Analytical Validation and Performance of a 7-Gene Next-Generation Sequencing Panel in Uveal Melanoma.

INTRODUCTION: Gene expression profiling (GEP) is widely used for prognostication in patients with uveal melanoma (UM). Because biopsy tissue is limited, it is critical to obtain as much genomic information as possible from each sample. Combined application of both GEP and next-generation sequencing (NGS) allows for analysis of RNA and DNA from a single biopsy sample, offers additional prognostic information, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of 7 genes commonly mutated in UM. METHODS: One hundred five primary UM tumors were analyzed, including 37 formalin-fixed paraffin-embedded (FFPE) and 68 fine-needle aspiration biopsy specimens. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of >500X per region of interest. RESULTS: The 7-gene panel achieved a positive percent agreement of 100% for detection of both single-nucleotide variants and insertions/deletions, with a technical positive predictive value of 98.8% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the assay's reproducibility and repeatability. DISCUSSION/CONCLUSION: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small-gauge needle biopsy sample or FFPE specimen.

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

BACKGROUND: The 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. METHODS: Inter-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. RESULTS: Robust 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 (R2 = 0.96). Inter-instrument concordance was 95%, and probability scores had a correlation R2 of 0.99 (p < 0.001). 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%. CONCLUSION: DecisionDx-Melanoma is a robust gene expression profile test that demonstrates strong reproducibility between experiments and has high technical reliability on clinical samples.

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

BACKGROUND: A 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. METHODS: Inter-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. RESULTS: Inter-assay concordance on 16 samples run on 3 consecutive days was 100%, and matched discriminant scores were strongly correlated (R2 = 0.9944). 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. CONCLUSIONS: These 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 (P = 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.

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.

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

BACKGROUND: A gene expression profile (GEP) test able to accurately identify risk of metastasis for patients with cutaneous melanoma has been clinically validated. OBJECTIVE: We 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. METHODS: Reverse 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. RESULTS: GEP 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. LIMITATIONS: Within 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. CONCLUSIONS: In this study cohort, GEP was an objective tool that accurately predicted metastatic risk in SLNB-eligible patients.

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.

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 (n = 36). 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 (n = 75). 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 (P = 0.0047, log-rank), respectively. For the validation set, 5-year metastasis-free survival rates were 97% and 30% for predicted low- and high-risk patients (P = 0.0004, 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 (P = 0.0537, 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 (P = 0.036). 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.

Resultados citogeneticos en adenocarcinoma de pancreas con la técnica convencional al GTG / Citogeneticos in pancreas adenocarcinoma with the conventional technique to the GTG

El cáncer de páncreas sigue siendo el causal de muerte debido a cáncer en los Estados Unidos. La enfermedad sigue un curso silencioso hasta que el paciente muestra un cuadro de ictericia, anorexia dolor abdominal y pérdida de peso entre otros síntomas. El simple hecho de que esta glándula sea relativamente inaccesible retroperitonealmente y por lo tanto, imposible de palpar clínicamente, hace que no se detecten anomalías en este órgano hasta etapas tardías, cuando en la mayoría de los casos ya ha involucrado estructuras adyacentes tales como estómago duodeno y conductos biliares. En el presente estudio se estudiaron citogenéticamente a seis líneas celulares pancreáticas, HS700t, HPAFIL, Su8686, HPAC, CFPAC-1 y 70259; siendo los rearreglos cromosómaticos clonales más comunes aquellos involucrando a los cromosomas 3,7,8,9,12,20,X. Las anomalías numéricas correspondieron a los cromosomas 1,3,5,7,9,11,12,16,21,-Y, resultados que corroboran hallazgos reportados en la literatura, pero nuevos estudios en tumores primarios de páncreas necesitan ser realizados para encontrar los cambios cromosómicos específicos a este tipo de neoplasia.

Identificación de un nuevo amplicón en el cromosoma 1q31 en cáncer de páncreas / Identification of a amplicon new in the chromosome 1q31 in pancreatic neoplasms

El adenocarcinoma de páncreas es un tumor altamente maligno que está incrementando en frecuencia y está situado actualmente en el quinto lugar como causa de muerte atribuida a cáncer en los EE. UU. Los pacientes con carcinoma pancreático tienen uno de los peores pronósticos dentro de los pacientes con cáncer, con un porcentaje de muertes de aproximadamente 75 por ciento del número total de casos. El uso de la libridación comparativa de los genomas (CGH), una nueva técnica de FISH, ha ganado uso ampliamente como una gran herramienta de diagnóstico y pronóstico en este tipo de carcinogénesis en donde solamente algunos casos han sido estudiados citogenéticamente, debido al hecho de que estos tumores son muy difíciles de cultivar "in vitro". Hemos examinado 14 líneas celulares pancreáticas con CGH. En 11 de ellas hemos encontrado un amplicón en el cromosoma 1q31, el cual no ha sido reportado previamente en cáncer de páncreas. Se necesita realizar estos estudios en tumores primarios para determinar el rol de 1q31 en este tipo de carcinogénesis.

