Publications by authors named "Ludmila Danilova"

64 Publications

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Cell 2021 Sep;184(19):5031-5052.e26

Department of Oncology, Johns Hopkins University, Baltimore, MD 21205, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2021.08.023DOI Listing
September 2021

Spatial UMAP and Image Cytometry for Topographic Immuno-oncology Biomarker Discovery.

Cancer Immunol Res 2021 Aug 25. Epub 2021 Aug 25.

Department of Pathology, The Johns Hopkins University School of Medicine, The Sidney Kimmel Comprehensive Cancer Center, and The Johns Hopkins Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Baltimore, Maryland.

Multiplex immunofluorescence (mIF) can detail spatial relationships and complex cell phenotypes in the tumor microenvironment (TME). However, the analysis and visualization of mIF data can be complex and time-consuming. Here, we used tumor specimens from 93 patients with metastatic melanoma to develop and validate a mIF data analysis pipeline using established flow cytometry workflows (image cytometry). Unlike flow cytometry, spatial information from the TME was conserved at single-cell resolution. A spatial uniform manifold approximation and projection (UMAP) was constructed using the image cytometry output. Spatial UMAP subtraction analysis (survivors vs. nonsurvivors at 5 years) was used to identify topographic and coexpression signatures with positive or negative prognostic impact. Cell densities and proportions identified by image cytometry showed strong correlations when compared with those obtained using gold-standard, digital pathology software (R > 0.8). The associated spatial UMAP highlighted "immune neighborhoods" and associated topographic immunoactive protein expression patterns. We found that PD-L1 and PD-1 expression intensity was spatially encoded-the highest PD-L1 expression intensity was observed on CD163 cells in neighborhoods with high CD8 cell density, and the highest PD-1 expression intensity was observed on CD8 cells in neighborhoods with dense arrangements of tumor cells. Spatial UMAP subtraction analysis revealed numerous spatial clusters associated with clinical outcome. The variables represented in the key clusters from the unsupervised UMAP analysis were validated using established, supervised approaches. In conclusion, image cytometry and the spatial UMAPs presented herein are powerful tools for the visualization and interpretation of single-cell, spatially resolved mIF data and associated topographic biomarker development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-21-0015DOI Listing
August 2021

Transfer learning between preclinical models and human tumors identifies a conserved NK cell activation signature in anti-CTLA-4 responsive tumors.

Genome Med 2021 Aug 11;13(1):129. Epub 2021 Aug 11.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.

Background: Tumor response to therapy is affected by both the cell types and the cell states present in the tumor microenvironment. This is true for many cancer treatments, including immune checkpoint inhibitors (ICIs). While it is well-established that ICIs promote T cell activation, their broader impact on other intratumoral immune cells is unclear; this information is needed to identify new mechanisms of action and improve ICI efficacy. Many preclinical studies have begun using single-cell analysis to delineate therapeutic responses in individual immune cell types within tumors. One major limitation to this approach is that therapeutic mechanisms identified in preclinical models have failed to fully translate to human disease, restraining efforts to improve ICI efficacy in translational research.

Method: We previously developed a computational transfer learning approach called projectR to identify shared biology between independent high-throughput single-cell RNA-sequencing (scRNA-seq) datasets. In the present study, we test this algorithm's ability to identify conserved and clinically relevant transcriptional changes in complex tumor scRNA-seq data and expand its application to the comparison of scRNA-seq datasets with additional data types such as bulk RNA-seq and mass cytometry.

Results: We found a conserved signature of NK cell activation in anti-CTLA-4 responsive mouse and human tumors. In human metastatic melanoma, we found that the NK cell activation signature associates with longer overall survival and is predictive of anti-CTLA-4 (ipilimumab) response. Additional molecular approaches to confirm the computational findings demonstrated that human NK cells express CTLA-4 and bind anti-CTLA-4 antibodies independent of the antibody binding receptor (FcR) and that similar to T cells, CTLA-4 expression by NK cells is modified by cytokine-mediated and target cell-mediated NK cell activation.

