR 50.7-77.9], p<0.0001). However, as the number of CD4+ T cells was increased in CML, the absolute number of CD4+PD1+ T cells of total cell population was 3-fold higher in CML BM than in NL BM (p=0.02). Both the proportion of OX40+CD4+ T cells (42.3% [IQR 28.7-51.6] vs. 18.1% [IQR 13.2-22.9], p=0.001) and OX40+CD8+ T cells (42.6% [IQR 25.8-60.7] vs. 12.7% [IQR 5.0-15.8], p<0.0001) were increased in leukemic patients. Interestingly, also the proportion of OX40+PD1+CD8+ T cells (25.7% [IQR 15.4-36.4] vs. 11.9% [IQR 5.0-15.8], p=0.0019) was higher in CML samples. Conclusion Multiplex IHC allows detailed characterization of immune cell subtypes and their phenotypes in BM biopsy samples. Our data show significant heterogeneity in immune cell subsets between individual patients. The CML BM is characterized with an increase in CD3+ T cells, especially helper T cells and CD45RO+ memory T cells, when compared to non-leukemic BM. Phenotypically, OX40+PD1neg T cells and OX40+PD1+ cytotoxic T cells were elevated in CML patients. The analysis of other immune cell subclasses, including inhibitory immune cells, and the correlation of histologic findings to prognostic data are ongoing. Together, they will provide a detailed understanding of BM immune cell composition in CML. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.
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Silverman, Andrew, Matthew Ingham, Robyn Denise Gartrell, Douglas Kanter Marks, Hojung Rachel Park, Thomas D. Hart, Camden L. Esancy, et al. "Interrogating the sarcoma immune microenvironment (iME) using multiplex immunohistochemistry (mIHC)." Journal of Clinical Oncology 36, no. 15_suppl (May 20, 2018): 11536. http://dx.doi.org/10.1200/jco.2018.36.15_suppl.11536.

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Ugolini, Filippo, Elisa Pasqualini, Sara Simi, Gianna Baroni, and Daniela Massi. "Bright-Field Multiplex Immunohistochemistry Assay for Tumor Microenvironment Evaluation in Melanoma Tissues." Cancers 14, no. 15 (July 28, 2022): 3682. http://dx.doi.org/10.3390/cancers14153682.

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The tumor microenvironment (TME) plays a crucial role in melanoma development, progression and response to treatment. As many of the most relevant TME cell phenotypes are defined by the simultaneous detection of more than two markers, the bright-field (BF) multiplex immunohistochemistry (IHC) technique has been introduced for the quantitative assessment and evaluation of the relative spatial distances between immune cells and melanoma cells. In the current study, we aimed to validate BF multiplex IHC techniques in the Ventana Discovery Ultra Immunostainer to be applied to the evaluation of the TME in variably pigmented melanoma tissues. The BF multiplex IHC staining was performed using different combinations of six immune-cell markers—CD3, CD4, CD8, CD20, CD68 and CD163—and the melanoma cell marker SOX10. Our results show that the BF double IHC Yellow/Purple protocol guarantees the maximum contrast in all the cell populations tested and the combination SOX10 (Green), CD8 (Yellow) and CD163 (Purple) of the BF triple IHC protocol ensures the best contrast and discrimination between the three stained cell populations. Furthermore, the labeled cells were clearly distinct and easily identifiable using the image analysis software. Our standardized BF IHC multiplex protocols can be used to better assess the immune contexts of melanoma patients with potential applications to drive therapeutic decisions within clinical trials.
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Ely, Scott, Peter Forsberg, Ruben Niesvizky, and Tomer Martin Mark. "Plasma cell proliferation by SynKii multiplex immunohistochemistry (mIHC) for clinical use in multiple myeloma (MM)." Journal of Clinical Oncology 34, no. 15_suppl (May 20, 2016): 8056. http://dx.doi.org/10.1200/jco.2016.34.15_suppl.8056.

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Kelly, Aubrey M., Brandon A. Fricker, and Kelly J. Wallace. "Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues." STAR Protocols 3, no. 4 (December 2022): 101672. http://dx.doi.org/10.1016/j.xpro.2022.101672.

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Kryeziu, Kushtrim, Christian H. Bergsland, Tormod K. Guren, Anita Sveen, and Ragnhild A. Lothe. "Multiplex immunohistochemistry of metastatic colorectal cancer and ex vivo tumor avatars." Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1877, no. 1 (January 2022): 188682. http://dx.doi.org/10.1016/j.bbcan.2022.188682.

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Aljakna, Aleksandra, Estelle Lauer, Sébastien Lenglet, Silke Grabherr, Tony Fracasso, Marc Augsburger, Sara Sabatasso, and Aurélien Thomas. "Multiplex quantitative imaging of human myocardial infarction by mass spectrometry-immunohistochemistry." International Journal of Legal Medicine 132, no. 6 (March 19, 2018): 1675–84. http://dx.doi.org/10.1007/s00414-018-1813-9.

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Chen, Ting, and Chukka Srinivas. "Group sparsity model for stain unmixing in brightfield multiplex immunohistochemistry images." Computerized Medical Imaging and Graphics 46 (December 2015): 30–39. http://dx.doi.org/10.1016/j.compmedimag.2015.04.001.

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MASUGI, Yohei. "Pancreatic cancer stromal heterogeneity characterized by multiplex immunohistochemistry-based image analyses." Suizo 38, no. 1 (February 28, 2023): 28–36. http://dx.doi.org/10.2958/suizo.38.28.

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Shidham, Vinod B., and Lester J. Layfield. "Cell-blocks and immunohistochemistry." Cytojournal 18 (January 30, 2021): 2. http://dx.doi.org/10.25259/cytojournal_83_2020.

