High Density Of CD8 Research Articles

Abstract 2821: Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer

Abstract Tumor infiltration by immune cells critically impacts on clinical outcome of human colorectal cancer (CRC). While high densities of CD8+ T cells and T-helper type 1 cells are associated with prolonged patient survival, the role of tumor-associated neutrophils (TANs) is debated. CRC arise in an environment heavily populated by microrganisms. Upon CRC oncogenesis, gut commensal bacteria translocate into the submucosa where they directly interact with residing immune cells. Neutrophils represent a front-line arm of the immune system in the response to bacteria. However, little is known about their interaction with commensal bacteria within CRC tissues. We investigated the interplay between neutrophils and commensal bacterial species present within the CRC microenvironment. Using an orthotopic CRC xenograft model, we found that commensal bacteria stimulate CRC cells to produce neutrophil recruiting chemokines. We then compared in vitro chemokine induction capacity of Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), two most abundant bacterial species of CRC microenvironment. Fninduced significantly higher levels of IL-8 by CRC cells than Bf, thus more effectively promoting neutrophil recruitment. Accordingly, in human CRC samples, abundance of Fn, but not Bf, significantly correlated with high infiltration by CD66b+ cells. Functional studies indicate that neutrophils cultured in the presence of Fn, but not Bf, lose their ability to enhance proliferation and cytokine release by CD8+ T cells undergoing antigenic stimulation. Moreover, neutrophils exposed to Fn, but not Bf, stimulate release of IL-6 by tumor-associated stromal cells, leading to enhanced tumor cell proliferation. Our data cumulatively suggest that distinct bacterial components of the human gut flora might differentially modulate functions of CRC infiltrating neutrophils, thus ultimately influencing their prognostic significance. Note: This abstract was not presented at the meeting. Citation Format: Valeria Governa, Eleonora Cremonesi, Diego Calabrese, Valentina Mele, Emanuele Trella, Raoul A. Droeser, Martin Bolli, Serenella Eppenberger-Castori, Salvatore Piscuoglio, Charlotte K. y. Ng, Jesus Francisco Glaus Garzon, Lubor Borsig, Klaus-Peter Janssen, Luigi M. Terracciano, Giulio Cesare Spagnoli, Dimitri Christoforidis, Pietro Edoardo Majno-Hurst, Giandomenica Iezzi. Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2821.

Abstract 2837: Fusobacterium nucleatum and T cells in colorectal cancer liver metastasis

Abstract Background: Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum has been shown to promote colorectal tumor growth and inhibit antitumor immune responses. Emerging evidence demonstrates an enrichment of Fusobacterium species in colorectal cancer live metastasis (CRLM). Aim: To test the hypothesis that higher amount of Fusobacterium nucleatum is associated with lower density of CD8+ T-cells in CRLM. Method: Genomic DNA was extracted from CRLM and adjacent normal liver tissues. We measured the amount of Fusobacterium nucleatum DNA in 164 CRLM tissues using a quantitative polymerase chain reaction assay. The density of CD8+ T-cells in CRLM was determined by immunohistochemical staining. Result: Fusobacterium nucleatum was detected in 8 (4.9%) of 164 CRLM cases. A higher density of CD8+ T-cells in CRLM was significantly associated with better relapse-free survival (P =0.036). Fusobacterium nucleatum-negative cases, Fusobacterium nucleatum-positive cases were inversely associated with the density of CD8+T-cells (P =0.020). Conclusions: The presence of Fusobacterium nucleatum is inversely associated with CD8+ T-cells density in CRLM. Upon validation, our data may have implications for preventing the formation of liver metastases in colorectal cancer through targeting intestinal microflora. Citation Format: Yuki Sakamoto, Kosuke Mima, Nobuya Daitoku, Yukiharu Hiyoshi, Katsunori Imai, Masaaki Iwatsuki, Takatsugu Ishimoto, Yoshifumi Baba, Shiro Iwagami, Yuji Miyamoto, Yoichi Yamashita, Naoya Yoshida, Hideo Baba. Fusobacterium nucleatum and T cells in colorectal cancer liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2837.