Conclusions: These data demonstrate a novel application of our transfer learning approach, which was able to identify cell state transitions conserved in preclinical models and human tumors. This approach can be adapted to explore many questions in cancer therapeutics, enhance translational research, and enable better understanding and treatment of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00944-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8356429PMC
August 2021

Systemic inhibition of PTPN22 augments anticancer immunity.

J Clin Invest 2021 Jul 20. Epub 2021 Jul 20.

Sidney Kimmel Comprehensive Cancer Center, John Hopkins University, Baltimore, United States of America.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI146950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409589PMC
July 2021

Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade.

Science 2021 06;372(6547)

The Mark Foundation Center for Advanced Genomics and Imaging, Johns Hopkins University, Baltimore, MD 21287, USA.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163PD-L1 myeloid cells and CD8FoxP3PD-1 T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aba2609DOI Listing
June 2021

Multi-omic profiling of lung and liver tumor microenvironments of metastatic pancreatic cancer reveals site-specific immune regulatory pathways.

Genome Biol 2021 05 13;22(1):154. Epub 2021 May 13.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 550 N Broadway Suite 1101E, Baltimore, MD, 21209, USA.

Background: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC.

Results: By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors.

Conclusions: Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-021-02363-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118107PMC
May 2021

DNA-methylation for the detection and distinction of 19 human malignancies.

Epigenetics 2021 Mar 5:1-11. Epub 2021 Mar 5.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The contribution of DNA-methylation based gene silencing to carcinogenesis is well established. Increasingly, DNA-methylation is examined using genome-wide techniques, with recent public efforts yielding immense data sets of diverse malignancies representing the vast majority of human cancer related disease burden. Whereas mutation events may group preferentially or in high frequency with a given histology, mutations are poor classifiers of tumour type. Here we examine the hypothesis that cancer-specific DNA-methylation reflects the tissue of origin or carcinogenic risk factor, and these methylation abnormalities may be used to faithfully classify tumours according to histology. We present an analysis of 7427 tumours representing 19 human malignancies and 708 normal samples demonstrating that specific tumour changes in methylation can correctly determine site of origin and tumour histology with 86% overall accuracy. Examination of misclassified tumours reveals underlying shared biology as the source of misclassifications, including common cell of origin or risk factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592294.2021.1890885DOI Listing
March 2021

Supervised mutational signatures for obesity and other tissue-specific etiological factors in cancer.

Elife 2021 Jan 25;10. Epub 2021 Jan 25.

Division of Biostatistics and Bioinformatics, Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins University School of Medicine, Baltimore, United States.

Determining the etiologic basis of the mutations that are responsible for cancer is one of the fundamental challenges in modern cancer research. Different mutational processes induce different types of DNA mutations, providing 'mutational signatures' that have led to key insights into cancer etiology. The most widely used signatures for assessing genomic data are based on unsupervised patterns that are then retrospectively correlated with certain features of cancer. We show here that supervised machine-learning techniques can identify signatures, called SuperSigs, that are more predictive than those currently available. Surprisingly, we found that aging yields different SuperSigs in different tissues, and the same is true for environmental exposures. We were able to discover SuperSigs associated with obesity, the most important lifestyle factor contributing to cancer in Western populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.61082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872524PMC
January 2021

Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells.

Front Cell Dev Biol 2020 9;8:698. Epub 2020 Sep 9.

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24-48 h. TEC-EV were also able to enhance the secretion of TGF-β1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.00698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509153PMC
September 2020

Viral status, immune microenvironment and immunological response to checkpoint inhibitors in hepatocellular carcinoma.

J Immunother Cancer 2020 04;8(1)

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Background And Aims: Immune checkpoint inhibitors (ICIs) targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway have clinical activity in hepatocellular carcinoma (HCC), but only a subset of patients respond to these therapies, highlighting a need for novel biomarkers to improve clinical benefit. HCC usually occurs in the setting of liver cirrhosis from chronic hepatitis B or C viral infection, but the effects of viral status on the tumor immune microenvironment and clinical responses to ICIs in HCC remains unclear.

Methods: We conducted a meta-analysis to estimate the objective response rates for PD-1/PD-L1 inhibitors in virally-infected and uninfected patients, and examined the effects of viral etiology on the tumor microenvironment using data from The Cancer Genome Atlas, as well as peripheral blood responses using an independent cohort of patients studied by mass cytometry (cytometry by time-of-flight (CyTOF)).

