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Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients.We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration.Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates.In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies.

PURPOSEAbout a third of relapsed or refractory classic Hodgkin lymphoma (r/r CHL) patients succumb to their disease after high-dose chemotherapy followed by autologous stem cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL.METHODSWe performed imaging mass cytometry (IMC) on 169 paired primary diagnostic and relapse biopsies using a marker panel specific for CHL biology. For each cell type in the TME, we calculated a ’spatial score’ measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within close interaction range. ‘Spatial scores’ were used as features in prognostic model development for post-ASCT outcomes.RESULTSHighly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early relapse/refractory CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single cell RNA sequencing data identified unique architecture defined by CXCR5+HRS cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay (‘RHL4S’) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk vs low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay.CONCLUSIONSWe identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma–specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma–associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II–deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma.Significance:We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell–like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327

Supplementary Methods, Supplementary Figures 1-19, Supplementary References

PDF file - 46K, Patient MHC class I alleles.

PDF file - 68K, Tested minimal peptides and corresponding MHC binding scores for Patient 1.

PDF file, 54KB, CA125 levels and NY-ESO-1 autoantibody titres before (Pre) and after (Post) primary surgery and chemotherapy for the 6 HGSC patients shown in Figure 4C. Titres represent mean values from three independent experiments.

HR pathway mutations

Supplementary Figure S14 shows the extent of TCR overlap between T-cell subsets isolated from primary HGSC tumor samples.

Purpose: CD103 is expressed in several immune cell types but in the context of the intratumoral immune response may be most important as a marker of antigen-activated CD8 T cells.Methods: We have examined the prognostic significance of CD103 TILs in breast cancer by IHC in a cohort of 424 breast cancer patients.Results: CD103 TILs were present in all subtypes but were more abundant in ER-negative tumors where CD103 TILs were preferentially localized to the intraepithelial compartment. CD103 was associated with tumor size, tumor grade, and ER/PR status (P < 0.05). CD103 TIL density and the epithelial to stromal ratio was highest in the basal-like tumors. Intraepithelial CD103 but not intrastromal CD103 was associated with better relapse-free and overall survival in basal-like subtype tumors [HR = 0.28; 95% confidence interval (CI), 0.17–0.72; P = 0.0047 and HR = 0.25; 95% CI, 0.17–0.66; P = 0.0017, respectively). CD8 status showed similar but less significant associations, but the combination of dual CD103+CD8+ TIL status was the most strongly prognostic combination for relapse-free and overall survival (HR = 0.10; 95% CI, 0.07–0.62; P = 0.006 and HR = 0.09; 95% CI, 0.07–0.57; P = 0.003, respectively).Conclusions: CD103 TILs are indicative of a good prognosis specifically within the basal-like subtype of breast cancer. Clin Cancer Res; 22(24); 6290–7. ©2016 AACR.

Supplementary Methods

Supplemental Table 1. A report concerning the source of the biospecimen and data using the BRISQ guidelines.

Purpose: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment.Experimental Design: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence.Results: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8+ T-cell response to the mutation hydroxysteroid dehydrogenase–like protein 1 (HSDL1)L25V was detected in one patient. In the primary tumor, the HSDL1L25V mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1L25V-specific CD8+ T-cell response. At second recurrence, the HSDL1L25V mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable.Conclusion: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy. Clin Cancer Res; 20(5); 1125–34. ©2013 AACR.

Supplementary Table S4 presents the list of differentially expressed genes identified between triple-positive and triple-negative Tregs and between triple-positive and other Tregs.

Supplemental Figure 2: Prognostic impact of CD103 and CD8 alone and combination in the Basel-like subset of breast cancers.

PDF file, 74KB, Clinical characteristics of patient cohort A.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569–80. ©2017 AACR.See related commentary by Peng and Mills, p. 508

List of Supplementary Figures and Tables, Supplementary Methods, and Supplementary References

Purpose: Tumor-infiltrating lymphocytes (TIL), in particular CD8+ T cells and CD20+ B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8+ TIL can mediate direct cytolytic activity against tumors, the role of CD20+ TIL is poorly understood. Here, we investigate the possible contributions of CD20+ TIL to humoral and cellular tumor immunity.Experimental Design: Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8+ and CD20+ TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA.Results: The vast majority of CD20+ TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20+ TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8+ TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20+ and CD8+ TIL correlated with increased patient survival compared with CD8+ TIL alone.Conclusions: In high-grade serous ovarian tumors, CD20+ TIL have an antigen–experienced but atypical CD27− memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8+ T cells. We propose that the association between CD20+ TIL and patient survival may reflect a supportive role in cytolytic immune responses. Clin Cancer Res; 18(12); 3281–92. ©2012 AACR.

Supplemental Table 2. Association of CD103 TIL and other markers of intra-tumoral immune response.

Supplemental Table 4. Univariate and multivariate analyses of clinical parameters and eCD103 or eCD8 status and their relation with recurrence free survival and overall survival in all cases.

Supplementary Tables S1-S6

PDF file - 72K, Tested minimal peptides and corresponding MHC binding scores for Patient 3.

