Immune Checkpoint Markers Research Articles

The prognostic value of immune checkpoints in oral squamous cell carcinoma.

Despite the importance of immune checkpoints in immunotherapy, the prognostic value of these molecules remains controversial in oral squamous cell carcinoma (OSCC). We performed a systematic review to investigate the prognostic significance of the immune checkpoints in OSCC. A systematic search was conducted in Ovid Medline, Scopus and Cochrane libraries, and all studies that evaluated the prognostic significance of immune checkpoints in OSCC were systematically retrieved. Twelve immune checkpoints/modulators were studied for their prognostic values in OSCC patients between 1985 and 2017. Seven immune checkpoints (FKBP51, B7-H4, B7-H6, ALHD1, PD-L1, B7-H3 and IDO1) were reported to be associated with poor patients' survival in at least one study, and five (CTLA-4, TLT-2, VISTA, PD-L2 and PD-1) did not have a significant prognostic value. PD-L1 results were controversial as it was reported to be associated with both better and worse patients' survival. Even though immune checkpoint markers had high expectation for OSCC prognostication, our systematic review revealed that the majority of them had been studied only once. The other molecules, which had been studied more than once, had controversial findings, except B7-H3.

A SERS microfluidic platform for targeting multiple soluble immune checkpoints

Immune checkpoint blockade therapies are promising next generation immunotherapeutic treatments for cancer. Whilst sequential solid biopsies are an invaluable source of prognostic information, they are not feasible for monitoring therapeutic outcomes over time. Monitoring soluble immune checkpoint markers expression in body fluids could potentially be a better alternative. Current methods (e.g. ELISA) for detecting immune-checkpoint proteins mostly rely on the use of monoclonal antibodies which are expensive and time-consuming to manufacture and isolate. Herein, we report an integrated surface enhanced Raman scattering (SERS)-microfluidics device for the detection of immune checkpoint proteins which involves the use of i) nano yeast single chain variable fragment (scFv) as a promising alternative to monoclonal antibodies providing high stability at relative low-cost and simplicity for production, ii) graphene oxide functionalised surface to reduces the bio functionalization steps, thus avoiding the general paradigm of biotin-streptavidin chemistry and iii) a microfluidic platform enabling alternating current electrohydrodynamics (ac-EHD) induced nanomixing to enhance the target scFv binding and minimize the non-specific interactions. Specific and multiplex detection of immune checkpoint biomarkers is achieved by SERS based spectral encoding. Using this platform, we successfully demonstrated the detection of clinically relevant soluble immune checkpoints PD-1, PD-L1 and LAG-3 from as low as 100 fg/mL of analytes spiked in human serum.

Molecular Targeting to Expand the Therapeutic Ratio in Women with Curable Cervical Cancer

