High Number Of Tumor-infiltrating Lymphocytes Research Articles

Negative prognostic impact of PD-L1 expression in tumor cells of undifferentiated (anaplastic) carcinoma with osteoclast-like giant cells of the pancreas: study of 13 cases comparing ductal pancreatic carcinoma and review of the literature.

Pancreatic carcinoma remains one of the leading cancer-related causes of death worldwide and is generally characterized by a dismal prognosis and limited potential for oncologic treatment. A rare subvariant of pancreatic cancer, undifferentiated carcinoma with osteoclast-like giant cells (UCOGC), has an unpredictable prognosis according to many previous studies, with unexpectedly long survival in individual cases. In this study, we collected, retrospectively, 13 cases of well-documented UCOGCs and performed immunohistochemistry focused on the expression of the programmed death-ligand 1 (PD-L1) and several other potential therapeutic and predictive markers (PanTRK, p53, MSH2, PMS2, and the number of tumor-infiltrating lymphocytes), to explore their correlation with the follow-up of the patients. As a control group, we examined 24 cases of conventional pancreatic ductal adenocarcinoma (PDAC). In our results, PanTRK was negative in all 24 cases. P53 did not show any significant differences between UCOGCs and PDACs, and the entire cohort was MSH2, MLH1, PMS2, and MSH6 positive. Significant differences were present in the analysis of PD-L1: UCOGCs were found to express PD-L1 significantly more frequently and have a higher number of tumor-infiltrating lymphocytes than PDAC. The expression of PD-L1 was related to significantly shorter survival in patients with UCOGC and in the entire cohort. Patients with PD-L1 negative UCOGCs displayed surprisingly long survival in comparison to PD-L1 positive UCOGCs and PDACs (both PD-L1+ and PD-L1-). We compared our results with previously published data, and, after statistical analysis, we were able to identify PD-L1 as an effective prognostic marker of UCOGC and suggest a strong need for a clinical trial of immune checkpoint immunotherapy in patients with advanced PD-L1 positive UCOGC.

Expression of Beclin-1, an autophagy-related protein, is associated with tumoral FOXP3 expression and Tregs in gastric adenocarcinoma: The function of Beclin-1 expression as a favorable prognostic factor in gastric adenocarcinoma

BackgroundAutophagy, which is stimulated by cellular or environmental stresses, is involved in several distinct biological processes, and the regulation mechanism is complex. Autophagy has been reported to modulate immune system components. In the present study, the expression of Beclin-1 and LC3, an autophagy-related proteins, and its relationship with FOXP3 expression were investigated in gastric adenocarcinoma cells and FOXP3+T cells (regulatory T cells). MethodsTissue samples were acquired from 182 cases of gastric adenocarcinoma that were surgically resected at Kyung Hee University Hospital at Gangdong from 2006 to 2012. Immunohistochemical staining for Beclin-1, LC3, FOXP3, and CD8 was performed. ResultsConsequently, positive Beclin-1 expression was significantly associated with a smaller tumor size, mixed histologic type, better histologic grade, lower T category, lower N category, lower recurrence rate, less lymphatic invasion, less vascular invasion, and less neural invasion. Positive tumoral FOXP3 expression was significantly associated with a lower T category, lower N category, lower recurrence rate, and less lymphatic invasion. The cases of more infiltrated Tregs (≥ 25/high power field, HPF) significantly correlated with a lower N category, lower recurrence rate, less lymphatic invasion, more infiltrated CD8+T cells, and a higher number of tumor-infiltrating lymphocytes. Beclin-1 and LC3 expression were positively correlated with tumoral FOXP3 overexpression. In addition, Beclin-1 expression was significantly associated with a greater number of infiltrated Tregs in gastric adenocarcinoma. Both the positive Beclin-1 expression and positive tumoral FOXP3 expression cases showed significantly better disease-free and overall survival. In addition, the patients with an increased number of infiltrated Tregs (≥ 25/HPF) showed better disease-free and overall survival rates. ConclusionIn conclusion, the autophagic function of Beclin-1 in gastric adenocarcinoma cells was associated with an increased number of infiltrated Tregs and tumoral FOXP3 containing tumor suppressor function. Therefore, the favorable effects of Beclin-1 expression in gastric adenocarcinoma is associated with the regulation of Tregs and tumoral FOXP3 expression.

