John F. Stone

Development of a Prognostic Genetic Signature to Predict the Metastatic Risk Associated with Cutaneous Melanoma

Purpose: The development of a genetic signature for the identification of high-risk cutaneous melanoma tumors would provide a valuable prognostic tool with value for stage I and II patients who represent a remarkably heterogeneous group with a 3% to 55% chance of disease progression and death 5 years from diagnosis. Experimental Design: A prognostic 28-gene signature was identified by analysis of microarray expression data. Primary cutaneous melanoma tumor tissue was evaluated by RT-PCR for expression of the signature, and radial basis machine (RBM) modeling was performed to predict risk of metastasis. Results: RBM analysis of cutaneous melanoma tumor gene expression reports low risk (class 1) or high risk (class 2) of metastasis. Metastatic risk was predicted with high accuracy in development (ROC = 0.93) and validation (ROC = 0.91) cohorts of primary cutaneous melanoma tumor tissue. Kaplan–Meier analysis indicated that the 5-year disease-free survival (DFS) rates in the development set were 100% and 38% for predicted classes 1 and 2 cases, respectively (P < 0.0001). DFS rates for the validation set were 97% and 31% for predicted classes 1 and 2 cases, respectively (P < 0.0001). Gene expression profile (GEP), American Joint Committee on Cancer stage, Breslow thickness, ulceration, and age were independent predictors of metastatic risk according to Cox regression analysis. Conclusions: The GEP signature accurately predicts metastasis risk in a multicenter cohort of primary cutaneous melanoma tumors. Preliminary Cox regression analysis indicates that the signature is an independent predictor of metastasis risk in the cohort presented. Clin Cancer Res; 21(1); 175–83. ©2015 AACR.

Gene expression profiling for molecular staging of cutaneous melanoma in patients undergoing sentinel lymph node biopsy.

BACKGROUNDnA gene expression profile (GEP) test able to accurately identify risk of metastasis for patients with cutaneous melanoma has been clinically validated.nnnOBJECTIVEnWe aimed for assessment of the prognostic accuracy of GEP and sentinel lymph node biopsy (SLNB) tests, independently and in combination, in a multicenter cohort of 217 patients.nnnMETHODSnReverse transcription polymerase chain reaction (RT-PCR) was performed to assess the expression of 31 genes from primary melanoma tumors, and SLNB outcome was determined from clinical data. Prognostic accuracy of each test was determined using Kaplan-Meier and Cox regression analysis of disease-free, distant metastasis-free, and overall survivals.nnnRESULTSnGEP outcome was a more significant and better predictor of each end point in univariate and multivariate regression analysis, compared with SLNB (P < .0001 for all). In combination with SLNB, GEP improved prognostication. For patients with a GEP high-risk outcome and a negative SLNB result, Kaplan-Meier 5-year disease-free, distant metastasis-free, and overall survivals were 35%, 49%, and 54%, respectively.nnnLIMITATIONSnWithin the SLNB-negative cohort of patients, overall risk of metastatic events was higher (∼30%) than commonly found in the general population of patients with melanoma.nnnCONCLUSIONSnIn this study cohort, GEP was an objective tool that accurately predicted metastatic risk in SLNB-eligible patients.

A Gene Signature to Determine Metastatic Behavior in Thymomas

Purpose Thymoma represents one of the rarest of all malignancies. Stage and completeness of resection have been used to ascertain postoperative therapeutic strategies albeit with limited prognostic accuracy. A molecular classifier would be useful to improve the assessment of metastatic behaviour and optimize patient management. Methods qRT-PCR assay for 23 genes (19 test and four reference genes) was performed on multi-institutional archival primary thymomas (nu200a=u200a36). Gene expression levels were used to compute a signature, classifying tumors into classes 1 and 2, corresponding to low or high likelihood for metastases. The signature was validated in an independent multi-institutional cohort of patients (nu200a=u200a75). Results A nine-gene signature that can predict metastatic behavior of thymomas was developed and validated. Using radial basis machine modeling in the training set, 5-year and 10-year metastasis-free survival rates were 77% and 26% for predicted low (class 1) and high (class 2) risk of metastasis (Pu200a=u200a0.0047, log-rank), respectively. For the validation set, 5-year metastasis-free survival rates were 97% and 30% for predicted low- and high-risk patients (Pu200a=u200a0.0004, log-rank), respectively. The 5-year metastasis-free survival rates for the validation set were 49% and 41% for Masaoka stages I/II and III/IV (Pu200a=u200a0.0537, log-rank), respectively. In univariate and multivariate Cox models evaluating common prognostic factors for thymoma metastasis, the nine-gene signature was the only independent indicator of metastases (Pu200a=u200a0.036). Conclusion A nine-gene signature was established and validated which predicts the likelihood of metastasis more accurately than traditional staging. This further underscores the biologic determinants of the clinical course of thymoma and may improve patient management.

Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas

Background We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. Results Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. Conclusions PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Clinical Performance and Management Outcomes with the DecisionDx-UM Gene Expression Profile Test in a Prospective Multicenter Study

Uveal melanoma management is challenging due to its metastatic propensity. DecisionDx-UM is a prospectively validated molecular test that interrogates primary tumor biology to provide objective information about metastatic potential that can be used in determining appropriate patient care. To evaluate the continued clinical validity and utility of DecisionDx-UM, beginning March 2010, 70 patients were enrolled in a prospective, multicenter, IRB-approved study to document patient management differences and clinical outcomes associated with low-risk Class 1 and high-risk Class 2 results indicated by DecisionDx-UM testing. Thirty-seven patients in the prospective study were Class 1 and 33 were Class 2. Class 1 patients had 100% 3-year metastasis-free survival compared to 63% for Class 2 (log rank test p = 0.003) with 27.3 median follow-up months in this interim analysis. Class 2 patients received significantly higher-intensity monitoring and more oncology/clinical trial referrals compared to Class 1 patients (Fishers exact test p = 2.1 × 10−13 and p = 0.04, resp.). The results of this study provide additional, prospective evidence in an independent cohort of patients that Class 1 and Class 2 patients are managed according to the differential metastatic risk indicated by DecisionDx-UM. The trial is registered with Clinical Application of DecisionDx-UM Gene Expression Assay Results (NCT02376920).

Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics

BackgroundA 15-gene expression profile test has been clinically validated and is widely utilized in newly diagnosed uveal melanoma (UM) patients to assess metastatic potential of the tumor. As most patients are treated with eye-sparing radiotherapy, there is limited tumor tissue available for testing, and technical reliability and success of prognostic testing are critical. This study assessed the analytical performance of the 15-gene expression test for UM and the correlation of molecular class with pathologic characteristics.MethodsInter-assay, intra-assay, inter-instrument/operator, and inter-site experiments were conducted, and concordance of the 15-gene expression profile test results and associated discriminant scores for matched tumor samples were evaluated. Technical success was determined from de-identified clinical reports from January 2010 - May 2016. Pathologic characteristics of enucleated tumors were correlated with molecular class results.ResultsInter-assay concordance on 16 samples run on 3 consecutive days was 100%, and matched discriminant scores were strongly correlated (R2xa0=xa00.9944). Inter-assay concordance of 46 samples assayed within a one year period was 100%, with an R2 value of 0.9747 for the discriminant scores. Intra-assay concordance of 12 samples run concurrently in duplicates was 100%; discriminant score correlation yielded an R2 of 0.9934. Concordance between two sites assessing the same tumors was 100% with an R2 of 0.9818 between discriminant scores. Inter-operator/instrument concordance was 96% for Class 1/2 calls and 90% for Class 1A/1B calls, and the discriminant scores had a correlation R2 of 0.9636. Technical success was 96.3% on 5516 samples tested since 2010. Increased largest basal diameter and thickness were significantly associated with Class 1B and Class 2 vs. Class 1A signatures.ConclusionsThese results show that the 15-gene expression profile test for UM has robust, reproducible performance characteristics. The technical success rate during clinical testing remains as high as first reported during validation. As molecular testing becomes more prevalent for supporting precision medicine efforts, high technical success and reliability are key characteristics when testing such limited and precious samples. The performance of the 15-gene expression profile test in this study should provide confidence to physicians who use the test’s molecular classification to inform patient management decisions.