Approximately 30-40% of cervical cancer patients treated with radio(chemo)therapy (RTCT) develop recurrence that can be difficult to treat. New approaches for overcoming treatment failures are needed. The CXCL12/CXCR4 chemokine pathway promotes tumor growth and metastasis in various tumor sites and we have observed the pathway is upregulated in RT treated cervical cancers. Plerixafor, an inhibitor of this pathway, is approved clinically and our initial studies showed the addition of plerixafor to RTCT enhanced primary tumor response. The current studies examined: 1. Different ways of sequencing RTCT and plerixafor to maximize efficacy 2. Biomarkers of response to RTCT and plerixafor 3. Effects of plerixafor with RTCT on intestinal toxicity, an important dose-limiting consequence of treatment in these patients. Orthotopic cervix xenografts were treated with RT (30 Gy; 2 Gy/day) and weekly cisplatin (4mg/kg) with or without plerixafor (5mg/kg/day). Plerixafor was administered concurrently with RTCT (3 wks), adjuvantly after RTCT (3 wks) or continuously (6 wks). Biomarker response was evaluated at the end of the concurrent and adjuvant treatments. Tumor growth delay was assessed at later times. Tumor control was assessed after 50 Gy (2Gy/day-5 wks) with cisplatin and plerixafor. Late intestinal toxicity was assessed by histologic examination of the colorectal junction 90 days after treatment. Plerixafor, whether administered concurrently or adjuvantly, prolonged tumor growth delay following RTCT (30 Gy). Adjuvant plerixafor was associated with longer growth delay. Tumor cure was achieved at a higher RT dose of 50 Gy with RTCT+plerixafor. RTCT alone increased CXCL12/CXCR4 signalling, PD-L1 (immune checkpoint marker) expression and the tumor accumulation of myeloid cells. The addition of plerixafor during or after RTCT abrogated these effects. This suggests: 1) RTCT upregulation of the CXCL12/CXCR4 pathway leads to recruitment of immune populations into the tumor that confer treatment resistance and 2) RTCT-induced increases in PD-L1 expression may promote an immunosuppressed tumor microenvironment that impairs treatment response. Plerixafor reduced RT-related intestinal injury, suggesting a protective effect that may relate to modifications of immune cell infiltrates involved with the mobilization of mesenchymal stem cells from the marrow. Further investigation is needed to assess mechanisms underlying these radio-protective effects. Adding plerixafor to RTCT blunts RTCT-induced upregulation of the CXCL12/CXCR4 pathway and reduces the increase in tumor-associated myeloid cells. These benefits may apply to other tumors where RT plays a curative role. Plerixafor protects normal tissue from RT injury. Few if any drugs have been identified previously that both improve the effectiveness of RT and prevent side effects. The combination of RT and plerixafor warrants testing in clinical trials to validate these findings.

Expression of programmed cell death ligand 1 and immune checkpoint markers in residual tumors after neoadjuvant chemotherapy for advanced high-grade serous ovarian cancer

ObjectiveTo investigate the prognostic value of the expressions of programmed cell death ligand 1 (PD-L1) and immune checkpoint markers in residual tumors after neoadjuvant chemotherapy (NAC) for advanced high-grade serous ovarian cancer (HGSOC). MethodsWe collected pre- and post-NAC tumor samples from patients with advanced HGSOC between 2006 and 2017. Post-NAC tumor tissue samples were available for immunostaining from 131 patients. The expressions of PD-L1 and immune checkpoint markers were assessed by immunohistochemical staining and the status of tumor-infiltrating lymphocytes (TILs) was also evaluated. We examined whether there are significant associations between protein expression status and patient outcomes and whether significant changes in protein expression levels occurred in response to NAC. ResultsPD-L1 expression in the tumor cells was evaluated in 113 patients, 12 (10.6%) of whom had high PD-L1 expression (≥25%) in post-NAC tissues. However, these high levels were not associated with progression-free survival (PFS; P = 0.348) or overall survival (OS; P = 0.699). Similarly, high stromal TILs [≥50%; n = 16 (15.0%)] among the 107 patients evaluated did not show any significant impact on PFS (P = 0.250) or OS (P = 0.800). Moreover, an abundance of TILs (intraepithelial, CD8+, and Foxp3+) and the expression of immune checkpoint markers (PD-1, ICOS, and LAG-3) in residual tumors did not confer any significant survival benefit. The impact of NAC on PD-L1 expression and stromal TILs varied considerably among individual patients. ConclusionAlthough the expression of PD-L1 and immune checkpoint markers in residual tumors after NAC had no prognostic impact on survival in patients with HGSOC, post-NAC evaluation of these proteins in chemoresistant tumors may help select patients for immunotherapy trials.

Characterization of the Neuroendocrine Tumor Immune Microenvironment.