Abstract P4-10-08: Absolute lymphocyte count (ALC) is a predictor of eribulin benefit in advanced or metastatic breast cancer (MBC)

Abstract Objective: This post hoc analysis assessed predictors for OS in patients (pts) treated with eribulin using data from 2 phase 3 studies: EMBRACE and Study 301. Background: Eribulin is a nontaxane synthetic inhibitor of microtubule dynamics approved for use in locally advanced or metastatic breast cancer after two previous lines of chemotherapy. In EMBRACE, 508 pts were randomized to eribulin monotherapy and 254 pts to the treatment of physician’s choice (TPC). Median OS of pts in the eribulin arm was significantly better than the TPC arm but no significant difference was seen in progression-free survival (PFS) by independent review. Study 301 randomized 554 pts to eribulin and 548 pts to capecitabine; no significant difference was seen in PFS. Kashiwagi et al, PloS one, 2017, suggested that cells such as tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment may be a predictive marker of eribulin efficacy—pts with a high number of TILs had significantly longer PFS than pts with low TIL counts. Miyagawa et al, Clin Breast Cancer, 2018, found neutrophil-to-lymphocyte ratio to be significantly associated with longer PFS with eribulin. Our analysis focused on ALC, a peripheral immune parameter. Methods: Pts from EMBRACE with baseline ALCs were included in this analysis. The Kaplan-Meier method was used to estimate OS distribution. Interaction analysis of OS was performed between treatment and baseline ALCs to explore differential cutoff values. Hazard ratios (HR) were estimated from the stratified Cox proportional hazard model with the randomization stratification factors: HER2/neu status, prior capecitabine treatment, and geographical region. Multivariate analyses for baseline factors were performed using the Cox proportional hazard model to identify predictors and to confirm baseline ALC effect for OS. Similar methods were used to analyze associations between baseline ALC and OS in Study 301. Results: EMBRACE included 500 pts in the eribulin arm and 251 in the TPC arm with an evaluable baseline ALC. Pts with high baseline ALC consistently experienced greater eribulin benefit vs pts with low baseline ALC across cutoff values from 1300-1750/μL. The greatest difference was observed at the cutoff value of 1500/μL. Median OS was higher in pts with baseline ALC ≥1500/µL vs baseline ALC <1500/µL in the eribulin arm (15.6 vs 11.6 months) but not in the TPC arm (Table). The eribulin group showed prolonged OS compared with the TPC group in pts with ALC ≥1500/µL (HR 0.586; 95% CI: 0.437-0.784; P<0.001). There was no significant difference by treatment for ALC <1500/µL (HR 1.002; 95% CI: 0.800-1.253; P=0.989). Study 301 included 553 pts on eribulin and 547 pts on capecitabine. Similar results were observed in the eribulin arm, median OS was higher in pts with baseline ALC ≥1500/µL vs pts with baseline ALC <1500/µL (19.6 vs 14.6 months). There was a treatment effect favoring eribulin vs capecitabine at ALC ≥1500/µL but not at ALC <1500/µL. Conclusion: ALC at baseline may potentially be an independent predictor for longer OS in pts treated with eribulin. This hypothesis-generating study speculates that baseline ALC may be useful in the selection of pts to be treated with eribulin. Table. Multivariate analysis of OS identified the following predictors for OS in the eribulin group: prior capecitabine, ECOG PS, HR status, number of organs involved, refractory to taxanes, and baseline ALCStudyBaseline ALCArmPatients, nMedian OS (months)HR (95% CI)EMBRACEALC ≥1500/µLEribulin19915.60.586 (0.437-0.784)TPC9211.4ALC <1500/µLEribulin30111.61.002 (0.800-1.253)TPC15910.3Study 301ALC ≥1500/µLEribulin23319.60.811 (0.662-0.993)Capecitabine26916.0ALC <1500/µLEribulin32014.60.907 (0.758-1.084)Capecitabine27812.5ALC, absolute lymphocyte count; CI, confidence interval; HR, hazard ratio; OS, overall survival; TPC, treatment of physician’s choice. Citation Format: Javier Cortes, Jose Manuel Perez-Garcia, Kenichi Nomoto, Karenza Alexis, Kenichi Saito, Yuta Yoshimura, Kenzo Muramoto, Yasuo Miyoshi. Absolute lymphocyte count (ALC) is a predictor of eribulin benefit in advanced or metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-08.

Prediction of Benefit from Checkpoint Inhibitors in Mismatch Repair Deficient Metastatic Colorectal Cancer: Role of Tumor Infiltrating Lymphocytes.