Validation of a Gene Expression Test for Mesothelioma Prognosis in Formalin-Fixed Paraffin-Embedded Tissues

A molecular test performed using fresh-frozen tissue was proposed for use in the prognosis of patients with pleural mesothelioma. The accuracy of the test and its properties was assessed under Clinical Laboratory Improvement Amendments-approved guidelines using FFPE tissue from an independent multicenter patient cohort. Concordance studies were performed using matched frozen and FFPE mesothelioma samples. The prognostic value of the test was evaluated in an independent validation cohort of 73 mesothelioma patients who underwent surgical resection. FFPE-based classification demonstrated overall high concordance (83%) with the matched frozen specimens, on removal of cases with low confidence scores, showing sensitivity and specificity in predicting type B classification (poor outcome) of 43% and 98%, respectively. Concordance between research and clinical methods increased to 87% on removal of low confidence cases. Median survival times in the validation cohort were 18 and 7 months in type A and type B cases, respectively (Pxa0= 0.002). Multivariate classification adding pathologic staging information to the gene expression score resulted in significant stratification of risk groups. The median survival times were 52 and 14 months in the low-risk (class 1) and intermediate-risk (class 2) groups, respectively. The prognostic molecular test for mesothelioma can be performed on FFPE tissues to predict survival, and can provide an orthogonal tool, in combination with established pathologic parameters, for risk evaluation.

Analytic validity of DecisionDx-Melanoma, a gene expression profile test for determining metastatic risk in melanoma patients

BackgroundThe DecisionDx-Melanoma test provides prognostic information for patients with cutaneous melanoma (CM). Using formalin-fixed paraffin-embedded primary tumor tissue, the RT-PCR-based test classifies patients into a low- (Class 1) or high-risk (Class 2) category for recurrence based on expression of 31 genes. The current study was designed to assess the analytical validity of this test.MethodsInter-assay, inter-instrument, and inter-operator studies were performed to evaluate reliability of the 31-gene expression test results, sample stability and reagent stability. From March 2013 through June 2016, the gene expression test was performed on 8244 CM tumors. De-identified data from Pathology Reports were used to assess technical success.ResultsRobust sample and reagent stability was observed. Inter-assay concordance on 168 specimens run on 2 consecutive days was 99% and matched probability scores were significantly correlated (R2u2009=u20090.96). Inter-instrument concordance was 95%, and probability scores had a correlation R2 of 0.99 (pu2009<u20090.001). From 8244 CM specimens submitted since 2013, 85% (7023) fulfilled pre-specified tumor content parameters. In these samples with sufficient tumor requirements, the technical success of the test was 98%.ConclusionDecisionDx-Melanoma is a robust gene expression profile test that demonstrates strong reproducibility between experiments and has high technical reliability on clinical samples.

Clinical Reliability and Reproducibility of a Prognostic 31-Gene Expression Profile Test for Cutaneous Melanoma, and Association of the Test with Standard Clinicopathologic Factors

Altman plot for 168 cases showing estimated bias (mean difference in discriminant scores, red line) and 95% confidence interval (dashed lines); C) instrument-to-instrument correlation analysis for 21 cases; D) BlandAltman plot for 21 cases showing estimated bias (mean difference in discriminant scores, red line) and 95% confidence interval (dashed lines) Background • The majority of metastases and death attributed to cutaneous melanoma (CM) occur in patients who are initially diagnosed with Stage I or Stage II disease. • A 31-gene expression profile (GEP) test that provides a molecular classification associated with risk of metastasis has been validated and clinically available since 2013. • The test determines a low risk (Class 1) or high risk (Class 2) of metastasis within five years of the primary diagnosis of CM with an area of reduced confidence identified from the true positives and negatives from the training set. • This study evaluated the analytical reliability and reproducibility of the 31-GEP test • We also report the technical experience of the test and the association of risk prediction with standard clinicopathologic factors linked to CM metastasis and death. Methods • Formalin-fixed paraffin-embedded tissue from primary melanoma tumors was successfully processed for 8,244 patients from 1,123 centers in the U.S. and Spain between March 2013 and June 2016 using the 31-GEP RT-PCR-based assay. • Metastatic risk class was determined using a proprietary predictive modeling algorithm which provides two results: a binary classification of Class 1 (low risk) or Class 2 (high-risk) tumor biology, and a quantitative discriminant score from 0 to 1.0, for which 0.5 represents the cutoff score between the binary classes. • Testing was repeated for a subset of the specimens to assess interassay variability and concordance of risk assignment. • Quality control and multiple gene failures were assessed, and pathology reports were evaluated for all specimens to evaluate association of the test results with clinical and pathologic characteristics of the samples.