The immune environment and the potential for neuroendocrine tumors (NETs) to respond to immune checkpoint inhibitors remain largely unexplored. We assessed immune checkpoint marker expression, lymphocytic infiltrate, and associated mutational profiles in a cohort of small intestine and pancreatic NETs. We assessed expression of PDCD1 (PD-1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) in archival tissue from 64 small intestine (SINETs) and 31 pancreatic NETs (pNET). We additionally assessed T-cell infiltrates, categorizing T-cell subsets based on expression of the T-cell markers CD3, CD8, CD45RO (PTPRC), or FOXP3. Finally, we explored associations between immune checkpoint marker expression, lymphocytic infiltrate, and tumor mutational profiles. Expression of PD-1 or PD-L1 in small intestine or pancreatic NET was rare, whereas expression of PD-L2 was common in both NET subtypes. T-cell infiltrates were more abundant in pNET than in SINET. We found no clear associations between immune checkpoint marker expression, immune infiltrates, and specific mutational profile within each tumor type. Our findings provide an initial assessment of the immune environment of well-differentiated NETs. Further studies to define the immunologic differences between pNET and SINET, as well as the role of PD-L2 in these tumors, are warranted.

TIM-3 expression in breast cancer

ABSTRACT Tumor-infiltrating lymphocytes (TILs) are predominantly present in breast cancer patients with estrogen receptor negative tumors, among whom increasing levels correlate with favorable outcomes. Nevertheless, currently available immune checkpoint inhibitors appear to benefit only a small number of women with breast cancer. Upregulation of additional immune checkpoint markers is one mechanism of resistance to current inhibitors that might be amenable to targeting with newer agents. T-cell Immunoglobulin and Mucin domain-containing molecule 3 (TIM-3) is an immune checkpoint receptor that is an emerging target for cancer immunotherapy. We investigated TIM-3 immunohistochemical expression in 3,992 breast cancer specimens assembled into tissue microarrays, linked to detailed outcome, clinico-pathological parameters and biomarkers including CD8, PD-1, PD-L1 and LAG-3. We scored and reported absolute counts for TIM-3+ intra-epithelial and stromal TILs (iTILs and sTILs), and find that breast cancer patients with TIM-3+ iTILs (≥ 1) represent a minority of cases (11%), with a predilection for basal-like breast cancers (among which 28% had TIM-3+ iTILs). TIM-3+ sTILs (≥ 2) represented 20% of cases and included more non-basal cases. The presence of TIM-3+ iTILs highly correlates with hematoxylin and eosin-stained stromal TILs and with other immune checkpoint markers (PD-1+ iTILs, LAG-3+ iTILs and PD-L1+ tumors). In prognostic analyses, early breast cancer patients with TIM-3+ iTILs have significantly improved breast cancer-specific survival whereas TIM-3+ sTILs did not reach statistical significance. In multivariate analyses, the presence of TIM-3+ iTILs is an independent favorable prognostic factor in the whole cohort as well as among ER negative patients. Our study supports TIM-3 as a target for breast cancer immunotherapy.

The clinical applications of The Cancer Genome Atlas project for bladder cancer

ABSTRACTIntroduction: Knowledge of the molecular subtypes of bladder cancer enables powerful generalizations involving the distinctive biology and pathways driving different disease subsets.Areas covered: In this review, we summarize the findings of a number of published studies exploring the molecular landscape of bladder cancer by analysis of genomic data from The Cancer Genome Atlas (TCGA). TCGA project has provided a comprehensive data resource of 412 muscle-invasive bladder cancers as characterized by multiple molecular analytical platforms. These data have been and will continue to be utilized in numerous subsequent studies aimed at better understanding the molecular basis of bladder cancer. The catalog of DNA-level alterations can greatly inform personalized and precision medicine approaches. Molecular subtypes of bladder cancer include distinct ‘basal/squamous’ and ‘luminal’ subtypes, cancers with papillary histology, disease subsets with prominent leukocyte infiltration and immune checkpoint marker expression, and a ‘neuronal’ subtype lacking small cell or neuroendocrine histology. The gene-level alterations and subtypes as revealed by TCGA data are relevant from the standpoint of both basic biology and clinical trial studies.Expert commentary: Multiple studies analyzing TCGA muscle-invasive bladder cancer cases point to the existence of five major expression-based molecular subtypes of the disease, with these subtypes having therapeutic implications.