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The interpretation of results on immunostained cell-block sections has to be compared with the cumulative published data derived predominantly from formalin-fixed paraffin-embedded (FFPE) tissue sections. Because of this, it is important to recognize that the fixation and processing protocol should not be different from the routinely processed FFPE surgical pathology tissue. Exposure to non-formalin fixatives or reagents may interfere with the diagnostic immunoreactivity pattern. The immunoprofile observed on such cell-blocks, which are not processed in a manner similar to the surgical pathology specimens, may not be representative resulting in aberrant results. The field of immunohistochemistry (IHC) is advancing continuously with the standardization of many immunomarkers. A variety of technical advances such as multiplex IHC with refined methodologies and automation is increasing its role in clinical applications. The recent addition of rabbit monoclonal antibodies has further improved sensitivity. As compared to the mouse monoclonal antibodies, the rabbit monoclonal antibodies have 10 to 100 fold higher antigen affinity. Most of the scenarios involve the evaluation of coordinate immunostaining patterns in cell-blocks with relatively scant diagnostic material without proper orientation which is usually retained in most of the surgical pathology specimens. These challenges are addressed if cell-blocks are prepared with some dedicated methodologies such as NextGen CelBloking™ (NGCB) kits. Cell-blocks prepared by NGCB kits also facilitate the easy application of the SCIP (subtractive coordinate immunoreactivity pattern) approach for proper evaluation of coordinate immunoreactivity. Various cell-block and IHC-related issues are discussed in detail.
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Meseck, Emily K., Bradley L. Njaa, Nicholas J. Haley, Edward H. Park, and Stephen C. Barr. "Use of a Multiplex Polymerase Chain Reaction to Rapidly Differentiate Neospora Caninum from Toxoplasma Gondii in an Adult Dog with Necrotizing Myocarditis and Myocardial Infarct." Journal of Veterinary Diagnostic Investigation 17, no. 6 (November 2005): 565–68. http://dx.doi.org/10.1177/104063870501700607.

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This report describes a 3-year-old male castrated Mastiff dog that died unexpectedly with locally extensive, acute, necrotizing myocarditis and myocardial infarction. Intralesional protozoal tachyzoites in the affected myocardium were confirmed to be Neospora caninum by a novel multiplex polymerase chain reaction (PCR) and immunohistochemistry. Protozoal organisms were not identified in other tissues by histology, immunohistochemistry, or PCR. The multiplex PCR assay was used to quickly provide preliminary results on fresh myocardium to differentiate N. caninum and Toxoplasma gondii. Neosporosis is an uncommon cause of myocarditis in adult dogs and differential diagnoses for myocarditis in this population of dogs are reviewed.
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Gorris, Mark A. J., Altuna Halilovic, Katrin Rabold, Anne van Duffelen, Iresha N. Wickramasinghe, Dagmar Verweij, Inge M. N. Wortel, Johannes C. Textor, I. Jolanda M. de Vries, and Carl G. Figdor. "Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment." Journal of Immunology 200, no. 1 (November 15, 2017): 347–54. http://dx.doi.org/10.4049/jimmunol.1701262.

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Ely, Scott, Peter Forsberg, Ihsane Ouansafi, Adriana Rossi, Alvin Modin, Roger Pearse, Karen Pekle, et al. "Cellular Proliferation by Multiplex Immunohistochemistry Identifies High-Risk Multiple Myeloma in Newly Diagnosed, Treatment-Naive Patients." Clinical Lymphoma Myeloma and Leukemia 17, no. 12 (December 2017): 825–33. http://dx.doi.org/10.1016/j.clml.2017.09.010.

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Gartrell, Robyn Denise, Douglas Kanter Marks, Thomas Hart, Edward Stack, Yan Lu, Camden Esancy, Camille Gerard, et al. "Characterizing the tumor microenvironment (TME) in primary melanomas using multiplex immunohistochemistry (mIHC)." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 9580. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.9580.

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9580 Background: Biomarkers are needed in primary melanoma to risk stratify for adjuvant trials. High levels of infiltrating cytotoxic (CD8+) T lymphocytes (CTLs) and low levels of CD68+ macrophages (MΦ) may correlate with prolonged survival but quantification methods are not standardized for clinical practice. HLA-DR is a marker of MΦ activation not expressed by suppressor myeloid cells. A novel pathology technique using mIHC allows for quantitative and spatial analysis of immune cell subsets. Methods: In a pilot set of stage II/III primary melanomas from Columbia University Medical Center (n = 94), clinical follow up is available for 51 cases. 32 had no evidence of recurrence at last follow up (minimum 2 years) while 19 died of melanoma. 5µm slides were stained using Opal multiplexed IHC (mIHC) for DAPI, CD3, CD8, CD68, SOX10, HLA-DR and Ki67. Tumor areas were pre-selected by a dermatopathologist, visualized using Mantra (Perkin Elmer) and analyzed using inForm (Perkin Elmer) and Spotfire (TIBCO). Results: In all patients (n = 94), CTLs are farther from tumor (SOX10+) cells when they are proliferating (Ki67+) (p < 0.0001***), while they are closer to MΦ when they are activated (HLA-DR+) (p = 0.0002***). Next, we evaluated impact on prognosis using disease specific survival (DSS) as an outcome based on median value (n = 51). In this exploratory study no correction for multiple comparisons was made. We find that CTL density correlates with prolonged DSS in tumor (p = 0.0185*) but not in stroma (p = 0.1630 ns). Ratio of density of CD8/CD68+HLA-DR- correlates with DSS in both tumor (p = 0.027*) and stroma (p = 0.017*). Finally, distance from CTLs to HLA-DR- MΦ was significantly greater in non-recurrent melanomas as compared to recurrent ones (p = 0.0167*). Conclusions: HLA-DR expression on MΦ and Ki67 expression on tumor cells correlate with position of CTLs in TME in primary melanoma. CTL density is a favorable prognostic marker while HLA-DR non-expressing MΦ may favor tumor progression. Quantitative mIHC allows for accurate spatial analysis of immune subsets within the TME and the development of novel, more accurate and potentially clinically relevant biomarkers.
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Tsujikawa, Takahiro, Rohan N. Borkar, Vahid Azimi, El Edward Rassi, Daniel R. Clayburgh, Sushil Kumar, Andrew J. Gunderson, Molly F. Kresz-Martin, Paul W. Flint, and Lisa M. Coussens. "Multiplex immunohistochemistry for immune profiling of HPV-associated head and neck cancer." Journal for ImmunoTherapy of Cancer 3, Suppl 2 (2015): P419. http://dx.doi.org/10.1186/2051-1426-3-s2-p419.

