Publications by authors named "Scott Rodig"

271 Publications

SMARCA4 and other SWI/SNF family genomic alterations in non-small cell lung cancer: Clinicopathological characteristics and outcomes to immune checkpoint inhibition.

J Thorac Oncol 2021 Apr 9. Epub 2021 Apr 9.

Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA. Electronic address:

Introduction: The SWI/SNF (SWitch/Sucrose Non-Fermentable) chromatin remodeling complex acts as a regulatory component of transcription, and inactivating mutations within the complex are implicated in genomic instability, higher tumor mutational burden (TMB), and an aggressive cancer phenotype. Whether SMARCA4 and other SWI/SNF alterations are independent prognostic factors or associated with clinical outcomes to immune checkpoint inhibitors (ICIs) in NSCLC remains unclear.

Methods: We collected clinicopathologic and genomic data from patients with NSCLC that underwent targeted next-generation sequencing (NGS) at the Dana-Farber Cancer Institute. Tumors were characterized based on the presence or absence of mutations across a set of 6 SWI/SNF genes (ARID1A, ARID1B, ARID2, PBRM1, SMARCA4, and SMARCB1).

Results: Of 2689 patients with NSCLC, 20.6% (N=555) had SWI/SNF genomic alterations. Compared to SWI/SNF wild-type (wt) NSCLC, patients with SWI/SNF mutant NSCLCs had a lower prevalence of concurrent targetable driver mutations (33.2% vs 22.2%; P<0.001), a higher TMB (median 8.5 vs 12.2 mutations/megabase; P<0.001), a shorter median overall survival (mOS) from the time of advanced disease diagnosis (25.0 vs 19.3 months; P=0.01); the detrimental effect in OS appeared to be largely driven by SMARCA4 mutations (mOS: 25.0 months for SMARCA4 wt vs 15.6 months for SMARCA4 mutant; P<0.001). Among 532 patients who received ICIs, 25.5% (N=136) harbored SWI/SNF mutations. From the start of immunotherapy, there was no difference in objective response rate (ORR 19.9% vs 25.0%; P=0.2), median progression-free survival (mPFS 3.0 vs 3.0 months; HR: 0.96 [95% CI: 0.77-1.18]; P=0.7), or mOS (13.1 vs 9.5 months; HR: 0.81 [95% CI: 0.64-1.02]; P=0.07) in SWI/SNF wt vs SWI/SNF mutant NSCLC, respectively. However, among KRAS-mutant NSCLCs treated with ICIs (N=176), a concurrent SWI/SNF mutation (N=39) conferred a numerically lower ORR (21.9% vs 12.8%; P=0.2), a significantly shorter mPFS (4.1 vs 1.8 months; HR: 0.57 [95%CI: 0.38-0.84]; P=0.005), and a significantly shorter mOS (15.5 vs 8.2 months; HR: 0.56 [95%CI: 0.36-0.86]; P=0.008). The deleterious effect on immunotherapy outcomes in KRAS-mutant NSCLC was most pronounced in the SMARCA4-mutant subset (N=17), with a lower ORR (22% vs 0%, P=0.03) a significantly shorter mPFS (4.1 vs 1.4 months; HR: 0.25 [95%CI: 0.14-0.42]; P<0.001), and a significantly shorter mOS (15.1 vs 3.0 months; HR: 0.29 [95%CI: 0.17-0.50]; P<0.001) compared to SMARCA4-wt KRAS-mutant NSCLCs.

Conclusions: Although there were no significant associations between SWI/SNF mutation status and immunotherapy efficacy in the overall NSCLC cohort, the presence of a SMARCA4 alteration may confer a worse outcome to immunotherapy among KRAS-mutant NSCLCs.
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http://dx.doi.org/10.1016/j.jtho.2021.03.024DOI Listing
April 2021

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

Haematologica 2021 Apr 1. Epub 2021 Apr 1.

Section of Hematology, Department of Medicine, University of Calgary, Calgary, AB.

B-cell non-Hodgkin's lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.
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http://dx.doi.org/10.3324/haematol.2020.274258DOI Listing
April 2021

Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.

Nat Cancer 2021 Jan 14;2(1):66-82. Epub 2020 Dec 14.

Breast Tumor Immunology Laboratory, Dana-Farber Cancer Institute, Boston, MA 02215.

Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors and is mediated by CD8 T-cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.
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http://dx.doi.org/10.1038/s43018-020-00148-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963404PMC
January 2021

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Blood 2021 Mar 15. Epub 2021 Mar 15.