Abstract 2112: Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% cases of lung cancer. It has poor prognosis as most NSCLC patients are at advanced stage when diagnosed. The development of immunotherapy blocking the PD-1/PD-L1 ligand pathway, in recent years, has shed light on new treatment for lung cancer. An in-depth understanding of the interaction between immune system and tumor will help to identify novel markers for lung cancer treatment. In this study, we performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples for circulating tumor cells (CTCs). The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization (IncellDx IncellPREPTM). The single cell suspensions were stained simultaneously with multiple (>20) immune check point markers (including PD-1*, PD-L1*, TIM-3*, LAG-3*, and CTLA-4*), cancer markers (such as EGFR* and ALK* fusion protein), and a cell cycle dye. The samples were interrogated on a novel, innovative, high parameter, spectral flow cytometer Cytek AuroraTM. Markers on subsets of immune cell and cancer cell populations were investigated. Our results showed the association of high levels of immune check point marker and cancer marker expressions with the high level of aneuploidy (indicated by DNA index >1.05), and the aggressiveness of the cancer (indicated by the number of CTCs). High numbers of CTCs were associated with aneuploidy, increased Post G0-G1%, and high expression of LAG-3, TIM-3, and PD-1. Multi-parametric flow cytometry allows simultaneous profiling of multiple immune and cancer markers on cancer samples at the single cell level. The knowledge acquired from these studies will enhance our understanding of cancer immune system, cancer cells, and the interaction between immune system and the cancer. It will potentially transform patient diagnosis, disease monitoring, and drug discovery. Our study demonstrated a powerful method to study solid tumors that may provide important information for successful precision cancer immunotherapy. *PD-1: programmed death-1; *PD-L1: programmed death-ligand 1; *TIM-3: mucin domain-3-containing molecule-3; *LAG-3: lymphocyte-activation gene-3; *CTLA-4: cytotoxic T-lymphocyte antigen-4; *EGFR: epidermal growth factor receptor; *ALK: anaplastic lymphoma kinase Citation Format: Xiaoyang Wang, Pin-I Chen, Maria Jaimes, Huimin Gu, Keith Shults, Fariba Fazeli, Janine Fernandez, Vishal Sharma, Kalyan Handique, Bruce K. Patterson. Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry [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 2112.

PO-364 Effect of exercise and immunotherapy on tumour immunogenicity and growth

IntroductionSeveral preclinical studies have shown that exercise can reduce the tumour growth across a range of tumour models. We recently demonstrated that this exercise-dependent suppression of tumour growth was due to an epinephrine-dependent mobilisation and IL-6-driven activation of cytotoxic cells. In line with this, exercise markedly increased intratumoral immune cell infiltration, thus promoting an immune-stimulatory intratumoral environment. Based on these original findings, we hypothesised that exercise may enhance the effect of immune checkpoint blockade treatment given the exercise-dependent increased intratumoral immune cell infiltration.Material and methodsWe used C57Black/C mice randomised to cages with or without running wheels (EX). After 4 weeks of running (with a running distance ranging from 1.8 km to 5.4 km per mice per day), the mice were inoculated with B16 melanoma cancer cells. In addition, a subset of mice was treated with αPD-L1 antibodies (100 µg per mouse three times per week starting on day 4 after tumour cell injection).Results and discussionsFirst, we observed that voluntary wheel running increased intratumoral expression of the immune checkpoint molecules, PD-L1, B7.1 and B7.2. In parallel, voluntary wheel running increased intratumoral infiltration of cytotoxic NK and T cells, and thus intratumoral expression of the receptors, PD-1 and CD28. Next, we tested the combination therapy of exercise and αPD-L1 antibody treatment. In this setup, wheel running had an overall suppressive effect (p=0.01, 2-way ANOVA). Post hoc analysis showed that αPD-L1 +EX reduced tumour growth by 82.6%, while EX alone reduced tumour growth by 72% with no significant difference between the two interventions. qPCR analysis of these tumours revealed that combination therapy further increased the expression of immune cell markers and immune check point molecules.ConclusionIn conclusion, our results show that exercise increases the expression of immune checkpoint markers and therefore, could be a good combination partner for immune checkpoint blockade treatment. However, more studies are needed to show an effect of this combination therapy on tumour growth.

