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Background: To improve identification of patients with cutaneous squamous cell carcinoma (SCC) at high risk for metastatic disease, the DecisionDx-SCC assay, a prognostic 40-gene expression profile (40-GEP) test, was developed and validated. The 40-GEP assay utilizes RT-PCR gene expression analysis on primary tumor biopsy tissue to evaluate the expression of 34 signature gene targets and 6 normalization genes. The test provides classifications of low risk (Class 1), moderate risk (Class 2A), and high risk (Class 2B) of metastasis within 3 years of diagnosis. The primary objective of this study was to validate the analytical performance of the 40-gene expression signature., Methods: The repeatability and reproducibility of the 40-GEP test was evaluated by performance of inter-assay, intra-assay, and inter-operator precision experiments along with monitoring the reliability of sample and reagent stability for class call concordance. The technical performance of clinical orders from September 2020 through July 2021 for the 40-GEP test was assessed., Results: Patient hematoxylin and eosin (H&E) stained slides were reviewed by a board-certified pathologist to assess minimum acceptable tumor content. Class specific controls (Class 1 and Class 2B) were evaluated with Levey-Jennings analysis and demonstrated consistent and reproducible results. Inter-assay, inter-operator and intra-assay concordance were all ≥90%, with short-term and long-term RNA stability also meeting minimum concordance requirements. Of the 2586 orders received, 93.5% remained eligible for testing, with 97.1% of all tested samples demonstrating actionable class call results., Conclusion: DecisionDx-SCC demonstrates a high degree of analytical precision, yielding high concordance rates across multiple performance experiments, along with exhibiting robust technical reliability on clinical samples., (© 2022. The Author(s).)

Sentinel lymph node (SLN) biopsy should be performed with the technical expertise required to correctly identify the sentinel node, in the context of understanding both the likelihood of positivity in a given patient and the prognostic significance of a positive or negative result. National Comprehensive Cancer Network guidelines recommend SLN biopsy for all cutaneous melanoma patients with primary tumor thickness greater than 1 mm and in select patients with thickness between 0.8 and 1 mm, yet admit a lack of consistent clarity in its utility for prognosis and therapeutic value in tumors < 1 mm and leave the decision for undergoing the procedure up to the patient and treating physician. Recent studies have evaluated specific patient populations, tumor histopathologic characteristics, and gene expression profiling and their use in predicting SLN positivity. These data have given insight into improving the physician's ability to potentially predict SLN positivity, shedding light on if and when omission of SLN biopsy in specific patients based on clinicopathological characteristics might be appropriate. This review provides discussion and insight into these recent advancements., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: K.M.A., L.M.S., K.R.C., S.M.A., J.K.W., J.F.S., S.B., K.M.O., F.A.M., and R.W.C. were employees and stockholders of Castle Biosciences, Inc. at the time of this study. J.W.H. is the inventor of intellectual property related to prognostic testing for uveal melanoma. He is a paid consultant for Castle Biosciences, Inc., Friendswood, TX, USA licensee of this intellectual property, and he receives royalties from its commercialization., (Copyright © 2021 by S. Karger AG, Basel.)