Abstract 4576: Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival

Abstract Introduction. PanNETs are characterized by heterogeneous but largely indolent growth, leading to advanced stage at diagnosis, difficulty predicting outcomes, and insufficiently effective treatments. A better understanding of PanNETs immune context is needed for rational immunotherapy strategies. The aim of this study was to characterize immune cell infiltrates within primary tumors, understand the correlation of immune infiltration with genes associated with PanNET development, and clinical-pathological features. Material and methods. Formalin-fixed paraffin-embedded (FFPE) surgically resected primary tumor specimens from 53 patients with metastatic PanNETs were evaluated for DAXX, ATRX, and immune-cell markers (CD8, CD4, CD45RO, FOXP3, ICOS, OX40, PD-1, LAG3, TIM-3, B7-H3, B7-H4, PD-L1, VISTA, and CD68) by immunohistochemistry (IHC). Intratumoral lymphocyte-enriched areas (LEA), defined by CD8 hot-spots, and macrophage-enriched areas (MEA), defined by CD68 hot spots, were selected for image analysis and cell densities were quantitated. We considered higher densities more than the third quartile value and low density as less or equal than the third quartile value for all the markers. 47 cases from the same FFPE tumor tissue blocks were used for paired-end RNA sequencing (HiSeq 4000 Sequencing System), and exome sequencing (T200 Platform) to MEN1, SETD2, MUTYH, CHEK2, BRCA2, ALT, DAXX, ATRX, PTEN, TSC1, and TSC2 genes. Differences between variables were analyzed by non-parametric t-test and Kaplan-Meier curves for time-to-event using SPSS statistical software version 24. Results. Overall, higher densities of CD8, CD4, CD68, and B7-H3 were found compared with the other markers. We found a significant correlation between CD8 in LEA with CD4 (r=0.7), FOXP3 (r=0.5), CD45RO (r=0.6), ICOS (r=0.5) and PD-1 (r=0.5) cell densities. In addition, CD68 in MEA had significant and positive correlation with TIM-3 (r=0.6) cell densities. Higher TIM-3 cell densities correlated with higher levels of TIM-3 (P=<0.00001), CD163 (P=0.004), and CSF1R (P=0.02) mRNAs. Patients with high CD68 and TIM-3 densities showed worse disease-specific survival (DSS). The ratio between CD68, TIM-3, B7-H3 and FOXP3 cell densities to CD8 positive cells was significantly higher in patients with loss of ATRX nuclear expression. We observed different gene mutations on those PanNET samples but only PTEN mutant tumors were associated with higher densities of CD68 and worst DSS compared with lowest CD68 and WT PTEN mutant tumors (P=0.002). Conclusions. TAMs were significantly correlated with inferior DSS in PanNETs. TAM depletion may, therefore, present an appealing and rational target for immunotherapeutic approaches in NETs. This work was funded by a Conquer Cancer Foundation of ASCO Career Development Award, and a grant from the Neuroendocrine Tumor Research Foundation. Citation Format: Alejandro Francisco-Cruz, Naohiro Uraoka, Suyu Liu, Edwin R. Parra, Luisa M. Solis, Barbara Mino, Arvind Dasari, Jaime Rodriguez-Canales, Michael J. Overman, Jonathan M. Loree, James C. Yao, Ignacio I. Wistuba, Daniel M. Halperin, Jeannelyn S. Estrella. Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4576.

Assessment of tumor-associated immune cells in laryngeal squamous cell carcinoma.

This study investigated the characteristics of tumor-associated immune cells (TAICs) in laryngeal squamous cell carcinoma (LSCC) and their correlation with clinicopathological variables. The immune cell infiltrates of 71 specimens of stages I-IV LSCC were examined. The density of TAICs expressing CD3, CD4, CD8, CD68, and CD163 was assessed using immunohistochemical staining and image analysis in peritumoral and intratumoral regions. Higher densities of CD3+ and CD8+ cell and lower densities of CD68+ and CD163+ cell infiltrations were found in early tumor stages than in late tumor stages. A higher percentage of patients with strong CD3+ and CD8+ immune cell infiltration and weak CD68+ cell infiltration in both tumor regions presented with T1 stage tumors compared with T4 stage tumors. Further, strong CD68+ cells infiltration in both regions was observed in a greater number of patients who had a relapse, while a weak CD3+ cells infiltration in both regions was found in a greater number of patients with nodal lymphatic metastasis. The univariate analysis showed that a high density of peritumoral CD3+ and CD8+ immune cells in both regions was significantly associated with a favorable overall survival (OS) (P = 0.004; P = 0.006; P = 0.042). In contrast, a high density of intratumoral CD68+ cells and peritumoral CD163+ cells was significantly associated with poor OS durations (P = 0.026; P = 0.030). The multivariate analysis demonstrated that a high density of peritumoral CD163+ cells correlated with poor OS after adjusting for tumor stage, recurrence, and nodal lymphatic metastasis (P = 0.034). This study found different patterns of TAIC infiltration in LSCC. The density and location of TAICs infiltration correlated with the clinicopathological characteristics of LSCC. A combined analysis of the density of TAICs and their location may help predict patient survival and response to checkpoint inhibitors.

Density of CD8+ and CD68+ lymphocyte cell infiltration is associated with clinic outcome in hepatocellular carcinoma.

e15612 Background: The achievements of immune checkpoint blockade strengthen the concept that tumor outgrowth and development are comprehensively regulated by immune system. The aim of the study was to explorer whether distinct infiltrated immune cell features differentially affect clinical outcome in hepatocellular carcinoma (HCC). Methods: We obtained respectable stage II HCC specimens, along with adjacent para-tumor tissues from 221 patients who underwent surgical resection at Eastern Hepatobiliary Surgery Hospital, (in Shanghai, China) from 2015 through April 2018. CD8+ and CD68+ tumor-infiltrating lymphocytes (TILs) in the cancer area (CA) and stroma area (SA), as well as PD-1/PD-L1 expression, were analyzed by multiple immunohistochemistry. Results: The density of CD8+ TILs were significantly associated with gender, tumor size, and cirrhosis; while the density of CD68+ TILs were associated with copies of HBV DNA and anti-viral treatment. Multivariate Cox regression analysis revealed that high densities of CD8+ TILs and low densities of CD68+/CD8+ ratios independently predicted better outcomes. Of note, a prognostic signature combining clinic features [portal vein tumor thrombus (PVTT) and tumor size (TS)] and CD8+ TILs densities discriminated HCC patients into four subtypes with increasing risk of mortality: PVTT-negative/TS-small/CD8-high (6.8% of cases), PVTT-negative/TS-small/CD8-low (45.7%), PVTT-negative/TS-large (34.4%) and PVTT-positive (13.1%). Further association studies suggested that the four subgroups correlated with gender, tumor envelope, microvascular invasion (MVI), and SA-PD1 expression. Similarly, a prognostic signature combining PVTT, TS and CD68+/CD8+ ratios discriminated HCC patients into four subtypes with increasing risk of recurrence. Conclusions: Combined immune features including CD8+ and CD68+ lymphocyte infiltration and clinic characteristics are useful prognostic biomarkers for HCC patients.