A Phase II Study of Pelareorep (REOLYSIN®) in Combination with Gemcitabine for Patients with Advanced Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

Immune checkpoint marker expression and clinicopathological factors in primary TNBC.

e13119Background: Immunotherapy emerges to a promising concept for patients with Triple-Negative Breast Cancer (TNBC). Immune Checkpoint Inhibitors (ICI) are expressed by tumor tissue to evade the ...

Immunohistochemical and Image Analysis-Based Study Shows That Several Immune Checkpoints are Co-expressed in Non-Small Cell Lung Carcinoma Tumors.

The understanding of immune checkpoint molecules' co-expression in non-small cell lung carcinoma (NCLC) is important to potentially design combinatorial immunotherapy approaches. We studied 225 formalin-fixed, paraffin-embedded tumor tissues from stage I-III NCLCs - 142 adenocarcinomas (ADCs) and 83 squamous cell carcinomas (SCCs) - placed in tissue microarrays. Nine immune checkpoint markers were evaluated; four (programmed death ligand 1 [PD-L1], B7-H3, B7-H4, and indoleamine 2,3-dioxygenase 1 [IDO-1]) expressed predominantly in malignant cells (MCs) and five (inducible T cell costimulator, V-set immunoregulatory receptor, T-cell immunoglobulin mucin family member 3, lymphocyte activating 3, and OX40) expressed mostly in stromal tumor-associated inflammatory cells (TAICs). All markers were examined using a quantitative image analysis and correlated with clinicopathologic features, TAICs, and molecular characteristics. Using above the median value as positive expression in MCs and high density of TAICs expressing those markers, we identified higher expression of immune checkpoints in SCC than ADC. Common simultaneous expression by MCs was PD-L1+ B7-H3+ IDO-1 in ADC and PD-L1+ B7-H3, or B7-H3+ B7-H4, in SCC. TAICs expressing checkpoint were significantly higher in current smokers than in never smokers. Almost all the immune checkpoint markers showed positive correlation with TAICs expressing inflammatory cell markers. KRAS-mutant ADC specimens showed higher expression of PD-L1 in MCs and of B7-H3, T-cell immunoglobulin mucin family member 3, and IDO-1 in TAICs than wild type. Kaplan-Meier survival curves showed worse prognosis in ADC patients with higher B7-H4 expression by MCs. We found frequent immunohistochemical co-expression of immune checkpoints in surgically resected NCLC tumors and correlated with tumor histology, smoking history, tumor size, and the density of inflammatory cells and tumor mutational status.

Abstract P3-05-12: VISTA expression on tumor-infiltrating lymphocytes in breast cancer: Clinical correlates and association with PD-1

Abstract Background: The V-domain containing Ig Suppressor of T Cell Activation (VISTA) is a recently discovered immune checkpoint receptor with homology to PD-1. VISTA expression on tumor-infiltrating lymphocytes (TILs) blocks their proliferation and effector functions. Recent pre-clinical data in cancer mouse models treated with anti-VISTA and anti-PD-L1 combinations showed promising results and non-redundant functions of VISTA and PD-L1 blockade. However, the expression and prognostic value of VISTA+ TILs in primary breast tumors has not been investigated in detail. Here we assess expression, prognostic value, and associations of VISTA+TILs with other immune checkpoint markers (PD-1/PD-L1, LAG-3, and IDO-1) and with H&E TIL counts. Methods: A tissue microarray consisting of breast carcinoma primary excision specimens (n=330) from the University of British Columbia hospital, linked to detailed clinicopathological data and outcomes, was used in this study. Patients from this cohort did not receive neoadjuvant treatment. A VISTA antibody (Clone D1L2G) was applied on a 4μm section of the tissue microarray by immunohistochemistry using a Ventana automated stainer. VISTA+TILs in direct contact with tumor nest were scored and reported as absolute count per 0.6mm core. Positive cases were defined as cases with VISTA+TILs≥1. All descriptive and survival analyses were conducted using SPSS software. Results: VISTA+TILs were present in 30% of cases and were significantly (p<0.05) associated with younger age (<50 years old), larger tumors (>2cm), hormone receptor negativity (ER/PR) and high Ki67proliferation index (≥13.25%). Almost half (48%) of basal-like breast cancers were positive for VISTA expressing TILs. No significant prognostic associations were observed in this cohort. Among the immune checkpoint receptors analyzed, VISTA+TILs were highly associated with PD-1+ TILs: 79% of cases positive for PD-1+TILs were also infiltrated by VISTA+TILs. Interestingly, we found that VISTA+TILs and PD-1+TILs were enriched in cases with otherwise low levels (<10%) of H&E TILs. Conclusions: Our study identifies the presence of VISTA+TILs in breast cancer patients and its strong association with PD-1+TILs. These results suggest that VISTA blockade could be a good candidate for combination therapy with other immune checkpoint inhibitors, a concept being tested in early phase clinical trials. Validation of these findings in a larger independent cohort powered for multivariate analysis is currently ongoing. Citation Format: Burugu S, Gao D, Nielsen TO. VISTA expression on tumor-infiltrating lymphocytes in breast cancer: Clinical correlates and association with PD-1 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-12.

