Brad H. Nelson

Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patients autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

The Prognostic Value of FoxP3+ Tumor-Infiltrating Lymphocytes in Cancer: A Critical Review of the Literature

CD8+ tumor-infiltrating lymphocytes (TIL) are associated with survival in a variety of cancers. A second subpopulation of TIL, defined by forkhead box protein P3 (FoxP3) expression, has been reported to inhibit tumor immunity, resulting in decreased patient survival. On the basis of this premise, several groups are attempting to deplete FoxP3+ T cells to enhance tumor immunity. However, recent studies have challenged this paradigm by showing that FoxP3+ T cells exhibit heterogeneous phenotypes and, in some cohorts, are associated with favorable prognosis. These discrepant results could arise from differences in study methodologies or the biologic properties of specific cancer types. Here, we conduct the first systematic review of the prognostic significance of FoxP3+ T cells across nonlymphoid cancers (58 studies from 16 cancers). We assessed antibody specificity, cell-scoring strategy, multivariate modeling, use of single compared with multiple markers, and tumor site. Two factors proved important. First, when FoxP3 was combined with one additional marker, double-positive T cells were generally associated with poor prognosis. Second, tumor site had a major influence. FoxP3+ T cells were associated with poor prognosis in hepatocellular cancer and generally good prognosis in colorectal cancer, whereas other cancer types were inconsistent or understudied. We conclude that FoxP3+ T cells have heterogeneous properties that can be discerned by the use of additional markers. Furthermore, the net biologic effects of FoxP3+ T cells seem to depend on the tumor site, perhaps reflecting microenvironmental differences. Thus, depletion of FoxP3+ T cells might enhance tumor immunity in some patient groups but be detrimental in others. Clin Cancer Res; 18(11); 3022–9. ©2012 AACR.

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.

Standard Treatments Induce Antigen-Specific Immune Responses in Prostate Cancer

Purpose: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. Experimental Design: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. Results: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. Conclusion: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

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.

Systematic Evaluation of Candidate Blood Markers for Detecting Ovarian Cancer

Background Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer. Methods and Findings We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer. Conclusions By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

A low carbohydrate, high protein diet slows tumor growth and prevents cancer initiation.

Since cancer cells depend on glucose more than normal cells, we compared the effects of low carbohydrate (CHO) diets to a Western diet on the growth rate of tumors in mice. To avoid caloric restriction-induced effects, we designed the low CHO diets isocaloric with the Western diet by increasing protein rather than fat levels because of the reported tumor-promoting effects of high fat and the immune-stimulating effects of high protein. We found that both murine and human carcinomas grew slower in mice on diets containing low amylose CHO and high protein compared with a Western diet characterized by relatively high CHO and low protein. There was no weight difference between the tumor-bearing mice on the low CHO or Western diets. Additionally, the low CHO-fed mice exhibited lower blood glucose, insulin, and lactate levels. Additive antitumor effects with the low CHO diets were observed with the mTOR inhibitor CCI-779 and especially with the COX-2 inhibitor Celebrex, a potent anti-inflammatory drug. Strikingly, in a genetically engineered mouse model of HER-2/neu-induced mammary cancer, tumor penetrance in mice on a Western diet was nearly 50% by the age of 1 year whereas no tumors were detected in mice on the low CHO diet. This difference was associated with weight gains in mice on the Western diet not observed in mice on the low CHO diet. Moreover, whereas only 1 mouse on the Western diet achieved a normal life span, due to cancer-associated deaths, more than 50% of the mice on the low CHO diet reached or exceeded the normal life span. Taken together, our findings offer a compelling preclinical illustration of the ability of a low CHO diet in not only restricting weight gain but also cancer development and progression.