Cancer cells expressing PD-1 ligands (PD-L1/PD-L2) inhibit immune-modulatory T-cell activation facilitating disease progression. Preliminary clinical trials exploring interruption of PD-1/PD-L1 signaling showed benefit in several cancer types. We analyzed the distribution of PD-1-positive tumor-infiltrating lymphocytes (TIL) and cancer cells' expression of PD-L1 in a molecularly profiled cohort of 437 malignancies (380 carcinomas, 33 sarcomas, and 24 melanomas). We showed that the presence of PD-1 þ TILs significantly varied among cancer types (from 0% in extraskeletal myxoid chondrosarcomas to 93% in ovarian cancer), and was generally associated with the increased number of mutations in tumor cells (P ¼ 0.029). Cancer cell expression of PD-L1 varied from absent (in Merkel cell carcinomas) to 100% (in chondro-and liposarcomas), but showed the inverse association with the number of detected mutations (P ¼ 0.004). Both PD-1 and PD-L1 expression were significantly higher in triple-negative breast cancers (TNBC) than in non-TNBC (P < 0.001 and 0.017, respectively). Similarly, MSI-H colon cancers had higher PD-1 and PD-L1 expression than the microsatellite stable tumors (P ¼ 0.002 and 0.02, respectively). TP53-mutated breast cancers had significantly higher PD-1 positivity than those harboring other driver mutations (e.g., PIK3CA; P ¼ 0.002). In non-small cell lung cancer, PD-1/PD-L1 coexpression was identified in 8 cases (19%), which lacked any other targetable alterations (e.g., EGFR, ALK, or ROS1). Our study demonstrated the utility of exploring the expression of two potentially targetable immune checkpoint proteins (PD-1/PD-L1) in a substantial proportion of solid tumors, including some aggressive subtypes that lack other targeted treatment modalities. Cancer Epidemiol Biomarkers Prev; 23(12); 2965-70. Ó2014 AACR.

Glioblastomas (GBM) are the most aggressive and prevalent form of gliomas with abysmal prognosis and limited treatment options. We analyzed clinically relevant molecular aberrations suggestive of response to therapies in 1035 GBM tumors. Our analysis revealed mutations in 39 genes of 48 tested. IHC revealed expression of PD-L1 in 19% and PD-1 in 46%. MGMT-methylation was seen in 43%, EGFRvIII in 19% and 1p19q co-deletion in 2%. TP53 mutation was associated with concurrent mutations, while IDH1 mutation was associated with MGMT-methylation and TP53 mutation and was mutually exclusive of EGFRvIII mutation. Distinct biomarker profiles were seen in GBM compared with WHO grade III astrocytoma, suggesting different biology and potentially different treatment approaches. Analysis of 17 metachronous paired tumors showed frequent biomarker changes, including MGMT-methylation and EGFR aberrations, indicating the need for a re-biopsy for tumor profiling to direct subsequent therapy. MGMT-methylation, PR and TOPO1 appeared as significant prognostic markers in sub-cohorts of GBM defined by age. The current study represents the largest biomarker study on clinical GBM tumors using multiple technologies to detect gene mutation, amplification, protein expression and promoter methylation. These data will inform planning for future personalized biomarker-based clinical trials and identifying effective treatments based on tumor biomarkers.

Angiosarcoma is a vascular malignancy associated with a poor prognosis and chemotherapy resistance. The tumor immune microenvironment of angiosarcoma has not been characterized. We investigated the expression of programmed death-ligand 1 (PD-L1) and programmed death 1 (PD-1) in angiosarcoma and correlated these findings with vascular endothelial growth factor (VEGF)-related gene expression and survival. Using archived formalin-fixed paraffin-embedded tissues of primary and metastatic angiosarcoma specimens, we characterized the immunohistochemical (IHC) expression of PD-L1 and PD-1. In addition, we extracted RNA from each tumor and quantified the expression of VEGF-related genes, and then tested if these genes were associated with PD-L1 and PD-1 expression and clinical outcomes. Retrospective review identified 27 angiosarcoma specimens collected between 1994 and 2012. IHC expression of tumor PD-L1, tumor-infiltrating immune cell PD-L1, and tumor-infiltrating immune cell PD-1 expression was identified in 5 (19%), 9 (33%), and 1 (4%) specimens, respectively. Expression of PD-L1 and PD-1 was not associated with VEGF-related gene expression or survival. PD-L1 tumor and tumor-infiltrating immune cells expression was identified in a large proportion of patients. Though neither was associated with VEGF-related gene expression or prognosis, targeting PD-1/PD-L1 may be of benefit for a significant proportion of angiosarcomas that do not respond to surgery, chemotherapy, or radiation.

ObjectivePatients with recurrent epithelial ovarian cancer (EOC) have limited treatment options. Studies have reported that biomarker profiling may help predict patient response to available treatments. This study sought to determine the value of biomarker profiling in recurrent EOC.ResultsPatients in the Matched cohort had a median OS of 36 months compared to 27 months for patients in the Unmatched cohort (HR 0.62, 95% CI 0.41-0.96; p < 0.03). Individual biomarkers were analyzed, with TUBB3, and PGP prognostic for survival. Biomarker analysis also identified a molecular subtype (positive for at least two of the following markers: ERCC1, RRM1, TUBB3, PGP) with particularly poor overall survival.Methods224 patients from a commercial registry (NCT02678754) with stage IIIC/IV EOC at diagnosis, or restaged to IIIC/IV EOC at the time of molecular profiling, were retrospectively divided into two cohorts based on whether or not the drugs they received matched their profile recommendations. The Matched cohort received no drugs predicted to be lack-of-benefit while the Unmatched cohort received at least one drug predicted to be lack-of-benefit. Profile biomarker/drug associations were based on multiple test platforms including immunohistochemistry, fluorescent in situ hybridization and DNA sequencing.ConclusionsThis report demonstrates the ability of multi-platform molecular profiling to identify EOC patients at risk of inferior survival. It also suggests a potential beneficial role of avoidance of lack-of-benefit therapies which, when administered, resulted in decreased survival relative to patients who received only therapies predicted to be of benefit.