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Chan, Ronald C. K., Joshua J. X. Li, W. Yeung, and Anthony W. H. Chan. "Virtual multiplex immunohistochemistry: Application on cell block of effusion and aspiration cytology." Diagnostic Cytopathology 48, no. 5 (May 2020): 417–23. http://dx.doi.org/10.1002/dc.24344.

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Tan, Wei Chang Colin, Sanjna Nilesh Nerurkar, Hai Yun Cai, Harry Ho Man Ng, Duoduo Wu, Yu Ting Felicia Wee, Jeffrey Chun Tatt Lim, Joe Yeong, and Tony Kiat Hon Lim. "Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy." Cancer Communications 40, no. 4 (April 2020): 135–53. http://dx.doi.org/10.1002/cac2.12023.

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Blessin, Niclas C., Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Markus Graefen, and Stefan Steurer. "Abstract 483: Automated Ki67-LI assessment in prostate cancer using artificial intelligence in multiplex fluorescence immunohistochemistry." Cancer Research 82, no. 12_Supplement (June 15, 2022): 483. http://dx.doi.org/10.1158/1538-7445.am2022-483.

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Abstract The Ki67 labeling index (Ki67-LI) is a strong prognostic marker in prostate cancer. Its analysis requires cumbersome manual quantification of Ki67 immunostaining in at least 200 tumor cells. To enable automated Ki67-LI assessment in routine clinical practice, we have developed and validated a framework for automated Ki67-LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry staining. The prognostic impact of the Ki67-LI was tested on a tissue microarray (TMA) containing one sample each of 12475 prostate cancers. A “heterogeneity TMA” containing 3 to 6 samples from different tumor areas was used to model Ki67 analysis of multiple different biopsies. The Ki67-LI provided strong and independent prognostic information in 11845 successfully analyzed prostate cancers (p&lt;0.001 each). The analysis of the heterogeneity TMA revealed that the Ki67-LI of the sample with the highest Gleason score (AUC:0.68) was similarly prognostic as the mean Ki67-LI of all six foci (AUC:0.71 [p=0.24]). The combined analysis of the Ki67-LI and Gleason grades obtained on identical tissue spots showed that the Ki67-LI added significant additional prognostic information in case of classical ISUP grades (AUC:0.82 [p=0.002]) and quantitative Gleason grades (AUC:0.83 [p=0.018]). The Ki67-LI is a powerful prognostic parameter in prostate cancer, which can be efficiently analyzed in multiplex fluorescence IHC. In case of multiple cancer positive biopsies, the sole analysis of the worst biopsy can be sufficient. Citation Format: Niclas C. Blessin, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Markus Graefen, Stefan Steurer. Automated Ki67-LI assessment in prostate cancer using artificial intelligence in multiplex fluorescence immunohistochemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 483.
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Pacia, Emmanuel, Xun Li, Ju Young Kim, Evelyn Diaz, Beiru Chen, Nathan Roscoe, Jason Hughes, et al. "47 Pathologists enhance interpretation of automated multiplex immunohistochemistry assays in cancer immunotherapy trials." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A50. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0047.