PS06.06 Immune Checkpoint Markers in Lung Large Cell Neuroendocrine Carcinomas (L- LCNEC): Topic: Medical Oncology

PS06.06 Immune Checkpoint Markers in Lung Large Cell Neuroendocrine Carcinomas (L- LCNEC): Topic: Medical Oncology

PD-L1 expression indicates favorable prognosis for advanced lung adenocarcinoma patients treated with pemetrexed.

Conventional chemotherapy for lung cancer exerts anti-tumor effects through cytotoxicity, and through immunologic regulation by reducing specific T cell subsets and inducing the expression of programmed death ligand 1 (PD-L1) on tumor cells. Even though pemetrexed has shown huge potential in combination with other targeted or immune therapies, there is still little information about the values of specific immune checkpoint markers for advanced lung adenocarcinoma treated with pemetrexed. In the present study, a total of 56 patients with advanced lung adenocarcinoma, who received pemetrexed-based chemotherapy, were included retrospectively. Immunohistochemistry was performed to assess PD-L1, programmed death 1 (PD-1), thymidylate synthase, and tumor infiltrating lymphocytes (TILs). In this cohort, the positive expression of PD-L1 and PD-1 were 26.8% and 33.9% respectively. PD-L1, PD-1, and thymidylate synthase expression were not significantly associated with any clinical features, while the expression of both PD-L1 and PD-1 were correlated with Ki-67 expression. Furthermore, the expression of PD-1 was significantly correlated with TILs. The progression-free survival (PFS) in patients with PD-L1+ specimens was significantly longer compared to PD-L1− specimens. Moreover, PD-L1 expression was an independent protective factor for PFS, and the smoking status was an independent risk factor. PD-L1 expression was significantly associated with better prognosis for patients with pemetrexed-based treatment. Our findings suggested that PD-L1 expression might be a favorable prognostic biomarker for pemetrexed-based regimen, which is a rationale for combining immunotherapy with chemotherapy for lung cancer.

Pan-urologic cancer genomic subtypes that transcend tissue of origin

Urologic cancers include cancers of the bladder, kidney, prostate, and testes, with common molecular features spanning different types. Here, we show that 1954 urologic cancers can be classified into nine major genomic subtypes, on the basis of multidimensional and comprehensive molecular characterization (including DNA methylation and copy number, and RNA and protein expression). Tissue dominant effects are first removed computationally in order to define these subtypes, which reveal common processes—reflecting in part tumor microenvironmental influences—driving cellular behavior across tumor lineages. Six of the subtypes feature a mixture of represented cancer types as defined by tissue or cell of origin. Differences in patient survival and in the manifestation of specific pathways—including hypoxia, metabolism, NRF2-ARE, Hippo, and immune checkpoint—can further distinguish the subtypes. Immune checkpoint markers and molecular signatures of macrophages and T cell infiltrates are relatively high within distinct subsets of each cancer type studied. The pan-urologic cancer genomic subtypes would facilitate information sharing involving therapeutic implications between tissue-oriented domains.