Patients in whom the standard of care has failed or who have uncommon tumors for which no standard of care exists are often treated with drugs selected based on the physician’s best guess. The rate of success for this method is generally low. With the advent of fast, affordable tumor profiling technologies, and a growth in the understanding of predictive biomarkers, it is now possible to identify drugs potentially associated with clinical benefit for such patients. We present the Caris approach to evidence-based tumor profiling and two patients with advanced ovarian and prostate cancer in whom standard of care had failed and tumor profiling identified an effective treatment schedule. To establish Caris Molecular IntelligenceTM (CMI), over 120,000 clinical publications were screened and graded to characterize the predictive value of biomarkers that form the panel of tests. CMI includes multiple technologies to measure changes in proteins, ribonucleic acid, and deoxyribonucleic acid and proprietary software that matches the test results with the published evidence. The CMI results enable physicians to select drugs that are more likely to benefit the patients, avoid drugs that are not likely to work, and find treatment options that otherwise would not be considered. Worldwide, over 60,000 cancer patients have undergone evidence-based tumor profiling with CMI. In the cases reported in this article, CMI identified treatments that would not have been routinely used in the respective clinical setting. The clinical outcomes observed help to illustrate the utility of this approach.

4013 Background: A great need exists for new therapeutic approaches for patients with pancreatic cancer. Methods: The study cohort included 1029 patients analyzedfor a.) up to 29 different immunohistochemical biomarkers – (e.g., COX-2, MGMT, PGP, RRM1, TOPOI, TOPOII, SPARC etc.)b.); in up to 450 patients’ specimens a whole genome expression analysis was performed using HumanHT-12 v4 beadChips ( Illumina Inc.,San Diego, CA) c.) in up to 695 patients FISH for c-Myc, EGFR, HER2 and TOPO2A gene copy amplifications; and d.) in up to 783 patients sequencing for KRAS, EGFR, PI3CA and BRAF, was performed. Results: IHC identified actionable targets included; 74% high COX-2; 57% negative ERCC1; 8% negative MGMT; 22% negative MRP1; 47% negative PGP; 77% low RRM1, 44% high SPARC; 30% high TOPO2A; 61% high TOPOI and 73% negative TS. Other biologically important findings by IHC for possible new therapeutics included 27% negative PTEN; and 20% high PDGFR. Microarray results presented multiple overexpressed targets for consideration including 36% of specimens with overexpressed adenosine deaminase; 28% asparagine synthase; 17% BCL2; 20% survivin; 23% carboxylesterase; 67% DNMT1; 40% thymidine phosphatase; 49% EPHA2 (and others in the src family of kinases); 57% FOLR2; 41% HDAC1; 62% HiF1α; 23% IL2RA (CD25); 46% NFkB1; 48% OGFR; 32% RARA; 26% VEGFR; and 43% vitamin D receptor. FISH yielded 2% amplified EGFR and 10% amplified Her2neu. Sequencing noted 73% mutated KRAS and 3% mutated PIK3CA. Conclusions: Examining actionable targets in patients’ pancreatic cancers (a)reiterates the commonality and importance of KRAS mutations in this disease (needs renewed targeting effort) (b)suggests that TOPO2 inhibitors (particularly if transport into tumor can be improved) should be examined in this disease (c)suggests other pathways to target including DNA repair, epigenetic, Src and inflammation (d) suggests protein turnover, amino acid targets and folate receptor2 as fresh areas to explore against the disease. Supported in part by a Stand Up To Cancer Dream Team Award.

Endothelial- and leukocyte-derived circulating microvesicles (cMV) represent a majority of the cMV present in the blood. The purpose of this study was to determine if depletion of these more common cMV populations is possible, allowing for the enrichment and analysis of the remaining subpopulations of cMV. Circulating microvesicles were isolated from the blood plasma of breast cancer patients. Magnetic beads conjugated to antibodies specific for either CD31 or CD45 were used to deplete the isolated endothelial- and leukocyte-derived cMV. The remaining cMV population was characterized with a multiplexed immunoassay against a panel of 20 different antigens. In addition to the expected depletion of the biomarker used to remove the targeted cMV in the remaining cMV population, a concomitant depletion of associated biomarkers was observed. In the case of the cMV population in which CD31 had been used to remove endothelial-derived cMV, for instance, there was a significant concomitant depletion of DLL4, a highly associated endothelial marker. This was not the case with general epithelial microvesicle markers, such as CD9, which were still very much present in the cMV population remaining after depletion. These findings suggest a potential method for a more complete characterization of biomarker profiles associated with cMV derived from disease-associated cells and provide the foundation for a novel cMV-based strategy for disease detection through a noninvasive blood test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2011-4060

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