NCI, Rockville, United States.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade (ICB) in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GvL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the ETCTN 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T cell populations and increased expression of markers of T cell activation and co-stimulation such as PD-1, HLA-DR and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GvL outcomes.
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http://dx.doi.org/10.1182/blood.2021010867DOI Listing
March 2021

Intrinsic immunogenicity of small cell lung carcinoma revealed by its cellular plasticity.

Cancer Discov 2021 Mar 11. Epub 2021 Mar 11.

Dana-Farber Cancer Institute.

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with de-repression of STING. Transient EZH2 inhibition expands these non-neuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine non-neuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0913DOI Listing
March 2021

Embedding Cultured Cells in Matrigel for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2021 03 1;2021(3). Epub 2021 Mar 1.

Adherent cells normally are prepared for cell staining by growing on a suitable support. Suspension cells can be fixed directly or can be attached to a solid support by centrifugation, chemical cross-linking, or simple dehydration. An alternative approach for cells grown in culture is to prepare a fixed cell pellet that is embedded in a scaffold, such as Matrigel, and then processed, embedded in paraffin, and stained in a manner identical to formalin-fixed tissue specimens.
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http://dx.doi.org/10.1101/pdb.prot099671DOI Listing
March 2021

Preparing Paraffin Tissue Sections for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2021 03 1;2021(3). Epub 2021 Mar 1.

Most histological studies are performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is immediately informative. Fixation and embedding procedures for preparation of paraffin tissue sections are described here. Because of the harsh fixation, embedding, and preparation conditions used in this procedure, many antigens are not well preserved. Thus, cell staining of paraffin-embedded tissue sections usually requires sensitive detection methods and may require amplification using multiple-layer techniques. The protein cross-linking associated with these fixation conditions can mask epitopes. To uncover them and thus improve antibody-antigen binding, the epitopes can be unmasked by reversing the protein cross-linking. One thermal method, heat-induced epitope retrieval, is presented here.
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http://dx.doi.org/10.1101/pdb.prot099663DOI Listing
March 2021

Preparing Frozen Tissue Sections for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2021 03 1;2021(3). Epub 2021 Mar 1.

Cell-staining studies on mammalian tissue are often performed on frozen sections. This is the gentlest method for the preparation of samples and gives good preservation of cell structure and antigens. Its principal disadvantages are that the specimens must be stored frozen and a special microtome, known as a cryostat, is required. In addition, many clinical specimens are not available in this form, and most classical histological descriptions of tissue structure and pathology are based on the use of formalin-fixed, paraffin-embedded (FFPE) material.
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http://dx.doi.org/10.1101/pdb.prot099655DOI Listing
March 2021

Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade.

Cancer Discov 2021 Feb 15. Epub 2021 Feb 15.

Medicine, Infectious Disease, Brigham and Women's Hospital.

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NF-kB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout (Traf3-KO) gene expression signature is associated with better survival in ICB-naive cancer patients and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified SMAC mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T-cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0812DOI Listing
February 2021

A Randomized Trial of Combined PD-L1 and CTLA-4 Inhibition with Targeted Low-Dose or Hypofractionated Radiation for Patients with Metastatic Colorectal Cancer.

Clin Cancer Res 2021 May 10;27(9):2470-2480. Epub 2021 Feb 10.

Dana-Farber Cancer Institute, Boston, Massachusetts.

Purpose: Prospective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade.

Patients And Methods: We performed a multicenter phase II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink.

Results: Eighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1-7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3-4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3-5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8 and CD8/PD-1/Ki-67 T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood.

Conclusions: We demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102320PMC
May 2021

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems.

Science 2021 01;371(6528)

Center for Cancer Genomics, Dana-Farber Cancer Institute, Boston, MA, USA.

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.
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http://dx.doi.org/10.1126/science.aax2656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900882PMC
January 2021

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Nat Med 2021 03 21;27(3):515-525. Epub 2021 Jan 21.

Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA, USA.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.
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http://dx.doi.org/10.1038/s41591-020-01206-4DOI Listing
March 2021

Preparing Cell Smears for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2020 12 1;2020(12). Epub 2020 Dec 1.

Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. In these procedures, a thin layer of cells is deposited on a dry slide by physical methods. The most important factor in obtaining good staining patterns is that the smear be only a single cell thick. Tissue smears do not preserve tissue architecture, but are useful for identifying pathological changes and infectious organisms in tissue samples. Cell smears are easily prepared and can be fixed readily by any of the methods used for attached cells.
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http://dx.doi.org/10.1101/pdb.prot099630DOI Listing
December 2020

Attaching Suspension Cells to Slides for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2020 12 1;2020(12). Epub 2020 Dec 1.

Suspension cells can be prepared for staining by several different methods. A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use of a cytocentrifuge. For surface staining, suspension cells can be attached to slides by cross-linking with poly-l-lysine. Lysine can be polymerized to any desired length, and poly-l-lysine will bind to most solid supports through its charged side chains. The positively charged polymer will provide a site for binding of cells (which carry an overall negative charge). Although this cross-link is not covalent, it is sufficiently strong for most cell-staining techniques.
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http://dx.doi.org/10.1101/pdb.prot099622DOI Listing
December 2020

Nodular primary cutaneous melanoma is associated with PD-L1 expression.

Eur J Dermatol 2020 Aug;30(4):352-357

Division of Clinical Immunology and Allergy, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 255, 8 andar, 05403-900, São Paulo SP, Brazil.

Background: In previous studies, patients with Stage III melanomas expressing PD-L1 in more than 5% of their neoplastic cells had improved recurrence-free survival with anti-PD1 adjuvant therapy.

Objectives: We examined PD-L1 expression as a possible biomarker of primary cutaneous melanomas in the vertical growth phase.

Materials And Methods: This was a retrospective study including 66 patients with invasive primary cutaneous melanomas. We assessed patient clinical and histopathological data and performed immunohistochemical assays with melanoma specimens from the patients to evaluate PD-L1, PD-1, CD3, CD8 and FoxP3 expression.

Results: We observed PD-L1 expression in 21% (14/66) of our samples, and this expression correlated with increased melanoma thickness (p = 0.002) and nodular-type melanoma (p = 0.001). After adjusting for tumor thickness using a logistic regression test, the association of PD-L1 with nodular-type melanoma persisted. Nodular-type melanoma was 6.48 times more likely to be positive for PD-L1 than other histological types (p = 0.014; 95% CI: 1.46-28.82). As expected, PD-L1 expression correlated with the number of PD-1-expressing cells in the tumor-infiltrating lymphocyte population (p = 0.04). No correlation with PD-L1 was observed for age, sex, tumor site, skin phototype, ulceration status, sentinel lymph node status, metastasis development or survival. Regarding the immune profile of the tumor-infiltrating lymphocytes of PD-L1-positive and -negative groups, no significant differences were observed in the numbers of CD3 + , CD8 + FoxP3-, CD8-FoxP3+ and CD8 + FoxP3+ cells by immunohistochemistry.

Conclusion: Nodular-type melanoma is associated with PD-L1 expression and may be a suitable candidate for adjuvant therapy of primary melanomas treated with immunotherapy.
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http://dx.doi.org/10.1684/ejd.2020.3846DOI Listing
August 2020

Pathomic Fusion: An Integrated Framework for Fusing Histopathology and Genomic Features for Cancer Diagnosis and Prognosis.

IEEE Trans Med Imaging 2020 Sep 3;PP. Epub 2020 Sep 3.

Cancer diagnosis, prognosis, and therapeutic response predictions are based on morphological information from histology slides and molecular profiles from genomic data. However, most deep learning-based objective outcome prediction and grading paradigms are based on histology or genomics alone and do not make use of the complementary information in an intuitive manner. In this work, we propose Pathomic Fusion, an interpretable strategy for end-to-end multimodal fusion of histology image and genomic (mutations, CNV, RNASeq) features for survival outcome prediction. Our approach models pairwise feature interactions across modalities by taking the Kronecker product of unimodal feature representations, and controls the expressiveness of each representation via a gatingbased attention mechanism. Following supervised learning, we are able to interpret and saliently localize features across each modality, and understand how feature importance shifts when conditioning on multimodal input. We validate our approach using glioma and clear cell renal cell carcinoma datasets from the Cancer Genome Atlas (TCGA), which contains paired wholeslide image, genotype, and transcriptome data with ground truth survival and histologic grade labels. In a 15-fold cross-validation, our results demonstrate that the proposed multimodal fusion paradigm improves prognostic determinations from ground truth grading and molecular subtyping, as well as unimodal deep networks trained on histology and genomic data alone. The proposed method establishes insight and theory on how to train deep networks on multimodal biomedical data in an intuitive manner, which will be useful for other problems in medicine that seek to combine heterogeneous data streams for understanding diseases and predicting response and resistance to treatment. Code and trained models are made available at: https://github.com/mahmoodlab/PathomicFusion.
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http://dx.doi.org/10.1109/TMI.2020.3021387DOI Listing
September 2020

Spatial signatures identify immune escape via PD-1 as a defining feature of T-cell/histiocyte-rich large B-cell lymphoma.