Purpose:Tumors with high mutation load are thought to engender stronger immune responses, which in turn promote prolonged patient survival. To investigate this, we assessed tumor-infiltrating lymphocytes (TILs) and immunosuppressive factors across the 4 molecular subtypes of endometrial cancer, which have characteristic mutation rates ranging from low to ultra-high.Experimental Design:A total of 460 endometrial cancers were stratified by ProMisE (Proactive Molecular Risk Classifier in Endometrial cancer) into 4 molecular subtypes: mismatch repair-deficient (MMRd), POLE mutant (POLE), p53 abnormal (p53abn), and p53 wild-type (p53wt). Immune markers (CD3, CD8, CD79a, CD138, PD-1, PD-L1, FoxP3, IDO-1) were quantified by multiplex IHC and tested for associations with ProMisE subtype, survival, and other clinicopathologic parameters.Results:Two major TIL patterns were observed. TILhigh tumors harbored dense T- and B-lineage infiltrates and multiple immunosuppressive features and were common in molecular subtypes associated with high mutation load (MMRd and POLE); however, equally strong responses were seen in significant numbers of p53abn and p53wt tumors, which have characteristically low mutation loads. TILlow tumors were generally devoid of immunologic features and were more prevalent in p53abn and p53wt endometrial cancers, yet were also seen in MMRd and POLE subtypes. In multivariable models involving ProMisE subtype, T-cell markers, and TIL clusters, only ProMisE showed independent prognostic significance.Conclusions:Immune response correlates with endometrial cancer molecular subtype but does not carry independent prognostic significance. Profound variation in immune response is seen across and within endometrial cancer molecular subtypes, suggesting that assessment of immune response rather than molecular subtype may better predict response to immunotherapy.See related commentary by Mullen and Mutch, p. 2366

Revised Supplementary Data, containing Supplementary Methods, Supplementary Figures, Supplementary Tables Supplementary Figure S1. Outline of cohort selection and analyses. Supplementary Figure S2. Clinical response and therapy course of 96 patients with exceptional responses to chemotherapy. Supplementary Figure S3. Distribution and type of TP53 mutations. Supplementary Figure S4. RB1 protein expression altered by genomic inactivation. Supplementary Figure S5. Characterization of CD8 and Ki-67 in tumors according to homologous recombination mutation status. Supplementary Table S2 Immunohistochemical analysis: primary antibodies and staining conditions Supplementary Table S3 Homologous recombination and DNA repair panel Supplementary Table S6 Comparison of molecular alteration prevalence between clinical subgroups Supplementary Table S7 Patient characteristics of tissue microarray cohort

PDF file - 248K, Patients 1 and 2 failed to respond to any of the mutant peptides at any of the time points.

PDF file, 56KB, B cells in HGSC tumors exhibit oligoclonal and somatically hypermutated immunoglobulin sequence.

Supplemental Table 3. CD103 positive TIL and association with clinical-pathological features.

Purpose:In vitro studies suggest that ovarian cancer evades immune rejection by fostering an immunosuppressive environment within the peritoneum; however, the functional responses of ovarian cancer–specific T cells have not been directly investigated in vivo. Therefore, we developed a new murine model to enable tracking of tumor-specific CD8+ T-cell responses to advanced ovarian tumors.Experimental Design: The ovarian tumor cell line ID8 was transfected to stably express an epitope-tagged version of HER-2/neu (designated NeuOT-I/OT-II). After i.p. injection into C57BL/6 mice, ID8 cells expressing NeuOT-I/OT-II gave rise to disseminated serous adenocarcinomas with extensive ascites. CD8+ T cells expressing a transgenic T-cell receptor specific for the OT-I epitope of NeuOT-I/OT-II were adoptively transferred into tumor-bearing mice, and functional responses were monitored. Cytokine signaling requirements were evaluated by comparing the responses of wild-type donor T cells with those with genetic deletion of the interleukin (IL)-2/IL-15 receptor β subunit (CD122) or the IL-2 receptor α subunit (CD25).Results: On adoptive transfer into tumor-bearing hosts, wild-type OT-I T cells underwent a striking proliferative response, reaching peak densities of ∼40% and ∼90% of CD8+ T cells in peripheral blood and ascites, respectively. OT-I cells infiltrated and destroyed tumor tissue, and ascites completely resolved within 10 days. By contrast, CD122−/− OT-I cells and CD25−/− OT-I cells proliferated in blood but failed to accumulate in ascites or tumor tissue or induce tumor regression.Conclusions: Contrary to expectation, advanced ovarian cancers can support extraordinary CD8+ T-cell proliferation and antitumor activity through an IL-2/IL-15–dependent mechanism.

Purpose: CD8+ tumor-infiltrating lymphocytes (TIL) are key mediators of antitumor immunity and are strongly associated with survival in virtually all solid tumors. However, the prognostic effect of CD8+ TIL is markedly higher in the presence of CD20+ B cells, suggesting that cooperative interactions between these lymphocyte subsets lead to more potent antitumor immunity.Experimental Design: We assessed the colocalization patterns, phenotypes, and gene expression profiles of tumor-associated T- and B-lineage cells in high-grade serous ovarian cancer (HGSC) by multicolor IHC, flow cytometry, and bioinformatic analysis of gene expression data from The Cancer Genome Atlas.Results: T cells and B cells colocalized in four types of lymphoid aggregate, ranging from small, diffuse clusters to large, well-organized tertiary lymphoid structures (TLS) resembling activated lymph nodes. TLS were frequently surrounded by dense infiltrates of plasma cells (PC), which comprised up to 90% of tumor stroma. PCs expressed mature, oligoclonal IgG transcripts, indicative of antigen-specific responses. PCs were associated with the highest levels of CD8+, CD4+, and CD20+ TIL, as well as numerous cytotoxicity-related gene products. CD8+ TIL carried prognostic benefit only in the presence of PCs and these other TIL subsets. PCs were independent of mutation load, BRCA1/2 status, and differentiation antigens but positively associated with cancer–testis antigens.Conclusions: PCs are associated with the most robust, prognostically favorable CD8+ TIL responses in HGSC. We propose that TLS facilitate coordinated antitumor responses involving the combined actions of cytolytic T cells and antibody-producing PCs. Clin Cancer Res; 22(12); 3005–15. ©2016 AACR.