Abstract LB-221: Tumor microenvironment predicts aggressive breast cancer: Combination of HERV-K, immune checkpoint and activation status of CD8+ T cells

Abstract The purpose of this study is to identify useful predictive biomarkers of anti-tumor cytotoxic T cell and immune checkpoint response, to optimize targeted therapy, chemotherapy, and immunotherapy of breast cancer (BC). Immune checkpoint markers (14 circulating proteins) in BC patients were determined using a magnetic bead assay. CD27, PDL-2, LAG-3, and TIM-3 levels in the plasma, obtained from patients with invasive ductal carcinoma (IDC: n=32), ductal carcinoma in situ (DCIS: n=11), other invasive BC (n=12), prophylactic surgery (BRCA1: n=7, and BRCA2 mutations: n=4, benign phyllodes tumor: n=2) were significantly enhanced compared with healthy controls (n=12), which supports a highly immune suppressed environment. Also, these markers were compared at T1 (before surgery), T2 (6 months post-surgery), and T3 (18 months post-surgery). LAG-3, CD27 (p=0.012), 4-1BB, CD80, PD-L2, PD-L1 (p=0.045), and IDO all had a decreased plasma concentrations post-surgery (6 months or 18 months) compared with pre-surgery. Interestingly, significantly higher concentrations of BTLA, IDO, LAG-3, PD-L2, TIM-3, and PD-L1 were detected in BC patients that had higher titers of circulating human endogenous retrovirus type K (HERV-K) antibodies. We characterized the subset distribution and phenotype of the immune cells of various BC patients. CD4+ or CD8+ T cells collected from breast tissues including tumor tissues (T) or uninvolved normal breast tissues (N) were analyzed for CD4 and CD8. Higher percentages of CD8+ tumor infiltrating lymphocyte (TIL) cells were detected in N compared with autologous T. The percentage of CD38+/HLA-DR+ CD8+ T cells was low in the PBMCs compared to percentages in TILs. Activated T cells were only detected in TIL (40% to 99%; n=6) but not in their PBMCs from the same patients. Several studies have demonstrated that regulatory T cells (Tregs) within the tumor environment lead to a decrease in patient survival. Higher percentages of Treg cells were observed in aggressive BC and in TIL obtained from tumor tissues compared to TIL cells obtained from uninvolved tissues. HERV-K specific CD8 T cells (K-T cells) enhanced the percentage of CD8 T cells (62.5%; n=8) in patients with aggressive BC, and were able to kill their autologous tumor cells or mammospheres. Significantly reduced tumor weights and metastases were demonstrated in MDA-MB-231 mice treated with K-T cells. The lower percentage of CD8 T cells and higher percentage of Treg T cells we observed in breast tumor tissues, coupled with elevated concentrations of circulating immune checkpoint proteins in BC patients, suggests a highly immune suppressed environment in these aggressive tumors that could be minimized with anti-HERV-K therapy. Thus, HERV-K T cell therapies combined with blockade of immune checkpoints may be a new regimen for treating aggressive BC. Citation Format: Feng Wang-Johanning, Jia Li, Gary Johanning, Ming Li, Raghu Chivukula, Guoqing Wu. Tumor microenvironment predicts aggressive breast cancer: Combination of HERV-K, immune checkpoint and activation status of CD8+ T cells [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 LB-221. doi:10.1158/1538-7445.AM2017-LB-221

Abstract 5597: Genomic profiling of syngeneic mouse cell lines and in vitro screen of the models against checkpoint inhibitors and target agents for preclinical application