Results: Meta-analysis comparing objective response rates (ORR) between virally-infected and uninfected patients showed no clinically meaningful difference (absolute difference of ORR in virally-infected vs uninfected=-1.4%, 95% CI: -13.5% to 10.6%). There was no relationship between viral etiology on features of the tumor immune microenvironment that are known to modulate responses to PD-1/PD-L1 inhibitors, and the tumor mutational burden was similar between virally-infected and uninfected HCC. RNA sequencing of tissue-resident T cell and B cell repertoires similarly showed no effect of viral status on their diversity. CyTOF analysis of peripheral blood specimens further demonstrated similar expression of immune-related markers in response to PD-1 inhibitor therapy in virally-infected and uninfected HCC.

Conclusion: There is no significant effect of viral etiology on the tumor immune microenvironment in HCC, and viral status should not be used as a criterion to select patients for PD-1/PD-L1 therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jitc-2019-000394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204805PMC
April 2020

Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer.

Clin Cancer Res 2020 07 9;26(14):3578-3588. Epub 2020 Apr 9.

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland.

Purpose: Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated -expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B).

Patients And Methods: Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates.

Results: Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7-8.6] and 6.1 (95% CI, 3.5-7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55-1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8 T cells and a decrease in CD68 myeloid cells, were observed in long-term survivors in Arm A only.

Conclusions: Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-3978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727397PMC
July 2020

Cytokines secreted by stromal cells in TNBC microenvironment as potential targets for cancer therapy.

Cancer Biol Ther 2020 06 26;21(6):560-569. Epub 2020 Mar 26.

Department of Pharmaceutical Science, Albany College of Pharmacy and Health Science, Albany, NY, USA.

In triple-negative breast cancer (TNBC), the lack of therapeutic markers and effective targeted therapies result in an incurable metastatic disease associated with a poor prognosis. Crosstalks within the tumor microenvironment (TME), including those between cancer and stromal cells, affect the tumor heterogeneity, growth, and metastasis. Previously, we have demonstrated that IL-6, IL-8, and CCL5 play a significant role in TNBC growth and metastasis. In this study, we performed a systematic analysis of cytokine factors secreted from four stromal components (fibroblasts, macrophages, lymphatic endothelial cells, and blood microvascular endothelial cells) induced by four TNBC cell types. Through bioinformatic analysis, we selected putative candidates of secreted factors from stromal cells, which are involved in EMT activity, cell proliferation, metabolism, and matrisome pathways. Among the candidates, LCN2, GM-CSF, CST3, IL-6, IL-8, and CHI3L1 are ranked highly. Significantly, Lipocalin-2 (LCN2) is upregulated in the crosstalk of stromal cells and four different TNBC cells. We validated the increase of LCN2 secreted from four stromal cells induced by TNBC cells. Using a specific LCN2 antibody, we observed the inhibition of TNBC cell growth and migration. Taken together, these results propose secreted factors as molecular targets to treat TNBC progression via crosstalk with stromal components.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15384047.2020.1739484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515526PMC
June 2020

Chromatin structure regulates cancer-specific alternative splicing events in primary HPV-related oropharyngeal squamous cell carcinoma.

Epigenetics 2020 09 22;15(9):959-971. Epub 2020 Mar 22.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine , Baltimore, MD, USA.

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumours. In this study, eleven ASEs (, and were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (, and showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592294.2020.1741757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518675PMC
September 2020

Revisiting the tumorigenesis timeline with a data-driven generative model.

Proc Natl Acad Sci U S A 2020 01 27;117(2):857-864. Epub 2019 Dec 27.