Combination of tissues analysis and immune infiltrate in localized colon cancer using artificial intelligence in PETACC8 study.

3574 Background: We used artificial intelligence to perform tissue classification and count CD3 and CD8 in each subclass and determined their role in outcome prediction in PETACC8 cohort of stage III colon cancer treated with FOLFOX or FOLFOX plus cetuximab. Methods: We developed artificial intelligence aimed to detect tumor, healthy mucosa, stroma and immune cells on whole slide of CD3 and CD8 staining. The invasive margin (IM) was also automatically determined. Using a lasso algorithm, the software was able to detect digital parameters within the tumor core (TC) which were related to patients’ outcome (variable called DGMate for DiGital tuMor pArameTErs). CD3 and CD8 lymphocytes density were also quantified automatically by the software in TC and at IM. Associations with disease-free survival (DFS) were evaluated by multivariable Cox regression adjusting for age, T/N stage, sidedness, KRAS/BRAF, DNA mismatch repair (MMR). Results: On 1220 samples collected, data could be generated for 1018 patients. We observed that a high IM stromal area and a high DGMate were associated with a poorer DFS [HR 5.65 (95% CI, 2.34, 13.67), p < 0.0001; HR 2.72 (95% IC, 1.92, 3.85), p<0.001 respectively for the continuous variable]. A higher density of CD3+ TC, CD3+ IM and CD8+ TC were significantly associated with a longer DFS (HR 0.75 (95% IC, .66, .87), p<0.0001; HR 0.78 (95% IC, .68, .88), p<0.0001; HR 0.83 (95% IC, .71, .96), p=0.01). All these immune variables were significantly correlated with each other. ANOVA test demonstrated that CD3+ TC gave a similar prognostic value compared to the classical CD3/CD8 immunoscore (p=0.44). The combination of IM stromal area, DGMate and CD3 outperformed the classical CD3/CD8 immunoscore to estimate patients’ prognosis (C-index= 0.601 vs 0.578, p-value=0.04). Adding this new variable to classical clinical prognostic parameters we generated a nomogram which predicted the risk of relapse of stage III colon cancer with a stronger predictive value compared to clinical parameters or the immunoscore. Conclusions: We propose a new fully automated method of whole slide analysis using a software based on artificial intelligence which classify tissue and determine tumor and immune parameters on one single slide stained with CD3 antibody. This valuable strategy outperforms immunoscore and clinical outcome prediction models.

Automated image analysis of NSCLC biopsies to predict response to anti-PD-L1 therapy

BackgroundImmune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies.MethodsArchival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software.ResultsFor patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients (p = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS (high: 67 months; low: 39.5 months, p = 0.0009) in this group.ConclusionsAn automated CD8xPD-L1 signature may help to identify NSCLC patients with improved response to durvalumab therapy. Our data also support the prognostic value of CD8+ TILS in NSCLC patients who do not receive ICT.Trial registrationClinicalTrials.gov identifier: NCT01693562.Study code: CD-ON-MEDI4736-1108.Interventional study (ongoing but not currently recruiting).Actual study start date: August 29, 2012.Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).

Prognostic value of tumor-infiltrating lymphocytes in hepatocellular carcinoma: A meta-analysis.

Background:In patients with hepatocellular carcinoma (HCC), the prognostic role of tumor-infiltrating lymphocytes (TILs) for survival is still controversial. A meta-analysis was performed to investigate the prognostic effect of TILs in HCC.Methods:We identify studies from PubMed, Embase, and the Cochrane Library to evaluate the prognostic value of TILs in patients with HCC. A meta-analysis was conducted to estimate overall survival and disease-free survival. The hazard ratio (HR) and 95% confidence interval (CI) were calculated employing fixed-effect or random-effect models depending on the heterogeneity of the included trials.Results:A total of 7905 patients from 46 observational studies were enrolled. For TILs subsets, the density of CD8+, FOXP3+, CD3+, and Granzyme B+ lymphocytes was significantly associated with improved survival (P < .05). The density of FOXP3+ TILs in intratumor (IT) was the most significant prognostic marker (pooled HR = 1.894; 95% CI = 1.659–2.164; P < .001). Patients with high infiltration of CD8+ TILs in IT (pooled HR = 0.676; 95% CI = 0.540–0.845; P = .001) or in margin of tumor (MT) (pooled HR = 0.577; 95% CI = 0.437–0.760; P < .001) had better OS. The pooled analysis revealed that high density of Granzyme B+ T-lymphocytes in IT was statistically significant associated with better OS (pooled HR = 0.621; 95% CI = 0.516–0.748; P < .001) and DFS (pooled HR = 0.678; 95% CI = 0.563–0.815; P < .001). It was interesting that high density of CD3+ in IT foreboded worse OS (pooled HR = 1.008; 95% CI = 1.000–1.015; P = .037), but better DFS (pooled HR = 0.596; 95% CI = 0.374–0.948; P = .029).Conclusion:Our findings suggested that some TIL subsets could serve as prognostic biomarkers in HCC. High-quality randomized controlled trials are needed to determine if these TILs could serve as targets for immunotherapy in HCC.