National guidelines recommend sentinel lymph node biopsy (SLNB) be offered to patients with > 10% likelihood of sentinel lymph node (SLN) positivity. On the other hand, guidelines do not recommend SLNB for patients with T1a tumors without high-risk features who have < 5% likelihood of a positive SLN. However, the decision to perform SLNB is less certain for patients with higher-risk T1 melanomas in which a positive node is expected 5%-10% of the time. We hypothesized that integrating clinicopathologic features with the 31-gene expression profile (31-GEP) score using advanced artificial intelligence techniques would provide more precise SLN risk prediction., Methods: An integrated 31-GEP (i31-GEP) neural network algorithm incorporating clinicopathologic features with the continuous 31-GEP score was developed using a previously reported patient cohort (n = 1,398) and validated using an independent cohort (n = 1,674)., Results: Compared with other covariates in the i31-GEP, the continuous 31-GEP score had the largest likelihood ratio (G 2 = 91.3, P < .001) for predicting SLN positivity. The i31-GEP demonstrated high concordance between predicted and observed SLN positivity rates (linear regression slope = 0.999). The i31-GEP increased the percentage of patients with T1-T4 tumors predicted to have < 5% SLN-positive likelihood from 8.5% to 27.7% with a negative predictive value of 98%. Importantly, for patients with T1 tumors originally classified with a likelihood of SLN positivity of 5%-10%, the i31-GEP reclassified 63% of cases as having < 5% or > 10% likelihood of positive SLN, for a more precise, personalized, and clinically actionable SLN-positive likelihood estimate., Conclusion: These data suggest the i31-GEP could reduce the number of SLNBs performed by identifying patients with likelihood under the 5% threshold for performance of SLNB and improve the yield of positive SLNBs by identifying patients more likely to have a positive SLNB., Competing Interests: John T. Vetto Employment: Gilead Sciences (I) Stock and Other Ownership Interests: Gilead Sciences (I), Roche/Genentech (I) Speakers' Bureau: Castle Biosciences Travel, Accommodations, Expenses: Castle Biosciences No other potential conflicts of interest were reported. John T. Vetto Employment: Gilead Sciences (I) Stock and Other Ownership Interests: Gilead Sciences (I), Roche/Genentech (I) Speakers' Bureau: Castle Biosciences Travel, Accommodations, Expenses: Castle Biosciences No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Competing Interests: Competing interest statement: C.L.S. is employed through Torreyana Corp, San Diego, CA; Blackhawk Genomics, Concord, CA; and Advagenix, Rockville, MD. Volunteer activities of C.L.S. include those for College of American Pathologists, Northfield, IL, and Clinical Laboratory Standards Institute, Wayne, PA. T.J.O. is a Member, Scientific Advisory Committee, MioDx and Integrated Nano-Technologies. A.W.I.L.’s laboratory receives sponsorships from AstraZeneca (HK) Ltd. and MSD (HK) Ltd. for providing selected companion diagnostic tests free to public patients. N.C. is an employee at Quest Diagnostics, Inc. F.A.M. is an employee and stock option holder at Castle Biosciences, Inc. A.B.C. is paid teaching faculty for the American Medical Informatics Association Clinical Informatics Board Review Course and receives small honoraria as well as travel reimbursement to speak at multiple scientific and professional medical society meetings. T.L.P. has been consulted (compensated) for Bio-Rad, Inc. and is Director of Pathology Strategies for the Sturge Weber Foundation (compensated). H.E.W. has employment through Viapath, a majority National Health Service-owned independent pathology service provider; has been a paid faculty member at Kingston University, accepted paid accommodation and subsistence as an invited speaker to Cytocell User Group meeting for the United Kingdom and Ireland, and accepted paid event registration as an invited speaker to Digital Pathology/Global Engage meeting. H.E.W.’s laboratory received scholarship funds from The International Council for Standardization in Haematology for providing JAK2 testing. The other authors (K.F., E.A.B., P.M., N.R.P., K.P.P., B.S., M.S., M.L.G., S.M.M., H.F., and S.O.) do not have any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this article.

Introduction: The prognostic 15-gene expression profile (15-GEP) test for uveal melanoma (UM) predicts metastatic risk based on primary tumor biology. Here we report outcomes from a prospective registry of 15-GEP-tested patients, and a meta-analysis with published cohorts., Objectives: Management and 5-year clinical outcomes following 15-GEP testing were evaluated., Methods: Eighty-nine patients with 15-GEP results were prospectively enrolled at four centers. Physician-recommended management plans were collected, and clinical outcomes tracked every 6 months., Results: Eighty percent of Class 1 (low-risk) patients underwent low-intensity management; all Class 2 (high-risk) patients underwent high-intensity management ( p < 0.0001). Median follow-up for event-free patients was 4.9 years. Five Class 1 (10%) and 23 Class 2 (58%) tumors metastasized ( p < 0.0001). Five-year Class 1 and 2 metastasis-free survival rates were 90% (81-100%) and 41% (27-62%; p < 0.0001), and melanoma-specific survival rates were 94% (87-100%) and 63% (49-82%; p = 0.0007). Class 2 was the only independent predictor of metastasis and was associated with increased risk for metastasis and mortality by meta-analysis., Conclusions: UM patient management is guided by 15-GEP testing. Class 2 patients were managed more intensely, in accordance with an observed metastatic rate of >50%; Class 1 patients were safely spared intensive surveillance, resulting in appropriate utilization of healthcare resources., Competing Interests: K.R.C., K.M.P., K.M.A., K.M.O., and F.A.M. are employees and shareholders of Castle Biosciences, Inc., (Copyright © 2020 by S. Karger AG, Basel.)