Abstract Cancer immunotherapy has provided substantial clinical benefit in a significant number of patients with advanced diseases. In pre-clinical studies, CrownBio has developed a large collection of syngeneic models to facilitate in vivo efficacy testing of immune-oncology agents in mice (MuScreenTM). To combine the ostensibly separate therapeutic strategies of activating immune cells against and targeting the unique genetic characteristics of a tumor model, we sought to thoroughly characterize the mutation profiles of these syngeneic mouse cell lines and examine drug response profiles of these cell line models. The goal of this work was to provide an in vitro system in evaluating combination effectiveness when targeting both immune checkpoint markers and oncogenic targets in preclinical studies. To this end, we investigated mutation and gene expression profiles of 18 mouse cancer cell lines out of the panel of 23 syngeneic mouse models included in the MuScreenTM for 50 well defined cancer-related mutations among 30,690 variants in exonic regions called by RNAseq (Illumina HiSeq X10). The somatic mutations revealed through the bioinformatics analysis include ALK (3 - frequency, same for the rest), BRAF (4), BRCA1 (7), BRCA2 (12), EGFR (3), ERBB2 (6), EGFR3 (2), FBXW7 (10), FLT3 (12), HRAS (1), KRAS (8), NRAS (1), PDGFRA (11), PTCH1 (9), PIK3CA (2), PTEN (6), RET (3), SETD2 (5), SMAD4 (3), SMO (13), TRP53 (13), TSC1 (3), and TSC2 (10). All of these oncogenic alterations are clinically actionable (Cancer Discovery 2, 82-93, 2012). The same set of genes was also subject to mRNA expression change analysis. We screened the 18 mouse cell lines with a few selected target therapy agents, and performed combination assays of these agents together with check point inhibitors such as aPD1, aPDL1, in a co-culture system with mouse T cells. An IncuCyte real-time imaging platform was used to distinguish activities on T cells and tumor cells. The results and the implications will be discussed. Citation Format: Frank Xing, Wubin Qian, Chunlan Dong, Xiaoxi Xu, Sheng Guo, Qian Shi. Genomic profiling of syngeneic mouse cell lines and in vitro screen of the models against checkpoint inhibitors and target agents for preclinical application [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 5597. doi:10.1158/1538-7445.AM2017-5597

Abstract 1469: Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors

Abstract Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA. Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples. Citation Format: Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors [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 1469. doi:10.1158/1538-7445.AM2017-1469

Abstract 1656: Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft model

Abstract The recent successes of immunotherapeutic approaches to the treatment of melanoma and the promise of similar treatments in a variety of other cancers underscore the importance of the immune system in cancer. Indeed, effective therapeutic design and evaluation require a comprehensive understanding of the interplay between the immune compartment and the proliferating tumor cells that comprise the tumor microenvironment. A humanized mouse strain engrafted with cancerous tissue from a patient derived xenograft (PDX) tumor provides researchers with a highly sophisticated tool, ideally suited to facilitate the design of treatment strategies that prevent tumor evasion of immune cells and that improve cytotoxic responses. Severely combined immunodeficient mice such as NOD scid gamma (NSGTM) and triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSGTM-SGM3) are proven hosts for the engraftment of human tumors and establishment of human immune system components following hematopoietic stem cell (CD34+) transplantation. The endogenous expression of cytokines that support the development of myeloid lineages and regulatory T cells potentially represents a substantial improvement over standard NSG mice. Here we employ four 14-color flow cytometry panels to perform a comprehensive and detailed analysis of the entire immune system. The four panels are designed to fully characterize specific branches of the immune system: 1) T cells 2) NK cells/dendritic cells/B cells 3) myeloid lineages, and 4) immune checkpoint markers. Blood, spleen, and bone marrow tissue from both NSG and NSG-SGM3 mice were evaluated at 9, 16, 21, and 31 weeks of age using each of the four phenotyping panels. Our results indicate that the triple transgenic NSG-SGM3 mice exhibit a more completely humanized immune system compared to NSG mice, with specific improvements in the distribution of T-cell subsets and overall representation of the myeloid lineage. NSG mice engrafted with allogeneic human tumors represent a valuable preclinical testing platform for immuno-oncology. Citation Format: Aaron J. Middlebrook, Eileen Snowden, Warren Porter, Friedrich Hahn, Mitchell Ferguson, Brian Soper, James Keck, Joan Malcolm, Shannon Dillmore, Smita Ghanekar, Rainer Blaesius. Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft model [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 1656. doi:10.1158/1538-7445.AM2017-1656