CD8+ tumor-infiltrating lymphocytes as a novel prognostic biomarker in lung sarcomatoid carcinoma, a rare subtype of lung cancer.

PurposeThe aim of this study was to investigate the degree of infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) including high and low density in lung sarcomatoid carcinoma (LSC) and their clinicopathological significance.Patients and methodsThe density of CD8+ TILs in paraffin-embedded tissue sections from 100 LSC patients was detected by immunohistochemical staining, and the relationship of CD8+ TILs with clinicopathological features and prognosis was analyzed.ResultsThe chi-squared test showed that the degree of infiltration of CD8+ TILs was significantly correlated with the clinicopathological stage and T stage of LSC (P<0.05). The univariate analysis demonstrated that tumor size, clinicopathological stage, T stage, N stage, M stage, and CD8+ TILs are risk factors that affect prognosis of the patients (P<0.05). The mean overall survival (OS) of LSC patients with a high density of CD8+ TILs was 92.3 months, which was significantly higher than 31.2 months in patients with a low density of CD8+ TILs (P<0.05). Cox regression multivariate analysis confirmed that the density of CD8+ TILs was an independent prognostic factor for OS time of LSC patients (hazard ratio=0.455, P<0.05).ConclusionCD8+ TILs could be used as an effective prognostic index for LSC patients, and a high density of CD8+ TILs in tumor tissue may predict a better outcome.

Abstract A069: Combination of immune status and tumor microvascularization provides strong prognostic markers for prostate cancer recurrence prediction

Abstract Introduction: The composition of different immune cell populations in the tumor microenvironment plays an important role for tumor progression in various cancer types. In particular, tumor-associated macrophages (TAMs) and tumor-infiltrating T cells (TILs) are relevant players. On the other hand, structural changes such as neoangiogenesis triggered by cancerous tumors to increase their nutrient supplies have also been associated with tumor progression. In this work we systematically quantified these factors in prostate cancer (PCa) and correlated them with clinical outcome data using a Tissue Phenomics approach. By investigating the prognostic relevance of TAMs (CD68/CD163), TILs (CD3/CD8), and microvessels (CD34) in the tumor, tumor microenvironment (TME), and stroma we identified strong prognostic markers for PCa recurrence prediction in patients after radical prostatectomy. Methods: In this study, we analyzed a cohort of 90 PCa patients, of whom 40 suffered from tumor progression measured by prostate cancer antigen (PSA) recurrence after prostatectomy. The cohort comprised low- and intermediate-risk PCa patients (Gleason-Score≤7b) since providing a reliable prognosis is particularly difficult for such grades. Tissue sections were immunohistochemically stained using the duplex stains CD68/CD163 for TAMs, CD3/CD8 for TILs, and CK18/p63 to identify and characterize glands as cancerous vs. noncancerous based on their expression level of p63 (in cancerous glands p63 is not expressed). To quantify tumor neoangiogenesis microvessels were stained by CD34. All sections were geometrically aligned per case (virtual multiplexing) to enable coanalysis of stains, and quantified within relevant regions-of-interest (tumor, TME, stroma) using fully automated computational methods (1, 2). In particular, we determined region-specific densities and average distances of TAMs, TILs, and microvessels, as well as ratios of all measures. We systematically analyzed the prognostic power of each measure by optimizing a cutoff with respect to the disease-free survival statistic (log-rank test) using cross-validation to avoid for overfitting. Results: The top-ranking prognostic markers regarding robustness and prediction performance were related to microvessel density combined with immune cell densities. In particular, we found that within the TME, a coverage of CD8(+) cytotoxic T cells larger than 10% of the coverage of CD34(+) microvessels is correlated with a good prognosis and long-term disease-free survival (cross-validated p &amp;lt;3.1•10-7, accuracy=83%). This corresponds to high densities of CD8(+) cells and/or low microvessel densities, which both have been shown to be associated with good prognosis in prostate cancer. In addition, we found that a larger average distance of CD68(+) M1 macrophages to CD34(+) microvessels above 75.7µm in the tumor region is associated with good prognosis (cross-validated p &amp;lt;2.9•10-8, accuracy=82%). Again, low tumor microvessel density seems to be beneficial as well as high densities of CD68(+) macrophages. The CD68(+) M1-polarized phenotype is associated with tumor-suppressing properties, indicating that a higher density of this population compared to the tumor-promoting M2-polarized phenotype fosters disease-free survival. Conclusion: Our results indicate a considerable prognostic potential of markers combining microvessel density with measures of TAMs and TILs to predict PSA recurrence in PCa. This application shows that systematic analysis as performed by Tissue Phenomics enables discovery of non-obvious combined prognostic markers characterizing the tumor landscape with high potential to improve patient treatment. In future work we aim to validate our findings on additional data from other clinical sites.