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BackgroundMultiplex fluorescence immunohistochemistry (mFIHC) enables simultaneous detection of multiple biomarkers on a single tissue section. Spatial patterns and differential expression of immune- and tumor cell biomarkers serve as powerful predictors of immunotherapies. In a recent meta-analyses of 8135 patients treated with PD1/L1 pathway blockers, mFIHC was found to provide highest predictive power (P<0.05) amongst commonly utilized biomarker modalities, namely, PD-L1 IHC, Tumor Mutation Burden and Gene Expression Profiling alone. [Lu et al., JAMA Oncol 2019]. As biomarkers in mFIHC assays are read by computer-aided algorithms, the role of pathologists in the digital workflow has been debated. Utilizing clinical cases representing multiple tumor indications, we illustrate the critical collaboration between pathologists (human intelligence, HI) and computer workflows (artificial intelligence, AI) required for accurate interpretation of mFIHC assays in cancer immunotherapy trials.MethodsIn our clinical trial laboratory, pathologists are involved in pre-analytical, analytical and post-analytical phases of clinical trial sample testing. In the pre-analytical phase, pathologist(s) perform histological examination of H&E stained tissue sections to annotate and confirm tissue types, diagnosis, tissue integrity and acceptance (including viable tumor component), followed by determination of Region of Interest (ROI) for subsequent analysis by computerized programs. In the analytical phase, pathologists identify specific areas of biological and/or clinical interest within ROI (tumor, non-tumor, invasive margin, and tumor-stromal interphase) in the computer scans, as well as exclude ROI containing necrosis, hemorrhage, blood vessels, and autofluorescence. Those pathologist-selected images are then quantified by digital pathology software such as Automated QUantitative Analyses (AQUA®) technology. Finally, pathologists also provide interpretation and summarize findings relevant to the clinical study during the post-analytical phase.ResultsCase studies representing distinct malignancies, such as melanoma, non-small cell lung cancer, squamous cell carcinoma of head and neck and diffuse large B-cell lymphoma, illustrating the role of pathologists and especially in rescuing challenging cases and interpreting biomarkers scores from mFIHC assays will be presented.ConclusionsWith the advancement in technologies to detect increasing number of biomarkers in a single tissue section and accompanied growth of mFIHC assays in immuno-oncology studies, there is a clear transition from conventional pathology (HI) to computer-aided pathology (AI+HI) that will ultimately ensure greater accuracy, reproducibility and standardization of clinical trial testing, and enable approval of more effective therapies and better patient care.
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Tsujikawa, Takahiro, Sushil Kumar, Rohan N. Borkar, Vahid Azimi, Guillaume Thibault, Young Hwan Chang, Ariel Balter, et al. "Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis." Cell Reports 19, no. 1 (April 2017): 203–17. http://dx.doi.org/10.1016/j.celrep.2017.03.037.

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Sorrelle, Noah, Debolina Ganguly, Adrian T. A. Dominguez, Yuqing Zhang, Huocong Huang, Lekh N. Dahal, Natalie Burton, Arturas Ziemys, and Rolf A. Brekken. "Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues." Journal of Immunology 202, no. 1 (December 3, 2018): 292–99. http://dx.doi.org/10.4049/jimmunol.1800878.

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Peter, Simon, Valery Volk, Charlotte Hoffmann, Melanie Bathon, Benjamin Goeppert, Thomas Longerich, Thomas Albrecht, et al. "Characterization and clinical correlation of the immune contexture in intrahepatic cholangiocarcinoma using multiplex immunohistochemistry." Journal of Hepatology 77 (July 2022): S927. http://dx.doi.org/10.1016/s0168-8278(22)02136-5.

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Arnold, Stephanie, Sarah Watts, Emrys Kirkman, Clive P. Page, and Simon C. Pitchford. "Single and Multiplex Immunohistochemistry to Detect Platelets and Neutrophils in Rat and Porcine Tissues." Methods and Protocols 5, no. 5 (September 19, 2022): 71. http://dx.doi.org/10.3390/mps5050071.

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Platelet–neutrophil complexes (PNCs) occur during the inflammatory response to trauma and infections, and their interactions enable cell activation that can lead to tissue destruction. The ability to identify the accumulation and tissue localisation of PNCs is necessary to further understand their role in the organs associated with blast-induced shock wave trauma. Relevant experimental lung injury models often utilise pigs and rats, species for which immunohistochemistry protocols to detect platelets and neutrophils have yet to be established. Therefore, monoplex and multiplex immunohistochemistry protocols were established to evaluate the application of 22 commercially available antibodies to detect platelet (nine rat and five pig) and/or neutrophil (four rat and six pig) antigens identified as having potential selectivity for porcine or rat tissue, using lung and liver sections taken from models of polytrauma, including blast lung injury. Of the antibodies evaluated, one antibody was able to detect rat neutrophil elastase (on frozen and formalin-fixed paraffin embedded (FFPE) sections), and one antibody was successful in detecting rat CD61 (frozen sections only); whilst one antibody was able to detect porcine MPO (frozen and FFPE sections) and antibodies, targeting CD42b or CD49b antigens, were able to detect porcine platelets (frozen and FFPE and frozen, respectively). Staining procedures for platelet and neutrophil antigens were also successful in detecting the presence of PNCs in both rat and porcine tissue. We have, therefore, established protocols to allow for the detection of PNCs in lung and liver sections from porcine and rat models of trauma, which we anticipate should be of value to others interested in investigating these cell types in these species.
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Pugh, Matthew, Ayse U. Akarka, Kelly Hunter, Stefan Dojcinov, and Teresa Marafioti. "Multiplex immunohistochemistry in lymphoma pathology: a research tool for study of the immune microenvironment." Diagnostic Histopathology 26, no. 9 (September 2020): 407–20. http://dx.doi.org/10.1016/j.mpdhp.2020.07.002.

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Ely, S., G. Lee, L. Menard, J. Yan, P. Fischer, B. Kakrecha, D. Locke, P. Patah, and K. Urbanska. "Multiplex chromogenic Immunohistochemistry (IHC) for spatial analysis of checkpoint-positive tumour infiltrating lymphocytes (TILs)." Annals of Oncology 30 (October 2019): v40. http://dx.doi.org/10.1093/annonc/mdz239.039.

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Hu, Weixian. "Multiplex immunohistochemistry/immunofluorescence is superior to tumor mutational burden and PD‐L1 immunohistochemistry for predicting response to anti‐PD‐1/PD‐L1 immunotherapy." Thoracic Cancer 11, no. 1 (November 12, 2019): 3–5. http://dx.doi.org/10.1111/1759-7714.13233.

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Gérus-Durand, Marie, Elena Baranova, Maroua Tliba, Sabine Iglesias, Manon Motte, Renaud Burrer, and Amanda Finan-Marchi. "Abstract 3871: Solid tumor analytical validation of a T-regulatory immunohistochemistry multiplex for clinical studies." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3871. http://dx.doi.org/10.1158/1538-7445.am2022-3871.