Immunotherapy with immune checkpoint inhibitors (ICIs) is highly effective in microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC); however, specific predictive biomarkers are lacking. Data and samples from 85 patients with MSI-H mCRC treated with ICIs were gathered. Tumor infiltrating lymphocytes (TILs) and tumor mutational burden (TMB) were analyzed in an exploratory cohort of "super" responders and "clearly" refractory patients; TILs were then evaluated in the whole cohort of patients. Primary objectives were the correlation between the number of TILs and TMB and their role as biomarkers of ICI efficacy. Main endpoints included response rate (RR), progression-free survival (PFS), and overall survival (OS). In the exploratory cohort, an increasing number of TILs correlated to higher TMB (Pearson's test, p = .0429). In the whole cohort, median number of TILs was 3.6 in responders compared with 1.8 in nonresponders (Mann-Whitney test, p = .0448). RR was 70.6% in patients with high number of TILs (TILs-H) compared with 42.9% in patients with low number of TILs (odds ratio = 3.20, p = .0291). Survival outcomes differed significantly in favor of TILs-H (PFS: hazard ratio [HR] = 0.42, p = .0278; OS: HR = 0.41, p = .0463). A significant correlation between higher TMB and increased number of TILs was shown. A significantly higher activity and better PFS and OS with ICI in MSI-H mCRC were reported in cases with high number of TILs, thus supporting further studies of TIL count as predictive biomarker of ICI efficacy. Microsatellite instability is the result of mismatch repair protein deficiency, caused by germline mutations or somatic modifications in mismatch repair genes. In metastatic colorectal cancer (mCRC), immunotherapy (with immune checkpoint inhibitors [ICIs]) demonstrated remarkable clinical benefit in microsatellite instability-high (MSI-H) patients. ICI primary resistance has been observed in approximately 25% of patients with MSI-H mCRC, underlining the need for predictive biomarkers. In this study, tumor mutational burden (TMB) and tumor infiltrating lymphocyte (TIL) analyses were performed in an exploratory cohort of patients with MSI-H mCRC treated with ICIs, demonstrating a significant correlation between higher TMB and increased number of TILs. Results also demonstrated a significant correlation between high number of TILs and clinical responses and survival benefit in a large data set of patients with MSI-H mCRC treated with ICI. TMB and TILs could represent predictive biomarkers of ICI efficacy in MSI-H mCRC and should be incorporated in future trials testing checkpoint inhibitors in colorectal cancer.

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Inside the USCAP Journals

Expression of lymphocyte immunoregulatory biomarkers in bone and soft-tissue sarcomas

Despite advances in our understanding of the underlying molecular drivers of sarcomas, few treatments are available with proven benefit for advanced metastatic sarcomas. Immunotherapy has value in this setting for some types of cancers, but sarcomas, with their multiplicity of rare types, have not been characterized in detail for their expression of targetable immune biomarkers. This study provides the most systematic evaluation to date of tumor-infiltrating lymphocytes and immune checkpoint biomarker expression in sarcomas. We examined by morphology and immunohistochemistry 1072 sarcoma specimens representing 22 types, in addition to 236 benign bone and soft-tissue tumors. Genomically-complex sarcoma types—those driven by mutations and/or copy-number alterations—had much higher numbers of tumor-infiltrating lymphocytes than translocation-associated sarcomas. Prior exposure to radiotherapy was associated with increased immune infiltrates. Higher lymphocytic infiltration was associated with better overall survival among the non-translocation-associated sarcomas. Expression of PD-1 and CD56 were associated with worse overall survival. LAG-3 and TIM-3, two emerging immune checkpoints, were frequently expressed in most sarcoma types. Indeed, most cases positive for PD-(L)1 coexpressed one or both of these novel biomarkers, providing a potential rationale in support for trials targeting LAG-3 and/or TIM-3 in conjunction with PD-1 inhibition.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.

1179P - Tumor-infiltrating lymphocytes expression in stage IIIc/IV of high-grade serous ovarian cancer: Variation with neoadjuvant chemotherapy and prognostic value