Blood 2021 Mar;137(10):1353-1364

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.
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http://dx.doi.org/10.1182/blood.2020006464DOI Listing
March 2021

Efficacy and safety results from CheckMate 140, a phase 2 study of nivolumab for relapsed/refractory follicular lymphoma.

Blood 2021 02;137(5):637-645

Mayo Clinic, Rochester, MN.

Nivolumab, an anti-programmed death-1 (PD-1) monoclonal antibody, showed promising activity in relapsed or refractory (R/R) follicular lymphoma (FL) in a phase 1 study. We conducted a phase 2 trial to further evaluate its efficacy and safety in patients with R/R FL and to explore biomarkers of response. Patients with R/R FL and at least 2 prior lines of therapy, each containing a CD20 antibody or an alkylating agent, were treated with nivolumab 3 mg/kg every 2 weeks. The primary end point was objective response rate (ORR) assessed by an independent radiologic review committee. Biomarker analyses included gene expression profiling and multiplex immunofluorescence studies of pretreatment tumor samples. A total of 92 patients were treated. After a minimum follow-up of 12 months, ORR was 4% (4 of 92 patients). Median progression-free survival (PFS) was 2.2 months (95% confidence interval [CI], 1.9-3.6 months). Median duration of response was 11 months (95% CI, 8-14 months). Exploratory analyses suggested that responders had significantly higher proportion of CD3+ T cells in the tumor microenvironment than nonresponders, but no significant differences in PD-1 or programmed death-ligand 1 expression were observed. High expression of a set of tumor-associated macrophage genes was associated with reduced PFS (hazard ratio, 3.28; 95% CI, 1.76-6.11; P = .001). The safety profile was consistent with previous reports of nivolumab. In conclusion, nivolumab monotherapy was associated with very limited activity in patients with R/R FL. Better understanding of the immune biology of this disease may facilitate the development of effective checkpoint-based strategies. This trial was registered at www.clinicaltrials.gov as #NCT02038946.
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http://dx.doi.org/10.1182/blood.2019004753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869188PMC
February 2021

Neoadjuvant Nivolumab or Nivolumab Plus Ipilimumab in Untreated Oral Cavity Squamous Cell Carcinoma: A Phase 2 Open-Label Randomized Clinical Trial.

JAMA Oncol 2020 10;6(10):1563-1570

Brigham and Women's Hospital, Boston, Massachusetts.

Importance: Novel approaches are needed to improve outcomes in patients with squamous cell carcinoma of the oral cavity. Neoadjuvant immunotherapy given prior to surgery and combining programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibitors are 2 strategies to enhance antitumor immune responses that could be of benefit.

Design, Setting, And Participants: In this randomized phase 2 clinical trial conducted at 1 academic center, 29 patients with untreated squamous cell carcinoma of the oral cavity (≥T2, or clinically node positive) were enrolled between 2016 to 2019.

Interventions: Treatment was administered with nivolumab, 3 mg/kg, weeks 1 and 3, or nivolumab and ipilimumab (ipilimumab, 1 mg/kg, given week 1 only). Patients had surgery 3 to 7 days following cycle 2.

Main Outcomes And Measures: Safety and volumetric response determined using bidirectional measurements. Secondary end points included pathologic and objective response, progression-free survival (PFS), and overall survival. Multiplex immunofluorescence was used to evaluate primary tumor immune markers.

Results: Fourteen patients were randomized to nivolumab (N) and 15 patients to nivolumab/ipilimumab (N+I) (mean [SD] age, 62 [12] years; 18 men [62%] and 11 women [38%]). The most common subsite was oral tongue (n = 16). Baseline clinical staging included patients with T2 (n = 20) or greater (n = 9) T stage and 17 patients (59%) with node-positive disease. Median time from cycle 1 to surgery was 19 days (range, 7-21 days); there were no surgical delays. There were toxic effects at least possibly related to study treatment in 21 patients, including grade 3 to 4 events in 2 (N), and 5 (N+I) patients. One patient died of conditions thought unrelated to study treatment (postoperative flap failure, stroke). There was evidence of response in both the N and N+I arms (volumetric response 50%, 53%; pathologic downstaging 53%, 69%; RECIST response 13%, 38%; and pathologic response 54%, 73%, respectively). Four patients had major/complete pathologic response greater than 90% (N, n = 1; N+I, n = 3). With 14.2 months median follow-up, 1-year progression-free survival was 85% and overall survival was 89%.