High expression of PD-1 and PD-L1 in ocular adnexal sebaceous carcinoma

ABSTRACTOcular adnexal sebaceous carcinoma (OASC) is an aggressive malignancy that frequently recurs locally and metastasizes. Surgical extirpation may produce significant aesthetic morbidity, and effective systemic therapies for locally advanced or metastatic disease are largely ineffective. Immune checkpoint inhibitors have shown efficacy in the management of several solid tumors where tumor cell PD-L1 expression correlates with improved response. To determine whether OASC might be amenable to immune checkpoint blockade, we performed comprehensive immune profiling for CD3, CD8, PD-1, FOXP3, and PD-L1 in 24 patients with primary OASC. The composition, distribution and density of the tumor associated immune infiltrate were quantified by automated image analysis and correlated with measures of clinical outcome. Tumor cells in 12 OASCs (50%) expressed PD-L1. Higher densities of CD3+ (p = 0.01), CD8+ (p = 0.006), and PD-1+ (p = 0.024) tumor-associated T cells were associated with higher T category (≥T3a per the 7th edition of the American Joint Committee on Cancer staging manual). Higher tumor cell expression of PD-L1 correlated with higher density of PD-1+ tumor-associated T cells (p = 0.021). Since a CD3+ CD8+ PD-1 + T-cell infiltrate represents a “suppressed T-cell phenotype” apparently permissive toward OASC progression, our findings provide a mechanistic rationale for the effective application of immune checkpoint blockade in OASC to abrogate PD-1/PD-L1 interaction and effectively unleash the immune infiltrate to treat higher-stage tumors.

Abstract 220: Intra-tumor immune heterogeneity is associated with prognosis of oropharyngeal head and neck squamous cell carcinoma

Abstract As cell-cell interactions among tumor and immune cells are known to contribute to tumor progression, in depth evaluation of tumor-immune heterogeneity will enable biomarker-guided patient stratification and improvement of treatment response. Here we examined head and neck squamous cell carcinomas (HNSCC) to reveal the prognostic significance of leukocytes in tumors with regards to their complexity, effector status and spatial characteristics via multiplex immunohistochemistry and image cytometry. To accomplish this, we examined oropharyngeal HNSCC (N = 38), where lymphoid, myeloid, and hypo-inflamed leukocyte complexity correlated with HPV-status (Tsujikawa et al. Cell Reports, 2017). In the present study, quantitative analysis of cell density and distribution of 14-distinct immune cell lineages (e.g., CD8+ T cells, regulatory T cells, Th1, Th2, Th17, Th0 lymphocytes, B cells, NK cells, CD163+ and CD163- macrophages, mature and immature dendritic cells, mast cells, granulocytes) was analyzed via immune cell density mapping and tissue segmentation algorithms. We revealed tropism of polarized Th1-type cells within tumor nests versus stroma in HPV-associated HNSCC. In addition, Cox regression analysis of cell density and distribution of the 14 immune populations revealed that CD66b+ granulocyte infiltration within tumor nests reflected a negative prognostic indicator for HNSCC outcome. Furthermore, cell-cell proximity analysis in HPV-associated HNSCC further revealed a correlation between PD-L1 positive immune cells, and micro-regionally polarized immune characteristics biased towards Th1, coincident with high density of CD8+ T cells. These results reveal intra-tumor immune heterogeneity is associated with micro-regional immune complexity profiles, and provide insight into in situ immune characteristics that may aid patient stratification for immune therapy going forward. Acknowledgement: This project was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Young Scientists (Start-up, 17H07016), Oregon Clinical and Translational Research Institute (OCTRI), grant number (UL1TR000128) from the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH), and P30 CA069533-17 OHSU Knight Cancer Institute. LMC acknowledges support from the NIH/NCI, DOD BCRP Era of Hope Scholar Expansion Award, Susan G. Komen Foundation, Stand Up To Cancer - Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant, Breast Cancer Research Foundation, and the Brenden-Colson Center for Pancreatic Health. Citation Format: Takahiro Tsujikawa, Guillaume Thibault, Young Hwan Chang, Edward El Rassi, Daniel R. Clayburgh, David Sauer, Akihito Arai, Molly F. Kulesz-Martin, Motomi Mori, Shigeru Hirano, Paul W. Flint, Lisa M. Coussens. Intra-tumor immune heterogeneity is associated with prognosis of oropharyngeal head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 220.

Abstract 4045: A novel transcriptomic-based immune classification of soft tissue sarcoma (STS) and its association with molecular characteristics, clinical outcome and response to therapy