Background: Ovarian cancer is a malignancy with a complex immune suppressive microenvironment mediated by the recruitment or induction of CD4+ regulatory T cell. The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIc/IV of high-grade serous ovarian carcinoma (HGSOC), and its relationship to treatment response. Methods: We retrospectively identified 33 patients diagnosed with HGSOC and treated with neoadjuvant platinum-paclitaxel from 2005-2014. Pre and post-neoadjuvant treatment tissue samples were submitted to immunohistochemical analyses with anti-CD3, CD4 and CD8 antibodies for the identification of tumor-infiltrating lymphocytes (TILs). Pathological response classification to NACT was made according to Steffen Bohm (JCO 2015). Response score system (CRS) was explicitly defined (CRS-1; No or minimal tumor response, CRS-2; Appreciable tumor response amid viable tumor that is readily identifiable, CRS-3; Complete or near-complete response). Results: The average age of patients was 63.44 years (46.53-84.14). BRCA-mutation status was negative in 78.8% of patients (26/33); BRCA-mutation was positive in 6.1% (2/33); and variant of uncertain significance was found in 15.1% (5/33). The majority of patients (78.8%) were stage IIIc. The area under the ROC curve of post-surgery TILs for complete pathological response were: CD4 (epithelial): [0.73 (0.5; 0.97), p: 0.084]; CD4 (stromal): [0.74 (0.51; 0.97), p: 0.077] and CD8 (epithelial): [0.81 (0.63; 1.0), p: 0.02]. The expression of epithelial CD4 TILs in pre-surgery samples (≤ 0.5 [OR: 0.7(0.01; 0.86), p: 0.038]) and epithelial CD8 TILs in post-surgery samples (≤ 5.4 [OR: 0.1(0.01; 1.19, p: 0.06]) proved to be a marker of good prognosis for pathological response. Survival analysis demonstrated that the expression of epithelial CD3 ≤ 4.3 in pre-surgery samples is a marker of poor prognosis. Conclusions: The high number of tumor-infiltrating lymphocytes in post-surgery samples was significantly associated with higher rates of complete pathological response and better prognosis. It is convenient to carry out further and multicentric studies to validate these results. Legal entity responsible for the study: Hospital 12 de Octubre. Funding: None Disclosure: All authors have declared no conflicts of interest.

Abstract 1654: Development of subtype specific mouse models of bladder cancer

Abstract Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition. Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs). Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p < 0.0001). Two BBN and two UPPL models were assessed for response to anti-PD-1 therapy in vivo as syngeneic tumors grown in wild type C57BL/6 mice. One of the BBN lines (BBN963) demonstrated robust control of tumor growth in some animals, including multiple complete responses (p = 0.0003), but also tumors that progressed, leading us to characterize BBN963 as a mixed response model. The marked response to PD-1 blockade in BBN963 was associated with significantly higher sharing of TCR CDR3 sequences among TILs compared to sequences of the other tumors (p = 0.003). In addition, analysis of BBN963 tumors by flow cytometry demonstrated naïve and memory T cell phenotypes correlated with increased and decreased tumor sizes, respectively. Closer examination of individual BBN963 tumor responses to PD-1 blockade revealed distinct responder and non-responder infiltrating immune cell phenotypes. Responders demonstrated a less diverse B cell repertoire (p = 0.0043) with increased BCR CDR3 sequence sharing (p < 0.0001). Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work] Citation Format: Christof C. Smith, Ryoichi Saito, Lisa M. Bixby, Takanobu Utsumi, Jordan Kardos, Shengjie Chai, Sara E. Wobker, Bhavani Krishnan, Jeffrey S. Damrauer, Jonathan S. Serody, David Darr, Benjamin G. Vincent, William Y. Kim. Development of subtype specific mouse models of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2017-1654

Fatty acid-binding protein 1 is preferentially lost in microsatellite instable colorectal carcinomas and is immune modulated via the interferon γ pathway

Fatty acid-binding protein 1 (FABP1) is an intracellular protein responsible for the transportation of long chain fatty acids. Aside from its functions in lipid metabolism and cellular differentiation, FABP1 also plays a role in inflammation through its interaction with peroxisome proliferator-activated receptors (PPARs). Previously, we compared expression of colonic epithelium genes in a subset of microsatellite instable (MSI) colorectal carcinomas (medullary carcinomas) to normal colonic mucosa and found that FABP1 expression was markedly decreased in the tumors. Further analysis of RNA expression in the colorectal subtypes and The Cancer Genome Atlas data set found that FABP1 expression is decreased in the CMS1 subset of colorectal carcinomas, which is characterized by microsatellite instability. As MSI colorectal carcinomas are known for their robust immune response, we then aimed to link FABP1 to the immune microenvironment of MSI carcinomas. To confirm the gene expression results, we performed immunohistochemical analysis of a cohort of colorectal carcinomas. FABP1 was preferentially lost in MSI carcinomas (123/133, 93%) compared with microsatellite stable carcinomas (240/562, 43%, P<0.0001). In addition, higher numbers of tumor-infiltrating lymphocytes were present in tumors with loss of FABP1 (P<0.0001). Decreased expression of the fatty acid storage and glucose regulator, PPARγ, was associated with the loss of FABP1 (P<0.0001). Colorectal cancer cell lines treated with interferon γ exhibited decreased expression of FABP1. FABP1 expression was partially recovered with the treatment of the cell lines with rosiglitazone, a PPARγ agonist. This study demonstrated that the loss of FABP1 expression is associated with MSI carcinomas and that interferon γ stimulation plays a role in this process via its interaction with PPARγ.

