Myriam Loyo

PD-L1 Expression in the Merkel Cell Carcinoma Microenvironment: Association with Inflammation, Merkel Cell Polyomavirus, and Overall Survival

Using paraffin-embedded specimens from 49 patients diagnosed with various stages of Merkel cell carcinoma (MCC), Lipson and colleagues found PD-L1 expression in approximately 50% of these rare tumors. PD-L1+ carcinomas were invariably associated with immune infiltrates and the presence of Merkel cell polyomavirus DNA. These findings suggest that an endogenous immune response, perhaps directed in part to MCC-related antigen, promotes PD-L1 expression in the tumor microenvironment and provide a rationale for investigating therapies blocking PD-1/PD-L1 for patients with MCC. Merkel cell carcinoma (MCC) is a lethal, virus-associated cancer that lacks effective therapies for advanced disease. Agents blocking the PD-1/PD-L1 pathway have shown objective, durable tumor regressions in patients with advanced solid malignancies and efficacy has been linked to PD-L1 expression in the tumor microenvironment. To investigate whether MCC might be a target for PD-1/PD-L1 blockade, we examined MCC PD-L1 expression, its association with tumor-infiltrating lymphocytes (TIL), Merkel cell polyomavirus (MCPyV), and overall survival. Sixty-seven MCC specimens from 49 patients were assessed with immunohistochemistry for PD-L1 expression by tumor cells and TILs, and immune infiltrates were characterized phenotypically. Tumor cell and TIL PD-L1 expression were observed in 49% and 55% of patients, respectively. In specimens with PD-L1(+) tumor cells, 97% (28/29) showed a geographic association with immune infiltrates. Among specimens with moderate-severe TIL intensities, 100% (29/29) showed PD-L1 expression by tumor cells. Significant associations were also observed between the presence of MCPyV DNA, a brisk inflammatory response, and tumor cell PD-L1 expression: MCPyV(-) tumor cells were uniformly PD-L1(-). Taken together, these findings suggest that a local tumor-specific and potentially MCPyV-specific immune response drives tumor PD-L1 expression, similar to previous observations in melanoma and head and neck squamous cell carcinomas. In multivariate analyses, PD-L1(-) MCCs were independently associated with worse overall survival [HR 3.12; 95% confidence interval, 1.28–7.61; P = 0.012]. These findings suggest that an endogenous immune response promotes PD-L1 expression in the MCC microenvironment when MCPyV is present, and provide a rationale for investigating therapies blocking PD-1/PD-L1 for patients with MCC. Cancer Immunol Res; 1(1); 54–63. ©2013 AACR.

Quantitative detection of Merkel cell virus in human tissues and possible mode of transmission.

Merkel Cell Virus (MCV) is a newly discovered polyomavirus, recently found in a rare skin cancer, Merkel cell carcinoma (MCC). However, MCV has also been detected in some normal tissue samples. We tested and compared the relative quantity of the MCV in a set of diverse human tissue samples with the MCC samples. The levels of MCV in MCCs were over 60 times higher than the highest values in all other tissues. Low quantities of MCV were detected in diverse tissue samples independently of malignant or benign histologic status. Higher levels of the virus were found in the upper aerodigestive tract, digestive system, and saliva compared to the lung and genitourinary system samples. These results confirm that MCV is widespread in the human body and suggest a possible fecal‐oral transmission route similar to the Hepatitis A virus. Despite widespread presence of the virus, it appears that only neuroendocrine skin cells are susceptible to transformation by MCV.

Activation of the NOTCH pathway in Head and Neck Cancer

NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation.

Background Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. Methodology/Principal Findings Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing. Conclusions/Significance Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.

Molecular Analysis of Plasma DNA for the Early Detection of Lung Cancer by Quantitative Methylation Specific PCR

Purpose: Aberrant promoter hypermethylation of tumor suppressor genes is a promising marker for lung cancer detection. We investigated the likelihood of detecting aberrant DNA methylation of tumor suppressor genes in plasma samples of patients with abnormalities of the lung detected upon computed tomography (CT) scan. Experimental Design: In a small evaluation cohort, four gene promoters (DCC, Kif1a, NISCH, and Rarb) were found to be methylated with increased frequency in samples from cancer patients specifically. We then examined DNA from 93 plasma samples from patients with abnormal findings in the lung detected upon CT scan for aberrant methylation of these four gene promoters by quantitative fluorogenic real-time PCR. The patients were divided into two groups, ground glass opacity (n = 23) and cancerous tumors (n = 70). Plasma DNA from age-matched nodule-free individuals were used as controls (n = 80). Results: In plasma, 73% of patients with cancerous tumors showed methylation of at least one gene with a specificity of 71% (P = 0.0001). Only 22% patients with ground glass opacity exhibited methylation of at least one gene. When smoking history was taken into account, 72% of cancer patients with no smoking history or those who smoked <20 pack-years showed methylation of at least one gene with 100% specificity (P = 0.05) when compared with matched controls. Among heavy smokers with 20+ pack-years of smoking history, 30% of the control group and 73% of the patients with cancerous tumors showed methylation (P = 0.0001). Conclusions: These biomarkers can distinguish between cancerous and noncancerous abnormal CT findings. Clin Cancer Res; 16(13); 3463–72. ©2010 AACR.