Division of Biostatistics and Bioinformatics, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205;

Cancer is driven by the sequential accumulation of genetic and epigenetic changes in oncogenes and tumor suppressor genes. The timing of these events is not well understood. Moreover, it is currently unknown why the same driver gene change appears as an early event in some cancer types and as a later event, or not at all, in others. These questions have become even more topical with the recent progress brought by genome-wide sequencing studies of cancer. Focusing on mutational events, we provide a mathematical model of the full process of tumor evolution that includes different types of fitness advantages for driver genes and carrying-capacity considerations. The model is able to recapitulate a substantial proportion of the observed cancer incidence in several cancer types (colorectal, pancreatic, and leukemia) and inherited conditions (Lynch and familial adenomatous polyposis), by changing only 2 tissue-specific parameters: the number of stem cells in a tissue and its cell division frequency. The model sheds light on the evolutionary dynamics of cancer by suggesting a generalized early onset of tumorigenesis followed by slow mutational waves, in contrast to previous conclusions. Formulas and estimates are provided for the fitness increases induced by driver mutations, often much larger than previously described, and highly tissue dependent. Our results suggest a mechanistic explanation for why the selective fitness advantage introduced by specific driver genes is tissue dependent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1914589117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969520PMC
January 2020

Multipanel mass cytometry reveals anti-PD-1 therapy-mediated B and T cell compartment remodeling in tumor-draining lymph nodes.

JCI Insight 2020 01 30;5(2). Epub 2020 Jan 30.

Sidney Kimmel Comprehensive Cancer Center.

Anti-programmed cell death protein 1 (anti-PD-1) therapy has become an immunotherapeutic backbone for treating many cancer types. Although many studies have aimed to characterize the immune response to anti-PD-1 therapy in the tumor and in the peripheral blood, relatively less is known about the changes in the tumor-draining lymph nodes (TDLNs). TDLNs are primary sites of tumor antigen exposure that are critical to both regulation and cross-priming of the antitumor immune response. We used multipanel mass cytometry to obtain a high-parameter proteomic (39 total unique markers) immune profile of the TDLNs in a well-studied PD-1-responsive, immunocompetent mouse model. Based on combined hierarchal gating and unsupervised clustering analyses, we found that anti-PD-1 therapy enhances remodeling of both B and T cell compartments toward memory phenotypes. Functionally, expression of checkpoint markers was increased in conjunction with production of IFN-γ, TNF-α, or IL-2 in key cell types, including B and T cell subtypes, and rarer subsets, such as Tregs and NKT cells. A deeper profiling of the immunologic changes that occur in the TDLN milieu during effective anti-PD-1 therapy may lead to the discovery of novel biomarkers for monitoring response and provide key insights toward developing combination immunotherapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.132286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098715PMC
January 2020

Differentially Methylated Super-Enhancers Regulate Target Gene Expression in Human Cancer.

Sci Rep 2019 10 21;9(1):15034. Epub 2019 Oct 21.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Current literature suggests that epigenetically regulated super-enhancers (SEs) are drivers of aberrant gene expression in cancers. Many tumor types are still missing chromatin data to define cancer-specific SEs and their role in carcinogenesis. In this work, we develop a simple pipeline, which can utilize chromatin data from etiologically similar tumors to discover tissue-specific SEs and their target genes using gene expression and DNA methylation data. As an example, we applied our pipeline to human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV + OPSCC). This tumor type is characterized by abundant gene expression changes, which cannot be explained by genetic alterations alone. Chromatin data are still limited for this disease, so we used 3627 SE elements from public domain data for closely related tissues, including normal and tumor lung, and cervical cancer cell lines. We integrated the available DNA methylation and gene expression data for HPV + OPSCC samples to filter the candidate SEs to identify functional SEs and their affected targets, which are essential for cancer development. Overall, we found 159 differentially methylated SEs, including 87 SEs that actively regulate expression of 150 nearby genes (211 SE-gene pairs) in HPV + OPSCC. Of these, 132 SE-gene pairs were validated in a related TCGA cohort. Pathway analysis revealed that the SE-regulated genes were associated with pathways known to regulate nasopharyngeal, breast, melanoma, and bladder carcinogenesis and are regulated by the epigenetic landscape in those cancers. Thus, we propose that gene expression in HPV + OPSCC may be controlled by epigenetic alterations in SE elements, which are common between related tissues. Our pipeline can utilize a diversity of data inputs and can be further adapted to SE analysis of diseased and non-diseased tissues from different organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-51018-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803762PMC
October 2019

PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

Epigenetics 2020 Jun - Jul;15(6-7):604-617. Epub 2019 Oct 13.