Abstract Soft tissue sarcomas (STS) form a group of rare cancers which accounts for around 1% of non-pediatric solid tumors. Recent clinical trials with check-point blockade immunotherapies reported a response in up to 15% of patients, but there are no biomarkers predicting response of STS to checkpoint blockade therapies yet. We analyzed transcriptomic data of publicly available cohorts, accounting for more than 600 complex genomic STS, including leiomyosarcoma (LMS, 35.4%), dedifferentiated liposarcoma (DDLPS, 33.9%) and undifferentiated pleomorphic sarcoma (UPS, 30.8%). Using a deconvolution method to estimate the tumor microenvironment composition with accurate scores depicting the immune profile of tumors, we established a robust immune classification that is consistent through different datasets. We divided the patients into 5 Sarcoma Immune Classes, labelled A1, A2, B, C1 and C2, which exhibit different extents and composition of immune infiltrate. Two groups (A1: 23.3% and A2: 27.1% of all tumors) exhibit a very low to low immune infiltrate for all cell types. Another class (B: 14.5% of all tumors) displays moderate immune infiltrate and a strong presence of endothelial cells. Finally, the remaining two groups (C1: 19.4% and C2: 15.6%) are highly infiltrated by all immune cell types. All histologies were found in all five immune classes, although LMS were enriched in classes A1 and A2. The immune-high groups exhibit a significantly prolonged overall survival compared to the other groups. Notably, the immune high group C2 is characterized by an extremely rich immune infiltrate containing both lymphocytes and myeloid cells, and is associated with a striking overexpression of several immune checkpoints genes: PDCD1 (PD-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), LAG3, HAVCR2 (TIM-3), CTLA4. This group also contains tumors with a strong expression of the B cell attractant chemokine CXCL13, often associated with the presence of tertiary lymphoid structures (TLS). We validated the relevance of TLS as a biomarker of this group by quantitative immunohistochemistry (IHC) using stainings for CD3, DC-Lamp, CD8, and CD20 on a 95-patient STS cohort (LMS, 34%; DDLPS, 33%; UPS, 34%). Tumors with visible TLS had a higher density of CD3+, CD8+ and CD20+ cells in the tumor core and the invasive margin, hence showing the possibility to identify the immune-high group by using IHC. The expression of immune checkpoint molecules was also detected by multiplex immunofluorescence for CD3, CD20, PD-1 and Lag3 in these tumors, confirming the presence of checkpoint molecules in the immune-high group. To check whether the immune-high group C2 was associated with enhanced response to checkpoint blockade therapy, we intend to analyze tumors from a recently published phase 2 clinical trial in which patients underwent treatment with anti-PD-1 pembrolizumab. Citation Format: Florent Petitprez, Tom Wei-Wu Chen, Cheng-Ming Sun, Julien Calderaro, Li-Ping Hsiao, Laetitia Lacroix, Ivo Natario, Maud Toulmonde, Carlo Lucchesi, Yec'han Laizet, Antoine Italiano, Aurélien de Reyniès, Catherine Sautès-Fridman, Wolf H. Fridman. A novel transcriptomic-based immune classification of soft tissue sarcoma (STS) and its association with molecular characteristics, clinical outcome and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4045.

Abstract 3122: Density of CD3+ and CD8+ tumor-infiltrating T lymphocytes as predictor of survival in colon cancer stage I-III

Abstract Introduction: The risk of recurrence after surgery for colon cancer is not only determined by cancer stage and molecular characteristics, but also by the tumor microenvironment. This study aims to assess the prognostic value of CD3+ and CD8+ T lymphocyte infiltration and the relation to mismatch repair (MMR) deficiency. Material and methods: The study cohort includes 289 patients with surgically treated colon cancer stage I-III (no adjuvant chemotherapy) with clinical- and follow-up data. Immunohistochemistry for CD3+ and CD8+ T lymphocytes and MMR proteins MLH1, MSH2, MSH 6 and PMS2 was performed on Tissue Micro Arrays. (Results were available from 242/258 patients, respectively). Tumors were characterized as MMR deficient/proficient according to expression pattern. Densities of CD3+ and CD8+ lymphocytes were assessed within the tumor margins (tumor infiltrating lymphocytes, TILs) and stroma &amp;lt;100 μm from tumor margin (stromal lymphocytes) and results were combined into semi-quantitative composite scores. In cases of intratumoral heterogeneity, the cylinder with the highest density of TILs was included in the analyses. Results were dichotomized at the median value into low and high density. Results: MMR deficiency (60 patients, 24.8%) was significantly associated with high density of CD3+ and CD8+ TILs and stromal lymphocytes (p&amp;lt;0.001, p=0.001 Chi square test), but no independent prognostic value of MMR deficiency was demonstrated in our multivariate models. CD3+ and CD8+ TILs and stromal CD3+ and CD8+ lymphocytes were analyzed in separate multivariate cox regression models adjusting for TNM-stage, sex, age, MMR status, histology and tumor differentiation. Low density of CD3+ and CD8+ TILs was an adverse independent prognostic marker of recurrence in colon cancer stages I-III (HR 4.63, CI 1.72-12.49, p=0.002) and the colon cancer stage III subgroup (HR 4.83, CI 1.06-21.97, p=0.042). TNM-stage and differentiation grade were other statistically significant variables in the colon cancer stage I-III model. The stromal lymphocyte score did not reach statistical significance in our multivariate models. Kaplan Meier plots illustrate marked differences in survival between groups of various TIL densities. The low TIL density group had a mean recurrence-free survival of 50.5 months (SE 1.89) versus 63.5 months (SE 1.11) for patients with high TIL density (p&amp;lt;0.001) in colon cancer stage I-III. Conclusions: Our results support the prognostic impact of tumor infiltrating CD3+ and CD8+ lymphocyte density in colon cancer stage I-III and indicate that a high TIL density is a stronger predictor of survival than MMR deficiency. Citation Format: Kjersti E. Hestetun, Luka Stanisavljević, Mette P. Myklebust, Olav Dahl. Density of CD3+ and CD8+ tumor-infiltrating T lymphocytes as predictor of survival in colon cancer stage I-III [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3122.