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Abstract While classic, chromogenic immunohistochemistry (IHC) remains a mainstay in clinical diagnostics, it can be limiting in the era of immuno-oncology. In the field of immuno-oncology, where access to tissue samples may be limited, there is a need for an accurate interpretation of the sociology and in-depth phenotyping of the cells in the tumor microenvironment that requires a higher level of targets to investigate. Cytotoxic T cells are the main effector cells against tumors. Conversely, T-regulatory cells play a more pro-tumor role by suppressing cytotoxic T cell activation. The importance of the balance and tumor infiltration of these two cell populations has repeatedly been demonstrated in the literature. Due to this, Cerba Research was driven to develop an immunofluorescent multiplex to allow cytotoxic and regulatory T cells detection on the same tissue section for use in solid tumor clinical trials. Following our standard workflow, we developed a CD3-CD8-FoxP3 IHC multiplex (Histoprofile®- T-reg light). This panel allows for analysis of cytotoxic T-cells (CD3+/CD8+/FoxP3-), T helper cells (CD3+/CD8-/FoxP3-) and T regulatory cells (CD3+/CD8-/FoxP3+). Then following our standard process, we performed an analytical validation on a range of formalin fixed paraffin embedded solid tumors (including but not limited to Non Small Cell Lung Cancer, Head and Neck Squamous Cell Carcinoma, Colorectal Cancer and Breast Cancer). Specificity was verified by a pathologist on control tonsil tissue. The robustness of the protocol on a range of solid tumors was verified by the pathologist on tissue blocks and tumor microarrays. Precision tests (intra- and inter-assay) demonstrated a high level of panel reproducibility in solid tumor tissues - CD3 (CV 21.24%), CD8 (CV 19.5%), FoxP3 (CV 13.13%). Using Halo® software (Indica Labs), spatial analysis of the populations of interest can be appreciated in the tumor and stroma compartments. Cerba Research’s Histoprofile®- T-reg light panel passed all the analytical validation criteria for use in clinical studies. Cerba Research can provide this off the shelf panel in solid tumor clinical studies, allowing an elevated analysis of multiple immune biomarkers on a single tissue slide. Citation Format: Marie Gérus-Durand, Elena Baranova, Maroua Tliba, Sabine Iglesias, Manon Motte, Renaud Burrer, Amanda Finan-Marchi. Solid tumor analytical validation of a T-regulatory immunohistochemistry multiplex for clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3871.
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Zito Marino, Federica, Giulio Rossi, Immacolata Cozzolino, Marco Montella, Mariacarolina Micheli, Giuseppe Bogina, Enrico Munari, Matteo Brunelli, and Renato Franco. "Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples." Journal of Clinical Pathology 73, no. 2 (September 27, 2019): 96–101. http://dx.doi.org/10.1136/jclinpath-2019-206152.

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AimsSeveral predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes.MethodsWe collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring ALK-rearrangement and ROS1-rearrangement. ALK and ROS1 status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 ALK-positive, 2 ROS1-positive and 53 ALK/ROS1-wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision.ResultsThe dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 ALK-positive and 2 ROS1-positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results.ConclusionMultiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide in cytological specimens with a small amount of biomaterial.
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De Lorenzo, Cíntia, Caroline P. de Andrade, Verônica S. L. Machado, Matheus V. Bianchi, Veronica M. Rolim, Raquel A. S. Cruz, and David Driemeier. "Piglet colibacillosis diagnosis based on multiplex polymerase chain reaction and immunohistochemistry of paraffin-embedded tissues." Journal of Veterinary Science 19, no. 1 (2018): 27. http://dx.doi.org/10.4142/jvs.2018.19.1.27.

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37

Park, Soon Sik, John Williams Gillespie, Bill James, and Michael Lebowitz. "Multiplex layered immunohistochemistry to predict response of HER2-positive breast cancer patients to trastuzumab therapy." Journal of Clinical Oncology 30, no. 27_suppl (September 20, 2012): 136. http://dx.doi.org/10.1200/jco.2012.30.27_suppl.136.

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136 Background: Since only 40% of HER2-positive breast cancer patients respond to trastuzumab therapy, there is a great clinical need for a test that will better predict which patients would be responders to trastuzumab therapy. Since no single biomarker will likely improve identification of responders, this study will determine the efficacy of multiple biomarkers to predict response of patients to trastuzumab therapy. Our company has developed a protein analysis platform, layered immunohistochemistry (L-IHC), for the analysis of multiple protein biomarkers from a single FFPE tissue section while retaining the histologic orientation. L-IHC is being used to develop a companion diagnostic test in which multiple protein biomarkers are analyzed in HER2-positive breast cancer specimens to predict which patients will respond to trastuzumab therapy. Methods: In this ongoing clinical exploratory study, analysis of pretreatment archival HER2-positive FFPE breast cancer tissue sections from patients whose subsequent therapy included trastuzumab and whose response to therapy (responders and non-responders) is known was performed using L-IHC. Tissue sections were probed using a panel of 14 biomarkers along the HER2 and mTOR pathways were analyzed. In a blinded fashion to the response data, the intensity of specific biomarker signals corresponding to cancer regions on an H&E was visually scored (0,1,2,3, & 4+). The pattern of biomarker expression was correlated with the responder status of the patient. Results: A total of 24 responders and 9 non-responders were analyzed. The sum of four of the 14 biomarkers including p-mTOR, p-4EBP1, pERK, and HIF1-alpha discriminated the responders and non-responders. Using 7 as a cutoff, this test correctly identified 21/24 responders and 7/9 non-responders for a sensitivity of 87%, specificity of 78%, and an accuracy of 82%. Conclusions: Results of this study strongly suggests that analysis of p-mTOR, p-4EBP1, pERK, and HIF1-alpha using L-IHC improves twofold the prediction of patient response to trastuzumab compared to Her2 alone.
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Bâeta, A. M., G. A. Putzu, D. Figarella-Branger, E. Cassote, and J. F. Pellissier. "Immunohistochemistry, immunoblot and PCR multiplex results in 16 patients with cramp/myalgia and/or rhabdomyolysis." Neuromuscular Disorders 6, no. 2 (March 1996): S30. http://dx.doi.org/10.1016/0960-8966(96)89049-3.