Aim: The Clinical Application of DecisionDx-UM Gene Expression Assay Results study aimed to evaluate the clinical utility of the prognostic 15-gene expression profile (15-GEP) test for uveal melanoma (UM) patients in a large, prospective multicenter cohort., Patients & Methods: Nine centers prospectively enrolled 138 UM patients clinically tested with the 15-GEP. Physician-recommended specialty referrals and metastatic surveillance regimens were collected., Results: A total of 93% of high-risk class 2 patients were referred to medical oncology for follow-up, compared with 51% of class 1 patients. A majority (62%) of class 2 patients were recommended overall high-intensity metastatic surveillance, while 85% of class 1 patients were recommended low-intensity metastatic surveillance., Conclusion: Treatment plan recommendations for UM patients are aligned with GEP-informed metastatic risk, consistent with prior studies., Competing Interests: Financial & competing interests disclosure Financial support/sponsorship for this study was provided by Castle Biosciences, Inc. The following authors are/were employees of Castle at the time of study enrollment: KM Plasseraud, KM Alsina and FA Monzon. AC Schefler has provided consulting services for Castle Biosciences. This work was also supported by the National Eye Institute grant NIH/NEI 5K08EY027464-02 [AB Daniels] and by a Career Development Award from the Research to Prevent Blindness Foundation [AB Daniels]. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript., (© 2020 Castle Biosciences, Inc.)

Aim: Can gene expression profiling be used to identify patients with T1-T2 melanoma at low risk for sentinel lymph node (SLN) positivity?, Patients & Methods: Bioinformatics modeling determined a population in which a 31-gene expression profile test predicted <5% SLN positivity. Multicenter, prospectively-tested (n = 1421) and retrospective (n = 690) cohorts were used for validation and outcomes, respectively., Results: Patients 55-64 years and ≥65 years with a class 1A (low-risk) profile had SLN positivity rates of 4.9% and 1.6%. Class 2B (high-risk) patients had SLN positivity rates of 30.8% and 11.9%. Melanoma-specific survival was 99.3% for patients ≥55 years with class 1A, T1-T2 tumors and 55.0% for class 2B, SLN-positive, T1-T2 tumors., Conclusion: The 31-gene expression profile test identifies patients who could potentially avoid SLN biopsy.

Background: Approximately 10% of pancreatic adenocarcinoma (PC) cases are attributed to hereditary causes. Individuals with PC and a personal history of another cancer associated with hereditary breast and ovarian cancer (HBOC) or Lynch syndrome (LS) may be more likely to carry germline mutations., Methods: Participants with PC and a history of cancer were selected from a pancreatic disease registry. Of 1296 individuals with PC, 149 had a relevant history of cancer. If banked DNA was available, a multigene panel was performed for individuals who had not 1) previously had a mutation identified through clinical testing or 2) undergone clinical multigene panel testing with no mutations detected., Results: Twenty-two of 124 individuals with PC and another HBOC- or LS-related cancer who underwent genetic testing had a mutation identified in a PC susceptibility gene (18%). If prostate cancer is excluded, the mutation prevalence increased to 23% (21/93). Mutation carriers were more likely to have more than 1 previous cancer diagnosis (P = .001), to have had clinical genetic testing (P = .001), and to meet National Comprehensive Cancer Network (NCCN) genetic testing criteria (P < .001). Approximately 23% of mutation carriers did not meet NCCN HBOC or LS testing guidelines based on their personal cancer history and reported cancer history in first-degree relatives., Conclusion: At least 18% of individuals with PC and a personal history of other HBOC- or LS-related cancers carry mutations in a PC susceptibility gene based on our data, suggesting that criteria for genetic testing in individuals with PC should include consideration of previous cancer history. Cancer 2018;124:1691-700. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

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 (R 2  = 0.96). Inter-instrument concordance was 95%, and probability scores had a correlation R 2 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.

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 (R 2  = 0.9944). Inter-assay concordance of 46 samples assayed within a one year period was 100%, with an R 2 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 R 2 of 0.9934. Concordance between two sites assessing the same tumors was 100% with an R 2 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 R 2 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.