Department of Hematology/Oncology, UPMC Hillman Cancer Center , Pittsburgh, PA, USA.

Signal Transducers and Activators of Transcription-3 (STAT3), a potent oncogenic transcription factor, is constitutively activated in lung cancer, but mutations in pathway genes are infrequent. Protein Tyrosine Phosphatase Receptor-T (PTPRT) is an endogenous inhibitor of STAT3 and PTPRT loss-of-function represents one potential mechanism of STAT3 hyperactivation as observed in other malignancies. We determined the role of PTPRT promoter methylation and sensitivity to STAT3 pathway inhibitors in non-small cell lung cancer (NSCLC). TCGA and Pittsburgh lung cancer cohort methylation data revealed hypermethylation of PTPRT associated with diminished mRNA expression in a subset of NSCLC patients. We report frequent hypermethylation of the PTPRT promoter which correlates with transcriptional silencing of PTPRT and increased STAT3 phosphorylation (Y705) as determined by methylation-specific PCR (MSP) and real time quantitative reverse transcription (RT)-PCR in NSCLC cell lines. Silencing of PTPRT using siRNA in H520 lung cancer cell line resulted in increased pSTAT3 and upregulation of STAT3 target genes such as Cyclin D1 and Bcl-X expression. We show this association of PRPRT methylation with upregulation of the STAT3 target genes and in patient derived lung tumour samples. We further demonstrate that promoter methylation associated with different levels of pSTAT3 in lung cancer cell lines had selective sensitivity to STAT3 pathway small molecule inhibitors (SID 864,669 and SID 4,248,543). Our data strongly suggest that silencing of PTPRT by promoter hypermethylation is an important mechanism of STAT3 hyperactivation and targeting STAT3 may be an effective approach for the development of new lung cancer therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592294.2019.1676597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574378PMC
May 2021

Mechanistically detailed systems biology modeling of the HGF/Met pathway in hepatocellular carcinoma.

NPJ Syst Biol Appl 2019 16;5:29. Epub 2019 Aug 16.

1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD USA.

Hepatocyte growth factor (HGF) signaling through its receptor Met has been implicated in hepatocellular carcinoma tumorigenesis and progression. Met interaction with integrins is shown to modulate the downstream signaling to Akt and ERK (extracellular-regulated kinase). In this study, we developed a mechanistically detailed systems biology model of HGF/Met signaling pathway that incorporated specific interactions with integrins to investigate the efficacy of integrin-binding peptide, AXT050, as monotherapy and in combination with other therapeutics targeting this pathway. Here we report that the modeled dynamics of the response to AXT050 revealed that receptor trafficking is sufficient to explain the effect of Met-integrin interactions on HGF signaling. Furthermore, the model predicted patient-specific synergy and antagonism of efficacy and potency for combination of AXT050 with sorafenib, cabozantinib, and rilotumumab. Overall, the model provides a valuable framework for studying the efficacy of drugs targeting receptor tyrosine kinase interaction with integrins, and identification of synergistic drug combinations for the patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41540-019-0107-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697704PMC
April 2020

Comparison of Biomarker Modalities for Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Review and Meta-analysis.

JAMA Oncol 2019 Aug;5(8):1195-1204

Department of Dermatology, Johns Hopkins Medical Institutions, Baltimore, Maryland.

Importance: PD-L1 (programmed cell death ligand 1) immunohistochemistry (IHC), tumor mutational burden (TMB), gene expression profiling (GEP), and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) assays have been used to assess pretreatment tumor tissue to predict response to anti-PD-1/PD-L1 therapies. However, the relative diagnostic performance of these modalities has yet to be established.

Objective: To compare studies that assessed the diagnostic accuracy of PD-L1 IHC, TMB, GEP, and mIHC/IF in predicting response to anti-PD-1/PD-L1 therapy.