Conclusions And Relevance: Treatment with N and N+I was feasible prior to surgical resection. We observed promising rates of response in both arms, supporting further neoadjuvant studies with these agents.

Trial Registration: ClinicalTrials.gov Identifier: NCT02919683.
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http://dx.doi.org/10.1001/jamaoncol.2020.2955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453348PMC
October 2020

A peripheral immune signature of responsiveness to PD-1 blockade in patients with classical Hodgkin lymphoma.

Nat Med 2020 09 10;26(9):1468-1479. Epub 2020 Aug 10.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

PD-1 blockade is highly effective in classical Hodgkin lymphomas (cHLs), which exhibit frequent copy-number gains of CD274 (PD-L1) and PDC1LG2 (PD-L2) on chromosome 9p24.1. However, in this largely MHC-class-I-negative tumor, the mechanism of action of anti-PD-1 therapy remains undefined. We utilized the complementary approaches of T cell receptor (TCR) sequencing and cytometry by time-of-flight analysis to obtain a peripheral immune signature of responsiveness to PD-1 blockade in 56 patients treated in the CheckMate 205 phase II clinical trial (NCT02181738). Anti-PD-1 therapy was most effective in patients with a diverse baseline TCR repertoire and an associated expansion of singleton clones during treatment. CD4, but not CD8, TCR diversity significantly increased during therapy, most strikingly in patients who had achieved complete responses. Additionally, patients who responded to therapy had an increased abundance of activated natural killer cells and a newly identified CD3CD68CD4GrB subset. These studies highlight the roles of recently expanded, clonally diverse CD4 T cells and innate effectors in the efficacy of PD-1 blockade in cHL.
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http://dx.doi.org/10.1038/s41591-020-1006-1DOI Listing
September 2020

Binding Antibodies to Attached Cells or Tissues in Preparation for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2020 08 3;2020(8):099705. Epub 2020 Aug 3.

Cells for staining are usually prepared from one of three sources: adherent cells, suspension cells, or whole tissues. Antibodies generally are applied directly to the area of the cells or tissues that is being studied. The antibodies can be labeled directly or they can be detected by using a labeled secondary reagent that will bind specifically to the primary antibody. Detection reagents for cell staining can be labeled with fluorochromes, enzymes, gold, or iodine.
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http://dx.doi.org/10.1101/pdb.prot099705DOI Listing
August 2020

Fixing Attached Cells for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2020 08 3;2020(8):099689. Epub 2020 Aug 3.

For cell staining, fixation methods decrease generally into two classes, organic solvents and cross-linking reagents. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, precipitating the proteins on the cellular architecture. Cross-linking reagents such as paraformaldehyde form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Choosing between fixation in organic solvents or cross-linking agents is empirical. There are no general rules to decide between the two and both procedures are described here. Both methods may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining. In some instances, anti-denatured-protein antibodies are the only ones that can work. Fixation in protein cross-linking reagents such as paraformaldehyde or glutaraldehyde preserves cell structure better than organic solvents but may reduce the antigenicity of some cell components. Simple fixation with paraformaldehyde or glutaraldehyde does not allow the antibody to access the specimen and therefore is followed by a permeabilization step using an organic solvent or nonionic detergent. Using the organic solvent is easy, but it can destroy certain elements of the cell architecture, although prior fixation with paraformaldehyde does help to preserve the cellular structure. If preservation of cell structure is important, the best first choice would be to use a nonionic detergent.
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http://dx.doi.org/10.1101/pdb.prot099689DOI Listing
August 2020

Growing Adherent Cells for Staining.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2020 08 3;2020(8):099614. Epub 2020 Aug 3.