Transcriptomic immune profiling of ovarian cancers in paraneoplastic cerebellar degeneration associated with anti-Yo antibodies

BackgroundParaneoplastic neurological syndromes are rare conditions where an autoimmune reaction against the nervous system appears in patients suffering from a tumour, but not linked to the spreading of the tumour. A break in the immune tolerance is thought to be the trigger.MethodsThe transcriptomic profile of 12 ovarian tumours (OT) from patients suffering from paraneoplastic cerebellar degeneration (PCD) linked to anti-Yo antibodies (anti-Yo PCD OT) was compared with 733 ovarian tumours (OT control) from different public databases using linear model analysis.ResultsA prominent significant transcriptomic over-representation of CD8+ and Treg cells was found in anti-Yo PCD OT, as compared to the OT control. However, the overall degree of immune cell infiltration was similar, according to the ESTIMATE immune score. We also found an under-representation of M2 macrophages in anti-Yo PCD OT. Furthermore, the differentially expressed genes were enriched for AIRE-related genes, a well-known transcription factor associated with a broad range of autoimmune diseases. Finally, we found that the differentially expressed genes were correlated to the transcriptomic profiling of the cerebellar structures.ConclusionsOur data pinpointed the enrichment of acquired immune response, particularly high density of CD8+ lymphocytes, and high-level expression of CDR-related antigens in anti-Yo PCD OT.

Programmed Death Ligand 1: A Step Toward Immunoscore for Esophageal Cancer

This study sought to evaluate the effect of tumor-infiltrating lymphocyte (TIL) density and programmed death ligand 1 (PD-L1) expression on the prognosis of esophageal cancer. Banked tissue specimens from 53 patients who underwent esophagectomies for malignancy at a single institution over a 6-year period were stained for cluster of differentiation 3 (CD3), CD8, and PD-L1. Tumors were characterized as staining high or low density for CD3 and CD8, as well as positive or negative for PD-L1. TIL density and PD-L1 expression were analyzed in the context of survival, recurrence, and perioperative characteristics. Median follow-up was 823 days, with 92.5% survival and 26.8% recurrence rates. All tumors were adenocarcinomas. Neoadjuvant chemotherapy was given in 56.6% of cases, and neoadjuvant radiotherapy was given in 37.7%. High CD3 density was found in 83%, whereas high CD8 density was found in 56.6%. A total of 18.9% of the tumors stained positive for PD-L1. Survival was significantly shorter in Kaplan-Meier analysis for patients with primary tumors staining positive for PD-L1 (log rank: p= 0.05). Multivariable analysis controlling for neoadjuvant therapy, TIL markers, PD-L1, age, and sex found no significant difference in recurrence or survival. Positive staining for PD-L1 may be a prognostic marker for decreased survival in esophageal adenocarcinoma. Additional TIL cell types should be investigated for creation of an esophageal cancer Immunoscore. PD-L1 has potential as a therapeutic target.

PD-L1 Expression and CD8+ T Cell Infiltration Predict a Favorable Prognosis in Advanced Gastric Cancer

Advanced gastric cancer (AGC) has high morbidity and mortality in East Asia, and it is urgent to explore new treatments to improve patient prognosis. Programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have exhibited remarkable activity in clinical trials and were approved by the FDA for clinical therapy in several types of tumors. Here, we evaluated PD-L1 expression and T cell infiltration in AGC. Positive tumor PD-L1 expression was detected in 171 AGCs (33.60%) out of 509 AGCs. PD-L1 expression was positively correlated with CD8+ T cell infiltration. Then, PD-L1 and CD8A mRNA expression was analyzed using gastric cancer data from the TCGA database, confirming a positive correlation. Patient survival was assessed according to PD-L1 status and the T cell infiltration density. PD-L1 expression and a high density of CD8+ T cells in AGCs were associated with improved prognosis, whereas no significant difference was noted between PD-1 and CD3 expression. In contrast, a high density of FOXP3+ T cells in AGCs indicated a poor prognosis. Multivariate Cox regression analysis revealed that CD8+ T cell density acts as an independent predictor of overall survival (OS) in AGC patients. Taken together, this study further highlights targets for immune checkpoint-based therapy in AGC.

CD3+/CD8+ T-cell density and tumoral PD-L1 predict survival irrespective of rituximab treatment in Chinese diffuse large B-cell lymphoma patients.

To investigate the prognostic value of tumor-infiltrating T-cell density and programmed cell death ligand-1 (PD-L1) expression in diffuse large B cell lymphoma (DLBCL). One-hundred-twenty-five Chinese DLBCL patients were enrolled in our study and provided samples; 76 of all cases were treated with rituximab (R). Tumor tissues were immunostained and analyzed for CD3+ and CD8+ tumor-infiltrating T-cell density, tumoral PD-L1, and microenvironmental PD-L1 (mPD-L1). The density of CD3 was rated as high in 33.6% cases, while 64.0% of DLBCLs were classified as high CD8 density. Of all cases, 16.8% were PD-L1+. Of the remaining PD-L1-DLBCLs, 29.8% positively expressed mPD-L1. Both CD3 high density and CD8 high density were associated with mPD-L1 positivity (P = 0.001 and P = 0.0001). In multivariate analysis, independently, high CD3 density predicted better OS (P = 0.023), while CD8 high density and PD-L1 positivity were both associated with prolonged PFS (P = 0.013 and P = 0.036, respectively). Even in the subgroup treated with R, univariate analyses indicated that high CD3 density and PD-L1 positivity were associated with better OS (P = 0.041) and PFS (P = 0.033), respectively. The infiltrating densities of CD3+ T-cells, CD8+ T-cells, and PD-L1 expression are predictive of survival in DLBCLs, irrespective of R usage.