Evidence Review: A search of PubMed (from inception to June 2018) and 2013 to 2018 annual meeting abstracts from the American Association for Cancer Research, American Society of Clinical Oncology, European Society for Medical Oncology, and Society for Immunotherapy of Cancer was conducted to identify studies that examined the use of PD-L1 IHC, TMB, GEP, and mIHC/IF assays to determine objective response to anti-PD-1/PD-L1 therapy. For PD-L1 IHC, only clinical trials that resulted in US Food and Drug Administration approval of indications for anti-PD-1/PD-L1 were included. Studies combining more than 1 modality were also included. Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines were followed. Two reviewers independently extracted the clinical outcomes and test results for each individual study.

Main Outcomes And Measures: Summary receiver operating characteristic (sROC) curves; their associated area under the curve (AUC); and pooled sensitivity, specificity, positive and negative predictive values (PPV, NPV), and positive and negative likelihood ratios (LR+ and LR-) for each assay modality.

Results: Tumor specimens representing over 10 different solid tumor types in 8135 patients were assayed, and the results were correlated with anti-PD-1/PD-L1 response. When each modality was evaluated with sROC curves, mIHC/IF had a significantly higher AUC (0.79) compared with PD-L1 IHC (AUC, 0.65, P < .001), GEP (AUC, 0.65, P = .003), and TMB (AUC, 0.69, P = .049). When multiple different modalities were combined such as PD-L1 IHC and/or GEP + TMB, the AUC drew nearer to that of mIHC/IF (0.74). All modalities demonstrated comparable NPV and LR-, whereas mIHC/IF demonstrated higher PPV (0.63) and LR+ (2.86) than the other approaches.

Conclusions And Relevance: In this meta-analysis, tumor mutational burden, PD-L1 IHC, and GEP demonstrated comparable AUCs in predicting response to anti-PD-1/PD-L1 treatment. Multiplex immunohistochemistry/IF and multimodality biomarker strategies appear to be associated with improved performance over PD-L1 IHC, TMB, or GEP alone. Further studies with mIHC/IF and composite approaches with a larger number of patients will be required to confirm these findings. Additional study is also required to determine the most predictive analyte combinations and to determine whether biomarker modality performance varies by tumor type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamaoncol.2019.1549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646995PMC
August 2019

Programmed Cell Death Ligand-1 (PD-L1) and CD8 Expression Profiling Identify an Immunologic Subtype of Pancreatic Ductal Adenocarcinomas with Favorable Survival.

Cancer Immunol Res 2019 06 1;7(6):886-895. Epub 2019 May 1.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Immune-checkpoint therapy has failed to demonstrate meaningful clinical benefit in unselected cases of pancreatic adenocarcinoma (PDAC), but a subset of PDACs are known to upregulate pathways involved in acquired immune suppression. Further delineation of immunologic subtypes of PDAC is necessary to improve clinical trial designs and identify patients who might benefit from immune-checkpoint therapy. We used clinical survival and RNA expression data from The Cancer Genome Atlas (TCGA) to investigate the relationship between immune-modulating pathways and immune subset markers and their impact on survival in PDAC patients. Of the adaptive immune-resistance pathways, expression of PD-L1 and IDO1 was individually associated with poor survival. Although CD8 expression alone was not correlated with survival, the combination of PD-L1 and high CD8 expression identified a subtype with favorable survival. We further extended these observations using an independent PDAC cohort from our institution via IHC, again observing that the PD-L1/CD8 subtype was associated with positive prognosis. Although PDAC is regarded as a poorly immunogenic cancer type, these findings infer that T-cell infiltration in the absence of adaptive immune-resistance pathways is a feature of long-term survival in PDAC and imply the importance of developing future immunotherapeutic strategies based on data-supported biomarkers to refine patient selection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-18-0822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548624PMC
June 2019

Persistent mutant oncogene specific T cells in two patients benefitting from anti-PD-1.

J Immunother Cancer 2019 02 11;7(1):40. Epub 2019 Feb 11.

Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD, USA.

Background: Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently.

Case Presentation: We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations - BRAF-N581I in the NSCLC and AKT1-E17K in the CRC - years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment.

Conclusions: These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40425-018-0492-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371497PMC
February 2019

Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

Clin Cancer Res 2019 05 5;25(9):2860-2873. Epub 2019 Feb 5.

