Katy Milne

Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer

IntroductionInfiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels.MethodsThe association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1.ResultsIn patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes.ConclusionsER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

Systematic Analysis of Immune Infiltrates in High-Grade Serous Ovarian Cancer Reveals CD20, FoxP3 and TIA-1 as Positive Prognostic Factors

Background Tumor-infiltrating T cells are associated with survival in epithelial ovarian cancer (EOC), but their functional status is poorly understood, especially relative to the different risk categories and histological subtypes of EOC. Methodology/Principal Findings Tissue microarrays containing high-grade serous, endometrioid, mucinous and clear cell tumors were analyzed immunohistochemically for the presence of lymphocytes, dendritic cells, neutrophils, macrophages, MHC class I and II, and various markers of activation and inflammation. In high-grade serous tumors from optimally debulked patients, positive associations were seen between intraepithelial cells expressing CD3, CD4, CD8, CD45RO, CD25, TIA-1, Granzyme B, FoxP3, CD20, and CD68, as well as expression of MHC class I and II by tumor cells. Disease-specific survival was positively associated with the markers CD8, CD3, FoxP3, TIA-1, CD20, MHC class I and class II. In other histological subtypes, immune infiltrates were less prevalent, and the only markers associated with survival were MHC class II (positive association in endometrioid cases) and myeloperoxidase (negative association in clear cell cases). Conclusions/Significance Host immune responses to EOC vary widely according to histological subtype and the extent of residual disease. TIA-1, FoxP3 and CD20 emerge as new positive prognostic factors in high-grade serous EOC from optimally debulked patients.

CD20+ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8+ T cells promote favorable prognosis in ovarian cancer.

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.

Tumor-infiltrating lymphocytes expressing the tissue resident memory marker CD103 are associated with increased survival in high-grade serous ovarian cancer

Background: The presence of CD8+ tumor-infiltrating lymphocytes (TIL) is associated with prolonged survival in high-grade serous ovarian cancer (HGSC) and other epithelial cancers. Survival is most strongly associated with intraepithelial versus intrastromal CD8+ TILs; however, the mechanisms that promote the intraepithelial localization of TILs remain poorly understood. We hypothesized that intraepithelial CD8+ TILs, like normal mucosal intraepithelial lymphocytes, might express CD103, a subunit of αE/β7 integrin, which binds E-cadherin on epithelial cells. Methods: A large collection of primary ovarian tumors (HGSC, endometrioid, mucinous, and clear cell) was analyzed by immunohistochemistry for the presence of TIL-expressing CD103. The activation and differentiation status of CD103+ TILs were assessed by flow cytometry. The prognostic significance of TIL subsets was evaluated by Kaplan–Meier analysis. Results: CD103+ TILs were present in all major ovarian cancer subtypes and were most abundant in HGSC. CD103+ TILs were preferentially localized to epithelial regions of tumors and were comprised predominantly of CD8+ T cells expressing activation (HLA-DR, Ki-67, PD-1) and cytolytic (TIA-1) markers, as well as CD56+ NK cells. Tumor infiltration by CD103+ TILs was strongly associated with patient survival in HGSC. Tumors containing CD8+ TILs that were CD103− showed poor prognosis equivalent to tumors lacking CD8+ TILs altogether. Conclusions: CD103+ TILs comprise intraepithelial, activated CD8+ T cells, and NK cells and are strongly associated with patient survival in HGSC. CD103 may serve as a useful marker for enriching the most beneficial subsets of TILs for immunotherapy. Clin Cancer Res; 20(2); 434–44. ©2013 AACR.

Surveillance of the Tumor Mutanome by T Cells during Progression from Primary to Recurrent Ovarian Cancer

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.

Clonal evolution of high‐grade serous ovarian carcinoma from primary to recurrent disease

High‐grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumours are highly sensitive to cytoreductive surgery and platinum‐ and taxane‐based chemotherapy, the majority of patients experience recurrence of treatment‐resistant tumours. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumour cells harvested from ascites at three time points (primary, first recurrence, and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumours were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumours from all three patients also harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control, and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumour with negligible accumulation of new mutations during standard treatment.

PD-L1 expression is associated with tumor-infiltrating T cells and favorable prognosis in high-grade serous ovarian cancer