National trends in thyroid surgery and the effect of volume on short-term outcomes.

To characterize contemporary patterns of thyroid surgical care and the effect of volume status on surgical care and short‐term outcomes.

Association between Lifestyle Factors and CpG Island Methylation in a Cancer-Free Population

Background: Many risk factors have been associated with cancer, such as age, family history, race, smoking, high-fat diet, and poor nutrition. It is important to reveal the molecular changes related to risk factors that could facilitate early detection, prevention, and overall control of cancer. Methods: We selected six cancer-specific methylated genes that have previously been reported in primary tumors and have also been detected in different bodily fluids of cancer patients. Here, we used quantitative fluorogenic real-time methylation-specific PCR in plasma DNA samples for the detection of methylation changes from an asymptomatic population who do not have any known cancer. Results: The promoter methylation frequencies of the studied genes were as follows: APC (7%), CCND2 (22%), GSTP1 (2%), MGMT (9%), RARβ2 (29%), and P16 (3%). Promoter methylation of at least one of the genes analyzed was observed in ∼46% (72 of 157) of the samples by binary dichotomization. Promoter hypermethylation of at least two genes was detected in 17% (26 of 157) of the samples. RARβ2 methylation was observed in 45% of subjects who had a high-fat diet in contrast with those who had a low-fat diet (23%; P = 0.007). Discussion: Our findings may help to elucidate early methylation changes that may lead to cancer development. These methylation changes could be due to exposure to risk factors and may be useful for cancer prevention measures such as changes in lifestyle. Longitudinal follow-up of a high-risk population is needed to understand the association of methylation of candidate genes in cancer development. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2984–91)

Endothelin receptor type B gene promoter hypermethylation in salivary rinses is independently associated with risk of oral cavity cancer and premalignancy.

Endothelin receptor type B (EDNRB) and kinesin family member 1A (KIF1A) are candidate tumor suppressor genes that are inactivated in cancers. In this study, we evaluated the promoter hypermethylation of EDNRB and KIF1A and their potential use for risk classification in prospectively collected salivary rinses from patients with premalignant/malignant oral cavity lesions. Quantitative methylation-specific PCR was performed to analyze the methylation status of EDNRB and KIF1A in salivary rinses of 191 patients. We proceeded to determine the association of methylation status with histologic diagnosis and estimate classification accuracy. On univariate analysis, diagnosis of dysplasia/cancer was associated with age and KIF1A or EDNRB methylation. Methylation of EDNRB highly correlated with that of KIF1A (P < 0.0001). On multivariable modeling, histologic diagnosis was independently associated with EDNRB (P = 0.0003) or KIF1A (P = 0.027) methylation. A subset of patients analyzed (n = 161) without prior biopsy-proven malignancy received clinical risk classification based on examination. On univariate analysis, EDNRB and risk classification were associated with diagnosis of dysplasia/cancer and remained significant on multivariate analysis (EDNRB: P = 0.047, risk classification: P = 0.008). Clinical risk classification identified dysplasia/cancer with a sensitivity of 71% and a specificity of 58%. The sensitivity of clinical risk classification combined with EDNRB methylation improved to 75%. EDNRB methylation in salivary rinses was independently associated with histologic diagnosis of premalignancy and malignancy and may have potential in classifying patients at risk for oral premalignant and malignant lesions in settings without access to a skilled dental practitioner. This may also potentially identify patients with premalignant and malignant lesions that do not meet the criteria for high clinical risk based on skilled dental examination. Cancer Prev Res; 3(9); 1093–103. ©2010 AACR.

A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In our study, we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumors biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation‐specific polymerase chain reaction (PCR) in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fishers exact test and quantitative values were optimized using cut off values derived from receiver–operator characteristic curves. The methylation status of AIM1, APC, CALCA, deleted in colorectal carcinomas (DCC), DLEC, deleted in liver cancer 1 (DLC1), estrogen receptor alpha (ESR), FHIT, KIF1A and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.