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Purpose: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. Experimental Design: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. Results: Coexpression of CD39, CD103, and PD-1 (“triple-positive” phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. Conclusions: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.

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

Purpose: Some forms of chemotherapy can enhance antitumor immunity through immunogenic cell death, resulting in increased T-cell activation and tumor infiltration. Such effects could potentially sensitize tumors to immunotherapies, including checkpoint blockade. We investigated whether platinum- and taxane-based chemotherapy for ovarian cancer induces immunologic changes consistent with this possibility. Experimental Design: Matched pre- and post-neoadjuvant chemotherapy tumor samples from 26 high-grade serous carcinoma (HGSC) patients were analyzed by immunohistochemistry (IHC) for a large panel of immune cells and associated factors. The prognostic significance of post-chemotherapy TIL patterns was assessed in an expanded cohort (n = 90). Results: Neoadjuvant chemotherapy was associated with increased densities of CD3+, CD8+, CD8+ TIA-1+, PD-1+ and CD20+ TIL. Other immune subsets and factors were unchanged, including CD79a+ CD138+ plasma cells, CD68+ macrophages, and MHC class I on tumor cells. Immunosuppressive cell types were also unchanged, including FoxP3+ PD-1+ cells (putative regulatory T cells), IDO-1+ cells, and PD-L1+ cells (both macrophages and tumor cells). Hierarchical clustering revealed three response patterns: (i) TILhigh tumors showed increases in multiple immune markers after chemotherapy; (ii) TILlow tumors underwent similar increases, achieving patterns indistinguishable from the first group; and (iii) TILnegative cases generally remained negative. Despite the dramatic increases seen in the first two patterns, post-chemotherapy TIL showed limited prognostic significance. Conclusions: Chemotherapy augments pre-existing TIL responses but fails to relieve major immune-suppressive mechanisms or confer significant prognostic benefit. Our findings provide rationale for multipronged approaches to immunotherapy tailored to the baseline features of the tumor microenvironment. Clin Cancer Res; 23(4); 925–34. ©2016 AACR.

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.

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.

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.

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.

Background: Mounting evidence indicates that tumor-specific host immunity is associated with favorable prognosis in a variety of cancer settings. However, the role of immunity in breast cancer remains controversial. The purpose of this study was to determine the significance of tumor infiltrating leukocytes (TIL) with respect to pathological features and patient survival in estrogen receptor alpha (ER) negative breast cancer. Patients and Methods: Tissue microarrays and immunohistochemistry were used to assess and categorize TIL in a cohort of 255 ER-negative invasive ductal breast carcinomas. Cases were registered by the Manitoba Breast Tumor Bank during the years 1988 to 2000 and had a minimum follow-up of 36 months. Stromal and intraepithelial TIL expressing the markers CD3, CD8, CD4, TIA1, CD25, FOXP3, CD20, CD68, and myeloperoxidase (MPO) were quantified by an experienced pathologist (PHW) and evaluated for associations with pathological parameters and survival. The median value for each marker was used as a cut-point to define high versus low cases. Results: TIL expressing CD4, FOXP3, TIA1, CD20, or MPO were significantly associated with improved overall and disease-free survival (DFS). A combination of high TIA1+ and FOXP3+ TIL, termed IR (immune response)-high, was superior to any single TIL marker and was significantly associated with DFS in multivariate analysis involving the following covariates: nodal status, patient age, tumor size, tumor grade, Her2/PR expression, and adjuvant treatment (HR = 0.478, 95% CI 0.246–0.932; P = 0.030). IR associated with DFS principally in patients with axillary lymph node metastasis (P = 0.0019 versus P = 0.2659 for node-negative patients) and in those who did not receive adjuvant radiation or chemotherapy (P < 0.0001 versus P = 0.0489 for adjuvant-treated patients). Similarly, nodal status and adjuvant therapy had prognostic value only for patients with low IR. Indeed, the outcomes of IR-high cases with or without lymph node metastasis were nearly identical (10 year DFS rates of 65% and 69%, respectively; P = 0.6503), as were those of IR-high patients who did or did not receive adjuvant therapy (10 year DFS rates of 68% and 67%, respectively; P = 0.7997). Unlike standard prognostic features such as nodal status (P = 0.7941), IR correlated with DFS at times greater than five years after diagnosis (P = 0.0084). Conclusions: High levels of TIL are indicative of favorable prognosis in ER-negative breast cancer. The prognostic effects of TIL are particularly relevant for node-positive patients and those not treated with adjuvant radiation or chemotherapy. These findings suggest that in ER-negative disease, the survival benefits of adjuvant therapy may be restricted to patients with poor tumor-specific immune responses. Assessment of TIL may improve overall prognostic prediction for ER-negative breast cancer and may be useful in identifying patients who are likely to respond to adjuvant regimens. Furthermore, our data suggest that ER-negative breast cancer may be a strong candidate for the design and deployment of novel immunotherapies. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B13