Tumor Biology Section, Head and Neck Surgery Branch, National Institute of Deafness and Other Communication Disorders, NIH, Bethesda, Maryland.

Purpose: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC).

Experimental Design: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen.

Results: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation . We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion . Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET . Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA ( = 0.002) and validation ( = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset.

Conclusions: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-18-0716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497577PMC
May 2019

Integrative Molecular Characterization of Malignant Pleural Mesothelioma.

Cancer Discov 2018 12 15;8(12):1548-1565. Epub 2018 Oct 15.

Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with and mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options. SIGNIFICANCE: Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with and mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene in epithelioid MPM...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-18-0804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310008PMC
December 2018

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

J Immunother Cancer 2018 10 1;6(1):99. Epub 2018 Oct 1.

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified.

Methods: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1.

Results: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1.

Conclusions: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, T, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40425-018-0404-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167897PMC
October 2018

Functional characterization of alternatively spliced GSN in head and neck squamous cell carcinoma.

Transl Res 2018 12 26;202:109-119. Epub 2018 Jul 26.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

We have recently performed the characterization of alternative splicing events (ASEs) in head and neck squamous cell carcinoma, which allows dysregulation of protein expression common for cancer cells. Such analysis demonstrated a high ASE prevalence among tumor samples, including tumor-specific alternative splicing in the GSN gene.In vitro studies confirmed that overall expression of either ASE-GSN or wild-type GSN (WT-GSN) isoform inversely correlated with cell proliferation, whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion. Additionally, a change in expression of either isoform caused compensatory changes in expression of the other isoform. Our results suggest that the overall expression and the balance between GSN isoforms are mediating factors in proliferation, while increased overall expression of ASE-GSN is specific to cancer tissues. As a result, we propose ASE-GSN can serve not only as a biomarker of disease and disease progression, but also as a neoantigen for head and neck squamous cell carcinoma treatment, for which only a limited number of disease-specific targeted therapies currently exist.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.trsl.2018.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218276PMC
December 2018

Discovery and development of differentially methylated regions in human papillomavirus-related oropharyngeal squamous cell carcinoma.

Int J Cancer 2018 11 21;143(10):2425-2436. Epub 2018 Sep 21.

Moores Cancer Center, University of California San Diego, La Jolla, CA.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) exhibits a different composition of epigenetic alterations. In this study, we identified differentially methylated regions (DMRs) with potential utility in screening for HPV-positive OPSCC. Genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV-positive OPSCC tissues and 25 normal tissues. Fifty-one DMRs were defined with maximal methylation specificity to cancer samples. The Cancer Genome Atlas (TCGA) methylation array data was used to evaluate the performance of the proposed candidates. Supervised hierarchical clustering of 51 DMRs found that HPV-positive OPSCC had significantly higher DNA methylation levels compared to normal samples, and non-HPV-related head and neck squamous cell carcinoma (HNSCC). The methylation levels of all top 20 DNA methylation biomarkers in HPV-positive OPSCC were significantly higher than those in normal samples. Further confirmation using quantitative methylation specific PCR (QMSP) in an independent set of 24 HPV-related OPSCCs and 22 controls showed that 16 of the 20 candidates had significant higher methylation levels in HPV-positive OPSCC samples compared with controls. One candidate, OR6S1, had a sensitivity of 100%, while 17 candidates (KCNA3, EMBP1, CCDC181, DPP4, ITGA4, BEND4, ELMO1, SFMBT2, C1QL3, MIR129-2, NID2, HOXB4, ZNF439, ZNF93, VSTM2B, ZNF137P and ZNF773) had specificities of 100%. The prediction accuracy of the 20 candidates rang from 56.2% to 99.8% by receiver operating characteristic analysis. We have defined 20 highly specific DMRs in HPV-related OPSCC, which can potentially be applied to molecular-based detection tests and improve disease management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.31778DOI Listing
November 2018

The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity.

Cancer Immunol Res 2018 08 12;6(8):888-899. Epub 2018 Jun 12.

The Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigen-specific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exome-guided prediction of MANAs. Following identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-18-0129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072595PMC
August 2018
-->