TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

Validation of Circulating Tumor DNA Assays for Detection of Metastatic Melanoma.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

In preclinical studies, heregulin (HRG) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG-positive and HRG-negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes (HMBS, IPO8, and EIF2B1), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

Cytoskeleton alterations in melanoma: aberrant expression of cortactin, an actin-binding adapter protein, correlates with melanocytic tumor progression.

Cortactin is a multidomain actin-binding protein important for the functions of cytoskeleton by regulating cortical actin dynamics. It is involved in a diverse array of basic cellular functions. Tumorigenesis and tumor progression involves alterations in actin cytoskeleton proteins. We sought to study the role of cortactin in melanocytic tumor progression using immunohistochemistry on human tissues. The results reveal quantitative differences between benign and malignant lesions. Significantly higher cortactin expression is found in melanomas than in nevi (P<0.0001), with levels greater in metastatic than in invasive melanomas (P<0.05). Qualitatively, tumor tissues often show aberrant cortactin localization at the cell periphery, corresponding to its colocalization with filamentous actin in cell cortex of cultured melanoma cells. This suggests an additional level of protein dysregulation. Furthermore, in patients with metastatic disease, high-level cortactin expression correlates with poor disease-specific survival. Our data, in conjunction with outcome data on several other types of human cancers and experimental data from melanoma cell lines, supports a potential role of aberrant cortactin expression in melanoma tumor progression and a rational for targeting key elements of actin-signaling pathway for developmental therapeutics in melanomas.