OBJECTIVE As a negative regulator of T cells, Programmed Death Ligand 1 (PD-L1) is both an indicator and inhibitor of anti-tumor immune responses, which has led to confusion about its prognostic significance. We investigated the primary source of PD-L1 expression in epithelial ovarian cancer and its relationship to tumor-infiltrating lymphocytes (TIL) and associated gene products. METHODS Tissue microarrays containing high-grade serous carcinomas (HGSC) and endometrioid, clear cell and mucinous ovarian cancers from optimally debulked patients were assessed by immunohistochemistry for expression of PD-L1 and other markers (CD68, CD3, CD8, PD-1, CD103, FoxP3 and CD25). The Cancer Genome Atlas was interrogated for associations between PD-L1 expression and immune-related transcriptional and genomic features of HGSC. RESULTS PD-L1 was primarily expressed by tumor-associated CD68(+) macrophages rather than tumor cells. PD-L1(+) cells frequently co-localized with CD8, CD4 and PD-1(+) TIL, CD25(+)FoxP3(+) Tregs, and other TIL subsets. PD-L1(+) cells were prognostically favorable in HGSC. Moreover, the presence of both PD-L1(+) cells and CD8 TIL was associated with better prognosis than CD8 TIL alone. PD-L1 gene expression was independent of BRCA status. At the transcriptional level, PD-L1 was associated with both cytolytic (granzyme B, T-bet and IFN-γ) and suppressive (PD-1, CTLA-4, LAG3 and IDO-1) gene products. CONCLUSIONS PD-L1 is primarily expressed by macrophages in ovarian cancer and is strongly associated with both cytolytic and regulatory TIL subsets, resulting in a net positive association with survival. Tumors containing PD-L1(+) macrophages appear caught in an immunological stalemate that may require multi-pronged immunotherapy to alleviate.

BRCA1 and BRCA2 mutations correlate with TP53 abnormalities and presence of immune cell infiltrates in ovarian high-grade serous carcinoma

We characterized BRCA1 and BRCA2 status (mutation/methylation) in a consecutive series of cases of ovarian carcinoma in order to identify differences in clinicopathological features, molecular characteristics, and outcome between the pelvic high-grade serous cancers with (i) germline or somatic mutations in BRCA1 or BRCA2, (ii) methylation of BRCA1, and (iii) normal BRCA1 or BRCA2. In all, 131 women were identified prospectively, who were undergoing surgical staging and agreed to germline testing for BRCA1 and BRCA2 mutations. Histopathology, germline and somatic BRCA1 or BRCA2 mutations, BRCA1 methylation, and BRCA1 and BRCA2 mRNA expression levels distinguished four subgroups. In all, 103 cases were high-grade serous carcinoma and of these 31 (30%) had germline or somatic BRCA1 or BRCA2 mutations (20% BRCA1 and 10% BRCA2) (group 1), 21 (20%) had methylation of BRCA1 (group 2), and in 51 (50%) there was no BRCA loss (group 3). Group 4 consisted of 28 cases of non-high-grade serous, none of which had BRCA loss. BRCA1 and BRCA2 mRNA expression levels correlated with designated group (P=0.0008). Among high-grade serous carcinomas, there were no differences between groups 1–3 with respect to stage, ascites, CA125 level, platinum sensitivity, cytoreduction rate, neoadjuvant chemotherapy, or survival. Tumors with BRCA1 or BRCA2 mutations had increased immune infiltrates (CD20 and TIA-1) compared with high-grade serous without mutations (P=0.034, 0.027). TP53 expression differed between groups (P<0.0001), with abnormal TP53 expression in 49/50 tumors from groups 1 and 2. Wild-type TP53 expression was associated with worse outcome in high-grade serous (P<0.001). BRCA loss (mutation/methylation) is a common event in the pelvic high-grade serous (50%). TP53 abnormalities and increased immune cell infiltrates are significantly more common in high-grade serous with germline and somatic mutations in BRCA1 or BRCA2, compared with tumors lacking BRCA abnormalities.

Tumor-Infiltrating Plasma Cells Are Associated with Tertiary Lymphoid Structures, Cytolytic T-Cell Responses, and Superior Prognosis in Ovarian Cancer

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.

Absolute lymphocyte count is associated with survival in ovarian cancer independent of tumor-infiltrating lymphocytes

BackgroundThe immune system strongly influences outcome in patients with ovarian cancer. In particular, the absolute lymphocyte count in peripheral blood (ALC) and the presence of tumor-infiltrating lymphocytes (TIL) have each been associated with favourable prognosis. However, the mechanistic relationships between ALC, TIL and prognosis are poorly understood. We hypothesized that high ALC values might be associated with stronger tumor immunity as manifested by increased TIL, decreased tumor burden and longer survival.MethodsALC values were collected from patient records ≥ 2 years before, during or after primary treatment for high-grade serous ovarian cancer (HGSC). Lymphocyte subsets were assessed in peripheral blood by flow cytometry. CD8+ and CD20+ TIL were assessed by immunohistochemistry.ResultsOverall, patients had normal ALC values two or more years prior to diagnosis of HGSC. These values were not predictive of disease severity or survival upon subsequent development of HGSC. Rather, ALC declined upon development of HGSC in proportion to disease burden. This decline involved all lymphocyte subsets. ALC increased following surgery, remained stable during chemotherapy, but rarely recovered to pre-diagnostic levels. ALC values recorded at diagnosis did not correlate with CD8+ or CD20+ TIL but were associated with progression-free survival.ConclusionsPatients with high intrinsic ALC values show no clinical or survival advantage upon subsequent development of HGSC. ALC values at diagnosis are prognostic due to an association with disease burden rather than TIL. Therapeutic enhancement of ALC may be necessary but not sufficient to improve survival in HGSC.