Role of TP53 mutations in triple negative and HER2-positive breast cancer treated with neoadjuvant anthracycline/taxane-based chemotherapy.

BackgroundTP53 mutations are frequent in breast cancer, however their clinical relevance in terms of response to chemotherapy is controversial.Methods450 pre-therapeutic, formalin-fixed, paraffin-embedded core biopsies from the phase II neoadjuvant GeparSixto trial that included HER2-positive and triple negative breast cancer (TNBC) were subjected to Sanger sequencing of exons 5-8 of the TP53 gene. TP53 status was correlated to response to neoadjuvant anthracycline/taxane-based chemotherapy with or without carboplatin and trastuzumab/lapatinib in HER2-positive and bevacizumab in TNBC. p53 protein expression was evaluated by immunohistochemistry in the TNBC subgroup.ResultsOf 450 breast cancer samples 297 (66.0%) were TP53 mutant. Mutations were significantly more frequent in TNBC (74.8%) compared to HER2-positive cancers (55.4%, P < 0.0001). Neither mutations nor different mutation types and effects were associated with pCR neither in the whole study group nor in molecular subtypes (P > 0.05 each). Missense mutations tended to be associated with a better survival compared to all other types of mutations in TNBC (P = 0.093) and in HER2-positive cancers (P = 0.071). In TNBC, missense mutations were also linked to higher numbers of tumor-infiltrating lymphocytes (TILs, P = 0.028). p53 protein overexpression was also linked with imporved survival (P = 0.019).ConclusionsOur study confirms high TP53 mutation rates in TNBC and HER2-positive breast cancer. Mutations did not predict the response to an intense neoadjuvant chemotherapy in these two molecular breast cancer subtypes.

Analysis of PD-L1, T-cell infiltrate and HLA expression in chondrosarcoma indicates potential for response to immunotherapy specifically in the dedifferentiated subtype

AbstractTherapies targeting the programmed cell death 1 (PD-1) or its ligand (PD-L1) promote antitumor T-cell activity, leading to unprecedented long-lasting tumor responses in some advanced cancers. Because of radiotherapy and chemotherapy resistance, no effective treatments have been defined for advanced chondrosarcomas. We here report an immunohistochemical analysis of PD-L1 expression in a large series of conventional, mesenchymal, clear cell and dedifferentiated chondrosarcomas using tissue microarrays. In the PD-L1-positive tumors, we analyzed the immune microenvironment (T-cell and macrophage infiltration as well as HLA class I expression) using whole sections. PD-L1 expression was absent in conventional (n=119), mesenchymal (n=19) and clear cell (n=20) chondrosarcomas. Forty-one percent (9 of the 22) of dedifferentiated chondrosarcomas displayed PD-L1 positivity. These results were confirmed in an independent cohort using whole tissue sections of dedifferentiated chondrosarcomas in which PD-L1 expression was detected in 52% (11 of the 21) of cases. PD-L1 expression was exclusively found in the dedifferentiated component and expression positively correlated with other immune parameters such as high number of tumor-infiltrating lymphocytes (P=0.014) and positive HLA class I expression (P=0.024) but not with patient overall survival (P=0.22). The presence of PD-L1 expression in association with immune-infiltrating cells and HLA class I expression in nearly 50% of the dedifferentiated chondrosarcomas provides rationale for including these patients in clinical trials with PD-1/PD-L1-targeted therapies.

Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors.

Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has a better prognosis than it's HPV negative (HPV(−)) counterpart. This may be due to the higher numbers of tumor-infiltrating lymphocytes (TILs) in HPV positive (HPV(+)) tumors. RNA-Sequencing (RNA-Seq) was used to evaluate whether the differences in clinical behaviour simply reflect a numerical difference in TILs or whether there is a fundamental behavioural difference between TILs in these two settings. Thirty-nine HNSCC tumors were scored for TIL density by immunohistochemistry. After the removal of 16 TILlow tumors, RNA-Seq analysis was performed on 23 TILhigh/med tumors (HPV(+) n=10 and HPV(−) n=13). Using EdgeR, differentially expressed genes (DEG) were identified. Immune subset analysis was performed using Functional Analysis of Individual RNA-Seq/ Microarray Expression (FAIME) and immune gene RNA transcript count analysis. In total, 1,634 DEGs were identified, with a dominant immune signature observed in HPV(+) tumors. After normalizing the expression profiles to account for differences in B- and T-cell number, 437 significantly DEGs remained. A B-cell associated signature distinguished HPV(+) from HPV(−) tumors, and included the DEGs CD200, GGA2, ADAM28, STAG3, SPIB, VCAM1, BCL2 and ICOSLG; the immune signal relative to T-cells was qualitatively similar between TILs of both tumor cohorts. Our findings were validated and confirmed in two independent cohorts using TCGA data and tumor-infiltrating B-cells from additional HPV(+) HNSCC patients. A B-cell associated signal segregated tumors relative to HPV status. Our data suggests that the role of B-cells in the adaptive immune response to HPV(+) HNSCC requires re-assessment.

CD8+ tumor‐infiltrating lymphocytes contribute to spontaneous “healing” in HER2‐positive ductal carcinoma in situ

We evaluated the associations between tumor‐infiltrating lymphocytes (TIL) including CD8‐positive [+] lymphocytes in ductal carcinoma in situ (DCIS) and histopathologic factors, particularly spontaneous “healing” and immunohistochemical (IHC)‐based subtypes, to clarify the effects of host immune response to cancer cells proliferation during early carcinogenesis for the breast cancer. This cohort enrolled 82 DCIS patients. We examined the relationships between clinicopathologic factors including age, DCIS architecture, Van Nuys classification, grade, comedo necrosis, apocrine features, TIL, CD8+ lymphocytes, healing, estrogen receptor and HER2 positivity, and IHC‐based subtypes [luminal, luminal‐HER2, HER2‐positive, triple negative (TN)]. The results were analyzed by univariate and multivariate analyses. High numbers of TIL (high‐TIL) and healing were seen in 30.5% and 39.0% of the cohort, respectively. The distributions of luminal, luminal‐HER2, HER2 and TN subtypes were 73.2%, 9.8%, 13.4%, and 3.6%, respectively. High Van Nuys grading, high‐grade, comedo necrosis, apocrine features, high‐TIL, high CD8+ lymphocytes and healing were significantly associated with HER2‐positive (luminal‐HER2, HER2), and TN subtypes. High‐TIL was significantly associated with high‐grade, comedo necrosis, apocrine features, healing, high CD8+ lymphocytes and HER2 and TN subtypes. Healing was significantly correlated with high CD8+ lymphocytes, high‐grade, comedo necrosis, apocrine features, and HER2‐positive and TN subtypes. Logistic regression analysis revealed a strong association between healing and TIL (odds ratio: 11.72, P = 0.024). High CD8+ lymphocytes was also significantly associated with healing (odds ratio: 9.26, P = 0.009). The results of this study suggested that the spontaneous healing phenomenon might be induced by CD8+ high‐TIL associated with high‐grade, comedo necrosis, apocrine features and HER2‐positive DCIS.