Hallazgos de regiones genomicas amplificadas en cancer de pamcreas a traves de una nueva técnica de fish: pintado al revés de los cromosomas / Discoveries of regions genomicas amplified in pancreas cancer through a new fisch technique: painted the other way around of the chromosomesDiscoveries of regions genomicas amplified in pancreas cancer through a new fisch technique: painted the other way around of the chromosomes

Una nueva técnica de FISH, llamada "Reverse Chromosome Painting" (Pintado al revés de los cromosomas) está siendo usada en algunos tumores en donde existen problemas tales como la falta de metafases de buena calidad, problemas en el cultivo de tumores primarios, bajo indice mitótico y problemas de contaminación, etc. Los tumores de páncreas pertenecen a este grupo, son muy muy dificiles de cultuvar "in vitro", lo cual minimiza la información citogenética disponible en este tipo de cáncer. Esta nueva técnica utiliza el ADN tumoral como (sonda) para una (hibridación) supresora in situ en metafases normales de un control varón (96,XY). Las secuencias de ADN que se encuentran amplificadas muestran unas señales específicas llamados Amplicones, revelando además la posición de éstos en los cromosomas normales. En el presente estadio, 14 lineas celulares han sido analizados con esta nueva técnica de FISH y nuestros resultados muestran los siguientes amplicones: 1p36.1-pter, 1q21.1, 1q31, en 5p y 12pter, los cuales son específicos para estas líneas celulares pancreáticos. Estudios posteriores en tumores primarios nos permitirán corroborar los resultados presente.

Uso de la técnica: pintado de todo el cromosoma (Whole chromosome painting), para identificar cromosomas marcadores en líneas celulares pancreáticas / I use of the technique: colored of the whole chromosome (Whole chromosome paintin), to identify chromosomes markers in pancreatic cellular lines

La hibridación in situ con fluorescencia, es un método altamente sensible para la detección de secuencias de ADN específicas en metafases preparadas en una lámina portaobjetos. Las aplicaciones de esta técnica de Fish se ven especialmente en la identificación de cromosomas marcadores en metafases de tumores sólidos y malignidades hematológicas. Los cromosomas marcadores son generalmente de origen no conocido y por lo tanto de significado clínico desconocido. La técnica denominada "Whole chromosoma paintin" o pintado de todo el cromosoma es una variante de la técnica de Fish que permite la identificación de cromosomas humanas o fragmentos de estos cromosomas no identificados con bandeo convencional. Este nuevo sistema íncluye una sonda que contiene secuencia única homólogas al ADN del cromosoma que deseamos identificar. Dicha sonda es marcada directamente con espectro o fluorócromo naranja o verde. Esta técnica ha tenido un gran impacto y como otra de las múltiples técnicas de Fish se ha convertido en una herramienta importante, en citogenética de tumores sólidos, en donde el progreso siempre ha sido bloqueado por dificultades técnicas como el obtener metafases de muy buena calidad, por la falta de crecimiento de algunos tumores y la contaminación de células normales con el parénquina tumoral. Sin embargo, esta técnica "Whole chromosoma painting" requiere metafases de alta calidad. Usamos en el presente estudio, sondas ONCOR para identificar cromosomas completos. Las sondas son desnaturalizadas e híbridadas a una metafase del tumor que se encuentra en una lámina portaobjetos. Después que se completa la hibridación , el exceso de sonda es removido mediante lavados. La sonda WCP es detectada por visualización con filtros especiales para espectros verde o naranja en un microscopio de fluorescencia. En el presente estudio, usamos esta técnica para identificar a algunos cromosomas en cierta líneas celulares pancreáticas: Pan-1, UACC462, HPAC, Hs700t, Mia Parca2. Nuestros resultados, corroboran estudios previso realizados en nuestro laboratorio, usando bandeo G y FISH convencional con sondas centroméricas.