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Maiques, Oscar, and Victoria Sanz-Moreno. "Multiplex chromogenic immunohistochemistry to stain and analyze paraffin tissue sections from the mouse or human." STAR Protocols 3, no. 4 (December 2022): 101879. http://dx.doi.org/10.1016/j.xpro.2022.101879.

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Tsujikawa, T., K. Yoshimura, J. Mitsuda, A. Arai, H. Ogi, K. Itoh, and S. Hirano. "Application of Machine Learning and Multiplex Immunohistochemistry to Tissue-immune Profiling for Head and Neck Cancer." Nihon Kikan Shokudoka Gakkai Kaiho 70, no. 2 (April 10, 2019): 177–78. http://dx.doi.org/10.2468/jbes.70.177.

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41

Rakaee, Mehrdad, Lill-Tove Rasmussen Busund, Simin Jamaly, Erna-Elise Paulsen, Elin Richardsen, Sigve Andersen, Samer Al-Saad, Roy M. Bremnes, Tom Donnem, and Thomas K. Kilvaer. "Prognostic Value of Macrophage Phenotypes in Resectable Non–Small Cell Lung Cancer Assessed by Multiplex Immunohistochemistry." Neoplasia 21, no. 3 (March 2019): 282–93. http://dx.doi.org/10.1016/j.neo.2019.01.005.

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42

Kuczkiewicz-Siemion, Olga, Kamil Sokół, Beata Puton, Aneta Borkowska, and Anna Szumera-Ciećkiewicz. "The Role of Pathology-Based Methods in Qualitative and Quantitative Approaches to Cancer Immunotherapy." Cancers 14, no. 15 (August 8, 2022): 3833. http://dx.doi.org/10.3390/cancers14153833.

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Immune checkpoint inhibitors, including those concerning programmed cell death 1 (PD-1) and its ligand (PD-L1), have revolutionised the cancer therapy approach in the past decade. However, not all patients benefit from immunotherapy equally. The prediction of patient response to this type of therapy is mainly based on conventional immunohistochemistry, which is limited by intraobserver variability, semiquantitative assessment, or single-marker-per-slide evaluation. Multiplex imaging techniques and digital image analysis are powerful tools that could overcome some issues concerning tumour-microenvironment studies. This novel approach to biomarker assessment offers a better understanding of the complicated interactions between tumour cells and their environment. Multiplex labelling enables the detection of multiple markers simultaneously and the exploration of their spatial organisation. Evaluating a variety of immune cell phenotypes and differentiating their subpopulations is possible while preserving tissue histology in most cases. Multiplexing supported by digital pathology could allow pathologists to visualise and understand every cell in a single tissue slide and provide meaning in a complex tumour-microenvironment contexture. This review aims to provide an overview of the different multiplex imaging methods and their application in PD-L1 biomarker assessment. Moreover, we discuss digital imaging techniques, with a focus on slide scanners and software.
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Gomez-Brouchet, Anne, Julia Gilhodes, Nathalie Van Acker, Regis Brion, Corinne Bouvier, Pauline Assemat, Nathalie Gaspar, et al. "Characterization of Macrophages and Osteoclasts in the Osteosarcoma Tumor Microenvironment at Diagnosis: New Perspective for Osteosarcoma Treatment?" Cancers 13, no. 3 (January 23, 2021): 423. http://dx.doi.org/10.3390/cancers13030423.

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Biological and histopathological techniques identified osteoclasts and macrophages as targets of zoledronic acid (ZA), a therapeutic agent that was detrimental for patients in the French OS2006 trial. Conventional and multiplex immunohistochemistry of microenvironmental and OS cells were performed on biopsies of 124 OS2006 patients and 17 surgical (“OSNew”) biopsies respectively. CSF-1R (common osteoclast/macrophage progenitor) and TRAP (osteoclast activity) levels in serum of 108 patients were correlated to response to chemotherapy and to prognosis. TRAP levels at surgery and at the end of the protocol were significantly lower in ZA+ than ZA− patients (padj = 0.0011; 0.0132). For ZA+-patients, an increase in the CSF-1R level between diagnosis and surgery and a high TRAP level in the serum at biopsy were associated with a better response to chemotherapy (p = 0.0091; p = 0.0251). At diagnosis, high CD163+ was associated with good prognosis, while low TRAP activity was associated with better overall survival in ZA− patients only. Multiplex immunohistochemistry demonstrated remarkable bipotent CD68+/CD163+ macrophages, homogeneously distributed throughout OS regions, aside osteoclasts (CD68+/CD163−) mostly residing in osteolytic territories and osteoid-matrix-associated CD68−/CD163+ macrophages. We demonstrate that ZA not only acts on harmful osteoclasts but also on protective macrophages, and hypothesize that the bipotent CD68+/CD163+ macrophages might present novel therapeutic targets.
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Jia, Keren, Yang Chen, Jiajia Yuan, Changsong Qi, Yanyan Li, Jifang Gong, Jing Gao, et al. "Abstract 6107: Multiplex immunohistochemistry defined the tumor immune microenvironment and immunotherapeutic outcome in CLDN18.2-positive gastric cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6107. http://dx.doi.org/10.1158/1538-7445.am2022-6107.