Abstract S1-02: Lymphocytic infiltration in invasive lobular breast cancer

Abstract Background: The presence and prognostic value of tumor infiltrating lymphocytes (TILs) in invasive breast carcinoma has been demonstrated in several studies, especially in the triple-negative and HER2-positive subtypes. So far, TILs have not been investigated with sufficient detail in invasive lobular breast cancer (ILBC). Here we therefore aimed at: first, assessing the distribution of stromal TILs in ILBC; second, correlating the presence of TILs with standard clinical and pathological markers; third, exploring associations of TILs with recurrent genomic alterations; and, fourth, comparing the lymphocytic composition of ER-positive/HER2-negative lobular to ER-positive/HER2-negative ductal tumors. Material and methods: The percentage of stromal TILs was independently assessed according to Salgado et al. (Ann Oncol 2015) by three pathologists on full-face hematoxylin and eosin slides in a well-annotated retrospective series of 614 primary ILBCs previously characterized at the genomic level. The median value of TILs was used for the analyses. For the association analyses, we focused on the more homogeneous group of ER-positive/HER2-negative ILBC (555/614). Breast cancer-free interval was used as survival endpoint and the analyses were censored at 12 years of follow-up. The comparison of the lymphocytic composition (relative percentage of CD45+ TILs which are CD4+, CD8+ or CD19+) was assessed by FACS in a separate prospective cohort of 51 ER-positive/HER2-negative lobular and 112 ER-positive/HER2-negative ductal tumors. Results: The intraclass correlation coefficient between the three pathologists was 0.71 (95%CI:0.65-0.76). The median percentage of stromal TILs was 5% and the interquartile range 5-10%, with only 9% of the samples having ≥ 20%. Greater numbers of TILs were significantly associated with younger age at diagnosis, axillary lymph node involvement, high proliferative tumors as assessed by Ki67, and with the mixed non-classic ILBC subtypes. Greater numbers of TILs were associated with worse prognosis (HR=1.22; 95%CI:1.07-1.38, p=0.003) only in the unadjusted analysis, as it lost significance after adjustment for standard clinical and pathological variables. Greater numbers of TILs were observed in tumors harboring ARID1A, BRCA2, KMT2C and TP53 mutations, as well as chr3p21.31 and chr8q24.23 (PTK2) loss; whereas lower numbers were observed in tumors with ERBB3 mutations as well as chr7p and chr11q14.1 (PAK1) gains. There were no significant differences in the relative proportion of CD4+, CD8+ or CD19+ lymphocytes between ER-positive/HER2-negative lobular and ductal tumors. Conclusion: In this work, which reports to our knowledge on the largest series of ILBC ever assessed for TILs, we showed that most ILBCs were characterized by low lymphocytic infiltration. Besides the association of TILs with clinical and pathological features of ILBC patients, we found that higher TIL levels were observed in the presence of specific mutations and copy number alterations. Higher numbers of TILs were associated with worse prognosis at the univariate analysis. Finally, based on the assessed markers, we have no evidence of differential lymphocytic composition between ER-positive/HER2-negative lobular and ductal tumors. Citation Format: Desmedt C, Salgado R, Buisseret L, Zoppoli G, Fornili M, Van den Eynden G, Garaud S, Gundem G, Rothé F, Brown D, Kheddoumi N, Rouas G, Galant C, Bertucci F, Piccart M, Campbell P, Viale G, Larsimont D, Willard-Gallo K, Biganzoli E, Pruneri G, Sotiriou C. Lymphocytic infiltration in invasive lobular breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S1-02.

Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.

Tertiary Lymphoid Structure-Associated B Cells are Key Players in Anti-Tumor Immunity.

It is now admitted that the immune system plays a major role in tumor control. Besides the existence of tumor-specific T cells and B cells, many studies have demonstrated that high numbers of tumor-infiltrating lymphocytes are associated with good clinical outcome. In addition, not only the density but also the organization of tumor-infiltrating immune cells has been shown to determine patient survival. Indeed, more and more studies describe the development within the tumor microenvironment of tertiary lymphoid structures (TLS), whose presence has a positive impact on tumor prognosis. TLS are transient ectopic lymphoid aggregates displaying the same organization and functionality as canonical secondary lymphoid organs, with T-cell-rich and B-cell-rich areas that are sites for the differentiation of effector and memory T cells and B cells. However, factors favoring the emergence of such structures within tumors still need to be fully characterized. In this review, we survey the state of the art of what is known about the general organization, induction, and functionality of TLS during chronic inflammation, and more especially in cancer, with a particular focus on the B-cell compartment. We detail the role played by TLS B cells in anti-tumor immunity, both as antigen-presenting cells and tumor antigen-specific antibody-secreting cells, and raise the question of the capacity of chemotherapeutic and immunotherapeutic agents to induce the development of TLS within tumors. Finally, we explore how to take advantage of our knowledge on TLS B cells to develop new therapeutic tools.

Tumor infiltrating lymphocytes (TIL) to predict response to neoadjuvant chemotherapy in breast cancer: A systemic review and meta-analysis.