Adherent cells are easily prepared for cell staining by growing on a suitable microscope slide, coverslip, or plastic tissue culture dish. For high-resolution studies, adherent cells should be grown on the highest available grade glass coverslips, because the controlled thickness, flatness, and good optical properties of a proper coverslip are required to produce the best images. In addition, the glass surface is compatible with all fixing and staining solutions. If many antibodies, different dilutions, or various controls are to be tested on the same cell type, plating the cells onto multiwell slides can be helpful. For low-resolution work, such as crude antigen detection, hybridoma screening, or antibody titration, cells for staining can be grown on regular tissue culture dishes.
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http://dx.doi.org/10.1101/pdb.prot099614DOI Listing
August 2020

Axicabtagene Ciloleucel in the Non-Trial Setting: Outcomes and Correlates of Response, Resistance, and Toxicity.

J Clin Oncol 2020 09 15;38(27):3095-3106. Epub 2020 Jul 15.

Dana-Farber Cancer Institute, Boston, MA.

Purpose: Axicabtagene ciloleucel (axi-cel) was approved by the Food and Drug Administration for relapsed aggressive B-cell non-Hodgkin lymphoma in part on the basis of durable remission rates of approximately 40% in a clinical trial population. Whether this efficacy, and the rates of toxicity, would be consistent in a postcommercial setting, with relaxed eligibility criteria and bridging therapy, is unknown. This study describes the efficacy and safety correlates and outcomes in this setting.

Patients And Methods: One hundred twenty-two patients from 7 medical centers in the United States were treated with axi-cel and were included in a modified intent-to-treat (mITT) analysis. Seventy-six patients (62%) were ineligible for the ZUMA-1 trial. Response and toxicity rates, duration of response (DOR), survival, and covariates are described on the basis of the mITT population. Correlative studies on blood and tumor samples were performed to investigate potential biomarkers of response and resistance.

Results: Median follow-up was 10.4 months. In the mITT population, the best overall and complete response (CR) rates were 70% and 50%, respectively. Median DOR and progression-free survival (PFS) were 11.0 and 4.5 months in all patients and were not reached (NR) in CR patients. Median overall survival (OS) was NR; 1-year OS was 67% (95% CI, 59% to 77%). Although response rates were similar in the ZUMA-1-eligible and ZUMA-1-ineligible groups (70% 68%), there was a statistically significant improvement in CR rate (63% 42%, = .016), DOR (median, NR 5.0 months; = .014), PFS (median, NR 3.3 months; = .020), and OS (1-year OS, 89% 54%; < .001) in patients who were ZUMA-1 eligible. Rates of grade ≥ 3 cytokine release syndrome and neurotoxicty were 16% and 35%, respectively.

Conclusion: Axi-cel yields similar rates of overall response and toxicity in commercial and trial settings, although CR rates and DOR were more favorable in patients eligible for ZUMA-1.
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http://dx.doi.org/10.1200/JCO.19.02103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499617PMC
September 2020

Neoadjuvant and Adjuvant Pembrolizumab in Resectable Locally Advanced, Human Papillomavirus-Unrelated Head and Neck Cancer: A Multicenter, Phase II Trial.

Clin Cancer Res 2020 Oct 14;26(19):5140-5152. Epub 2020 Jul 14.

Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts.

Purpose: Pembrolizumab improved survival in patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)-unrelated HNSCC.

Patients And Methods: Neoadjuvant pembrolizumab (200 mg) was administered and followed 2 to 3 weeks later by surgical tumor ablation. Postoperative (chemo)radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (<10%), pTR-1 (10%-49%), and pTR-2 (≥50%). Coprimary endpoints were pTR-2 among all patients and 1-year relapse rate in patients with high-risk pathology (historical: 35%). Correlations of baseline PD-L1 and T-cell infiltration with pTR were assessed. Tumor clonal dynamics were evaluated (ClinicalTrials.gov NCT02296684).

Results: Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3-4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among 18 patients with high-risk pathology was 16.7% (95% confidence interval, 3.6%-41.4%). pTR ≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFNγ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0 and confirmed clonal loss in some patients.

Conclusions: Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The 1-year relapse rate in patients with high-risk pathology was lower than historical.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547532PMC
October 2020

Activation of CAR and non-CAR T cells within the tumor microenvironment following CAR T cell therapy.

JCI Insight 2020 06 18;5(12). Epub 2020 Jun 18.

Center for Immuno-Oncology and.

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.
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http://dx.doi.org/10.1172/jci.insight.134612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406247PMC
June 2020

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

J Immunother Cancer 2020 05;8(1)

Providence Portland Medical Center, Portland, Oregon, USA.

Objectives: The 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.

Methods: The 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.

Results: Representative 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.

Conclusions: mIHC/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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http://dx.doi.org/10.1136/jitc-2019-000155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239569PMC
May 2020