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Abstract Introduction: The FAST study ignited the hope of CLDN18.2 as a promising novel therapeutic target for gastric cancer (GC). However, the tumor immune-microenvironmental features of CLDN18.2-positive GC is still unclear, making it difficult to develop/optimize CLDN18.2-targeted treatments. Methods: Multiplex immunohistochemistry (mIHC) was used to decipher the density and spatial structure of T cells, B cells, macrophages, neutrophils in FFPE tumor tissues of 80 GC patients (60 of them received anti-PD-1/PD-L1 treatment). Tumor immune-microenvironmental features and survival distributions stratified by CLDN18.2 were analyzed with two independent-sample t-test and Log-rank test, respectively. Results: We considered mIHC-stained samples with moderate-to-strong CLDN18.2 expression ≥40% of tumor cells as the cutoff for positivity, which shared a high concordance with IHC-based CLDN18.2 staining. The density of CD8+PD-1-, CD8+LAG-3-, and CD8+TIM-3- T cells were significantly higher in CLDN18.2-positive compared with negative tumors (346.28 vs. 204.86/mm2, P=0.011; 436.83 vs. 278.64/mm2, P=0.024; 391.85 vs. 260.72/mm2, P=0.039). In addition, the density of neutrophils (CD66b+) was higher in CLDN18.2-positive than negative group (725.17 vs. 496.23/mm2, P=0.043), while the density of M1 (CD68+CD163-HLA-DR+), M2 macrophages (CD68+CD163+HLA-DR-) and B cells (CD20+) were comparable between CLDN18.2-positive and negative group. In view of spatial analysis, average numbers of CD8+PD1-, CD8+LAG3-, CD8+TIM3-T cells surrounding tumor cells within a 20 μm range were higher in CLDN18.2-positive tumors than in negative group (0.16 vs. 0.09, P=0.011; 0.20 vs. 0.12, P=0.029; 0.18 vs. 0.12, P=0.047). Also, in CLDN18.2-positive group, tumor cells surrounded by CD8+PD1-, CD8+LAG3- T cells or M1 macrophages within a 20 μm range accounted for a higher proportion of all tumor cells than CLDN18.2-negative group (10.79% vs. 6.60%, P=0.015; 12.68% vs. 8.70%, P=0.049; 9.08% vs. 6.56%, P=0.033). These findings suggested that CLDN18.2-positive GC harbored complex immune-microenvironmental features. Additionally, CLDN18.2-positive group had shorter OS/irOS than CLDN18.2-negative group (median OS: 23.33 vs.36.6 months, P&lt;0.001; median irOS: 10.03 vs. 20.13 months, P=0.012). The impact of CLDN18.2-related microenvironmental features on prognosis deserved further investigation. Conclusion: CLDN18.2-positive GC displayed unique immune-microenvironmental characteristics, which provided a phenotypic view for the biological role of CLDN18.2 in GC, and is of great significance for the development of CLDN18.2-targeted therapies. Citation Format: Keren Jia, Yang Chen, Jiajia Yuan, Changsong Qi, Yanyan Li, Jifang Gong, Jing Gao, Xiaotian Zhang, Jian Li, Cheng Zhang, Lin Shen. Multiplex immunohistochemistry defined the tumor immune microenvironment and immunotherapeutic outcome in CLDN18.2-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6107.
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Bedeir, Mai, Yuzuru Ninoyu, Takashi Nakamura, Takahiro Tsujikawa, Shigeru Hirano, and Bedeir. "Multiplex immunohistochemistry and image cytometry reveals an impact of cochlear resident macrophages on Cisplatin-induced hearing loss." Proceedings for Annual Meeting of The Japanese Pharmacological Society 95 (2022): 1—YIA—28. http://dx.doi.org/10.1254/jpssuppl.95.0_1-yia-28.

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46

Cowan, Matthew, Ping Xie, Siqi Chen, Horacio Cardenas, Masha Kocherginsky, Bin Zhang, and Daniela Matei. "Immune markers of response to pembrolizumab and guadecitabine in platinum resistant ovarian cancer utilizing multiplex immunohistochemistry (mIHC)." Gynecologic Oncology 162 (August 2021): S28—S29. http://dx.doi.org/10.1016/s0090-8258(21)00699-5.

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47

Humphries, Matthew P., Victoria Bingham, Fatima Abdullahi Sidi, Stephanie G. Craig, Stephen McQuaid, Jacqueline James, and Manuel Salto-Tellez. "Improving the Diagnostic Accuracy of the PD-L1 Test with Image Analysis and Multiplex Hybridization." Cancers 12, no. 5 (April 29, 2020): 1114. http://dx.doi.org/10.3390/cancers12051114.