138 Background: Whether tumor-infiltrating lymphocytes (TILs) predict response to neoadjuvant chemotherapy (NAC) remains elusive. Methods: A systematic review and meta-analysis was undertook to establish the relationship between TIL and pathological complete response (pCR) rate in NAC of breast cancer. A PubMed and Web of Science literature search was designed. Studies were included, in which the predictive significance of intratumoral and/or stromal TIL, and/or CD3+, CD4+, CD8+, and FOXP3+ lymphocytes were determined . Pooled ORs and publication bias was evaluated by STATA software. Results: A total of 13 published studies (including 3,555 patients) were eligible. In pooled analysis, higher number of TIL in pre-treatment biopsy was correlated with higher pCR rate of neoadjuvant chemotherapy, and odds ratio (OR) was 3.82 (95% confidence interval (CI), 3.10-4.70), no matter tested in intratumor (OR=3.32, 95% CI: 2.52-4.37), in stroma (OR=4.15,95% CI: 2.94-5.86), or in combined sites (OR=8.98, 95% CI: 3.79,21.30). Moreover, TIL predicts higher pCR rate in triple negative (OR=5.03,95% CI: 2.31-10.97) and HER2+ (OR=5.54,95% CI: 1.39-22.12) patients, but not in hormonal receptor (HR) +/HER2- patients (OR=2.57, 95% CI: 0.20-33.24). For TIL subsets, CD8+ T-lymphocytes predict better pathological response to NAC no matter in pre- (OR=3.36,95%CI: 1.15-9.85) or post-NAC (OR=4.71,95%CI: 1.29-17.27) tissue, while FOXP3+ T-lymphocytes have similar predictive roles only when tested after NAC (OR=4.26, 95%CI: 1.83-9.92).With limited study, the predictive role of CD3+ and CD4+T-lymphoctes were unclear, more perspective studies were needed in future to establish the relationship. Conclusions: High level of TIL in pre-treatment biopsy could be a good marker indicates better pathological response to NAC in triple-negative and HER2+ breast cancer patients. Different subsets have different predictive roles in the pCR rate to NAC. However, significant heterogeneity and an insufficient number of studies underscore the need for further prospective studies on subsets of T lymphocytes and different subtypes of breast cancer to increase the robustness of the analyses.

Clinicopathological analysis of colorectal cancer: a comparison between emergency and elective surgical cases

BackgroundApproximately 15 to 30% of colorectal cancers present as an emergency, most often as obstruction or perforation. Studies report poorer outcome for patients who undergo emergency compared with elective surgery, both for their initial hospital stay and their long-term survival. Advanced tumor pathology and tumors with unfavorable histologic features may provide the basis for the difference in outcome. The aim of this study was to compare the clinical and pathologic profiles of emergency and elective surgical cases for colorectal cancer, and relate these to gender, age group, tumor location, and family history of the disease. The main outcome measure was the difference in morphology between elective and emergency surgical cases.MethodsIn total, 976 tumors from patients treated surgically for colorectal cancer between 2004 and 2006 in Stockholm County, Sweden (8 hospitals) were analyzed in the study. Seventeen morphological features were examined and compared with type of operation (elective or emergency), gender, age, tumor location, and family history of colorectal cancer by re-evaluating the histopathologic features of the tumors.ResultsIn a univariate analysis, the following characteristics were found more frequently in emergency compared with elective cases: multiple tumors, higher American Joint Committee on Cancer (AJCC), tumor (T) and node (N) stage, peri-tumor lymphocytic reaction, high number of tumor-infiltrating lymphocytes, signet-ring cell mucinous carcinoma, desmoplastic stromal reaction, vascular and perineural invasion, and infiltrative tumor margin (P<0.0001 for AJCC stage III to IV, N stage 1 to 2/3, and vascular invasion). In a multivariate analysis, all these differences, with the exception of peri-tumor lymphocytic reaction, remained significant (P<0.0001 for multiple tumors, perineural invasion, infiltrative tumor margin, AJCC stage III, and N stage 1 to 2/3).ConclusionsColorectal cancers that need surgery as an emergency case generally show a more aggressive histopathologic profile and a more advanced stage than do elective cases. Essentially, no difference was seen in location, and therefore it is likely there would be no differences in macro-environment either. Our results could indicate that colorectal cancers needing emergency surgery belong to an inherently specific group with a different etiologic or genetic background.

Microsatellite Instability in Colorectal Cancer: Time to Stop Hiding!

Colorectal cancer (CRC) is the second cause of cancer-related death worldwide. Surgery constitutes the primary therapy for these tumors, together with chemotherapy that is usually recommended in patients with metastatic primary CRC. Although molecularly distinct entities arising from different physiopathogenic mechanisms - microsatellite (MSI) and chromosomal instability (also called microsatellite stable, MSS) - have been characterized in CRC, there is still no specific therapeutic approach that takes into account disease’s molecular heterogeneity [1]. MSI is observed in 1015% of sporadic CRCs. MSI CRCs displayed particular morphologic features, with greater predilection for the right colon, mucinous histology, low metastatic power, poorer differentiation and higher numbers of tumorinfiltrating lymphocytes. They have been consistently reported to show an improved prognosis and a different response to chemotherapeutic agents. In a recent article in Nature Medicine, we have reported the specific mutation of the molecular chaperone HSP110 in MSI CRCs and how the presence of this mutant may constitute a first step towards the understanding of their particular clinical characteristics [2].