VISTA expression associated with CD8 confers a favorable immune microenvironment and better overall survival in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) often arises in the setting of chronic inflammation with multiple inhibitory immune signals. V-domain Ig suppressor of T cell activation (VISTA) is identified as a novel negative checkpoint regulator. This study sought to determine the expression and prognostic value of VISTA in HCC and classify tumor microenvironments (TMEs) based on VISTA and CD8+ tumor-infiltrating lymphocytes (TILs).MethodsThe expression of VISTA and CD8 proteins was assessed in 183 HCC tissue microarrays (TMAs) by immunohistochemistry (IHC). VISTA and CD8A mRNA extracted from 372 patients with HCC in The Cancer Genome Atlas (TCGA) database was included as a validation cohort. Associations between the VISTA, clinicopathological variables, and survival were analyzed.ResultsVISTA expression was detected in 29.5% HCC tissues, among which 16.4% tissues were positive for tumor cells (TCs), and 16.9% tissues were positive for immune cells (ICs). VISTA expression was significantly associated with tissues with a high pathological grading (p = 0.038), without liver cirrhosis (p = 0.011), and with a high density of CD8 + TILs (p < 0.001). Kaplan-Meier curves demonstrated that patients with VISTA-positive staining in TCs (p = 0.037), but not in ICs, (p = 0.779) showed significantly prolonged overall survival (OS) than those with VISTA-negative expression. Classification of HCC TME-based VISTA and CD8 + TILs showed 4 immune subtypes: VISTA+/CD8+ (16.9%), VISTA+/CD8- (12.6%), VISTA-/CD8+ (16.4%), and VISTA-/CD8+ (54.1%). The dual positive VISTA+/CD8+ subtype showed significantly prolonged OS than other subtypes (p = 0.023).ConclusionsVISTA protein expression in HCC showed cell specific and displayed different prognosis. VISTA expression was significantly associated with CD8 + TILs, Dual positive VISTA+/CD8+ showed favorable TME and better OS.

PD57-08 TUMORAL CD8+ T CELLS CORRELATE WITH FAVORABLE OUTCOME IN METASTATIC RENAL CELL CARCINOMA TREATED WITH TYROSINE KINASE INHIBITORS

You have accessJournal of UrologyKidney Cancer: Advanced (including Drug Therapy) III1 Apr 2018PD57-08 TUMORAL CD8+ T CELLS CORRELATE WITH FAVORABLE OUTCOME IN METASTATIC RENAL CELL CARCINOMA TREATED WITH TYROSINE KINASE INHIBITORS Jiajun Wang, Li Liu, Wei Xi, Jiejie Xu, and Jianming Guo Jiajun WangJiajun Wang More articles by this author , Li LiuLi Liu More articles by this author , Wei XiWei Xi More articles by this author , Jiejie XuJiejie Xu More articles by this author , and Jianming GuoJianming Guo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2652AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES CD8+ T cells play a central role in cancer immune elimination, and tyrosine kinase inhibitor (TKI) therapy could enhance their anti-tumor function. We aim to investigate the predictive value of CD8+ T cells for TKI therapy in metastatic renal cell carcinoma (mRCC) patients. METHODS One hundred and thirty-eight metastatic mRCC patients treated with TKIs were retrospectively scanned, and 111 eligible patients were finally enrolled. CD8+ T cell density and major histocompatibility complex (MHC) class I expression in tumor tissues was assessed by immunohistochemistry. Associations between CD8+ T cell density, MHC class I expression and local immune cytolytic activity were also assessed in the TCGA cohort. RESULTS CD8+ T cells were observed in mRCC samples (median density 45 cells/mm2, range 0-2750 cells/mm2). Patients with high density of CD8+ T cells showed higher response rate (P=0.002), longer overall survival (OS) (hazard ratio=0.510, P=0.005) and longer progression-free survival (PFS) (hazard ratio=0.591, P=0.012) under TKI therapy. Multivariate Cox analyses identified high CD8+ T cell density as an independent prognostic factor for both OS (hazard ratio=0.559, P=0.027) and PFS (hazard ratio=0.574, P=0.017). We also identified close relationship between high CD8+ T cell infiltration, activated antigen presentation mechanism and enhanced immune cytolytic activity. Moreover, mRCC patients with both high tumoral CD8+ T cell density and high MHC class I expression showed significantly prolonged survival (P<10-4). CONCLUSIONS Tumoral CD8+ T cell density could serve as a predictive marker for TKI therapy in mRCC patients, especially in those with high MHC class I expression. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1069-e1070 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Jiajun Wang More articles by this author Li Liu More articles by this author Wei Xi More articles by this author Jiejie Xu More articles by this author Jianming Guo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...