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Targeting of the programmed cell death protein (PD-1)/programmed death-ligand 1 (PD-L1) axis with checkpoint inhibitors has changed clinical practice in non-small cell lung cancer (NSCLC). However, clinical assessment remains complex and ambiguous. We aim to assess whether digital image analysis (DIA) and multiplex immunofluorescence can improve the accuracy of PD-L1 diagnostic testing. A clinical cohort of routine NSCLC patients reflex tested for PD-L1 (SP263) immunohistochemistry (IHC), was assessed using DIA. Samples of varying assessment difficulty were assessed by multiplex immunofluorescence. Sensitivity, specificity, and concordance was evaluated between manual diagnostic evaluation and DIA for chromogenic and multiplex IHC. PD-L1 expression by DIA showed significant concordance (R² = 0.8248) to manual assessment. Sensitivity and specificity was 86.8% and 91.4%, respectively. Evaluation of DIA scores revealed 96.8% concordance to manual assessment. Multiplexing enabled PD-L1+/CD68+ macrophages to be readily identified within PD-L1+/cytokeratin+ or PD-L1-/cytokeratin+ tumor nests. Assessment of multiplex vs. chromogenic IHC had a sensitivity and specificity of 97.8% and 91.8%, respectively. Deployment of DIA for PD-L1 diagnostic assessment is an accurate process of case triage. Multiplex immunofluorescence provided higher confidence in PD-L1 assessment and could be offered for challenging cases by centers with appropriate expertise and specialist equipment.
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Abdullahi Sidi, Fatima, Victoria Bingham, Stephanie G. Craig, Stephen McQuaid, Jacqueline James, Matthew P. Humphries, and Manuel Salto-Tellez. "PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics." Cancers 13, no. 1 (December 23, 2020): 29. http://dx.doi.org/10.3390/cancers13010029.

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Multiplex immunofluorescence (mIF) and digital image analysis (DIA) have transformed the ability to analyse multiple biomarkers. We aimed to validate a clinical workflow for quantifying PD-L1 in non-small cell lung cancer (NSCLC). NSCLC samples were stained with a validated mIF panel. Immunohistochemistry (IHC) was conducted and mIF slides were scanned on an Akoya Vectra Polaris. Scans underwent DIA using QuPath. Single channel immunofluorescence was concordant with single-plex IHC. DIA facilitated quantification of cell types expressing single or multiple phenotypic markers. Considerations for analysis included classifier accuracy, macrophage infiltration, spurious staining, threshold sensitivity by DIA, sensitivity of cell identification in the mIF. Alternative sequential detection of biomarkers by DIA potentially impacted final score. Strong concordance was observed between 3,3’-Diaminobenzidine (DAB) IHC slides and mIF slides (R2 = 0.7323). Comparatively, DIA on DAB IHC was seen to overestimate the PD-L1 score more frequently than on mIF slides. Overall, concordance between DIA on DAB IHC slides and mIF slides was 95%. DIA of mIF slides is rapid, highly comparable to DIA on DAB IHC slides, and enables comprehensive extraction of phenotypic data and specific microenvironmental detail intrinsic to the sample. Exploration of the clinical relevance of mIF in the context of immunotherapy treated cases is warranted.
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Christopherson, Cindy, Monica Chang, David A. Eberhard, John J. Sninsky, Steven M. Anderson, Alice M. Wang, Shirley Kwok, and Benjamin Calhoun. "Comparison of immunohistochemistry (IHC) and quantitative RT-PCR: ER, PR, and HER2 receptor status." Journal of Clinical Oncology 30, no. 27_suppl (September 20, 2012): 47. http://dx.doi.org/10.1200/jco.2012.30.27_suppl.47.

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47 Background: IHC is the present standard for measuring estrogen (ER) and progesterone receptor (PR) expression for breast cancer. However, the lack of concordance between testing laboratories and the critical patient treatment decisions made with results prompted ASCO/CAP to recommend guidelines and proficiency testing requirements to ensure accuracy. Quantitative RT-PCR (qRT-PCR) is an alternative method for ER and PR testing and while concordance with IHC has been reported, additional testing is merited. This study compares ER, PR and HER2 status determined by IHC and qRT-PCR. Methods: FFPE tissues of ER(+) tumors collected and tested at the Blumenthal Cancer Center were studied. Expression levels of ESR1, PGR, and ERBB2 were determined by a multiplex qRT-PCR TaqMan assay and by the Oncotype Dx assay. Pre-established cutpoints were used to determine positivity. Only samples with qRT-PCR and corresponding IHC data were used, resulting in 144 ER, 128 PR, and 107 HER2 comparisons. ESR1 and PGR expression levels determined by the two qRT-PCR assays were also compared. Results: Of the144 IHC ER(+) samples, 142 were positive and 2 were negative by both qRT-PCR assays. All 120 IHC PR(+) samples were positive by both qRT-PCR assays. Of the 8 IHC PR(-) samples, 5 were negative and 3 were positive by both qRT-PCR assays. Of the 107 IHC HER2(-) samples, all but 2 were negative by qRT-PCR. One sample was positive by both qRT-PCR assays; one was positive by Oncotype Dx only. The expression levels determined by the qRT-PCR assays showed good correlation for ESR1 (r=0.85) and PGR (r=0.9). Conclusions: Concordance between qRT-PCR and IHC was 98.6% for ER and 97.7% for PR. Except for a single HER2 determination, there was 100% concordance between the two qRT-PCR assays. The discordant sample was HER2 (+) by multiplex qRT-PCR and “equivocal” by Oncotype Dx. Excellent correlation was also observed in the mRNA expression levels. The absence of HER2(+) and ER(-) samples are limitations of this study. These results suggest that qRT-PCR is a promising alternative method to IHC for determining hormone receptor status. Additional testing of samples with endocrine therapy outcomes including ER(-) samples would be beneficial.
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Taube, Janis M., Guray Akturk, Michael Angelo, Elizabeth L. Engle, Sacha Gnjatic, Shirley Greenbaum, Noah F. Greenwald, et al. "The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation." Journal for ImmunoTherapy of Cancer 8, no. 1 (May 2020): e000155. http://dx.doi.org/10.1136/jitc-2019-000155.

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ObjectivesThe interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment.MethodsThe Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms.ResultsRepresentative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed.ConclusionsmIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.
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