Purpose Molecular testing of colorectal cancers (CRCs) to improve patient care and outcomes of targeted and conventional therapies has been the center of many recent studies, including clinical trials. Evidence-based recommendations for the molecular testing of CRC tissues to guide epidermal growth factor receptor (EGFR) -targeted therapies and conventional chemotherapy regimens are warranted in clinical practice. The purpose of this guideline is to develop evidence-based recommendations to help establish standard molecular biomarker testing for CRC through a systematic review of the literature. Methods The American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and the American Society of Clinical Oncology (ASCO) convened an Expert Panel to develop an evidence-based guideline to help establish standard molecular biomarker testing, guide targeted therapies, and advance personalized care for patients with CRC. A comprehensive literature search that included over 4,000 articles was conducted to gather data to inform this guideline. Results Twenty-one guideline statements (eight recommendations, 10 expert consensus opinions and three no recommendations) were established. Recommendations Evidence supports mutational testing for genes in the EGFR signaling pathway, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize molecular testing for predictive and prognostic molecular biomarkers involve selection of assays, type of specimens to be tested, timing of ordering of tests and turnaround time for testing results. Additional information is available at: www.asco.org/CRC-markers-guideline and www.asco.org/guidelineswiki.

Objectives: - To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens., Methods: - The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted., Results: - Twenty-one guideline statements were established., Conclusions: - Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

This Editorial provides readers additional insight on the colorectal guideline appearing in this issue., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. Methods: The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. Results: Twenty-one guideline statements were established. Conclusions: Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

Objective: DecisionDx-Melanoma * is a 31-gene expression profile test that predicts the risk of metastasis in patients with primary cutaneous melanoma (CM). This study was designed to ascertain clinical management changes determined by the test outcome, which classifies CM patients being at low (Class 1) or high (Class 2) risk for recurrence., Research Design and Methods: Medical charts were reviewed from 156 CM patients from six institutions (three dermatology and three surgical oncology practices) who were consecutively tested between May 2013 and December 2015. Clinical management data that were compiled and compared before and after receipt of the 31-gene expression test result included frequency of physical exams, frequency and modality of imaging, and referrals to surgical and medical oncologists., Results: Forty-two percent of patients were Stage I, 47% were Stage II and 8% were Stage III. Overall, 95 patients (61%) were Class 1 and 61 (39%) were Class 2. Documented changes in management were observed in 82 (53%) patients, with the majority of Class 2 patients (77%) undergoing management changes compared to 37% of Class 1 patients (p < 0.0001 by Fisher's exact test). The majority (77/82, 94%) of these changes were concordant with the risk indicated by the test result (p < 0.0001 by Fisher's exact test), with increased management intensity for Class 2 patients and reduced management intensity for Class 1 patients., Conclusions: Molecular risk classification by gene expression profiling has clinical impact and influences physicians to direct clinical management of CM patients. The vast majority of the changes implemented after the receipt of test results were reflective of the low or high recurrence risk associated with the patient's molecular classification. Because follow-up data was not collected for this patient cohort, the study is limited for the assessment of the impact of gene expression profile based management changes on healthcare resource utilization and patient outcome.

Importance: Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown., Objective: To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors., Design: Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children's hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record., Main Outcomes and Measures: Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations., Results: Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%)., Conclusions and Relevance: Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Mutations in SETD2, a histone H3 lysine trimethyltransferase, have been identified in clear cell renal cell carcinoma (ccRCC); however it is unclear if loss of SETD2 function alters the genomic distribution of histone 3 lysine 36 trimethylation (H3K36me3) in ccRCC. Furthermore, published epigenomic profiles are not specific to H3K36me3 or metastatic tumors. To determine if progressive SETD2 and H3K36me3 dysregulation occurs in metastatic tumors, H3K36me3, SETD2 copy number (CN) or SETD2 mRNA abundance was assessed in two independent cohorts: metastatic ccRCC (n=71) and the Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma data set (n=413). Although SETD2 CN loss occurs with high frequency (>90%), H3K36me3 is not significantly impacted by monoallelic loss of SETD2. H3K36me3-positive nuclei were reduced an average of ~20% in primary ccRCC (90% positive nuclei in uninvolved vs 70% positive nuclei in ccRCC) and reduced by ~60% in metastases (90% positive in uninvolved kidney vs 30% positive in metastases) (P<0.001). To define a kidney-specific H3K36me3 profile, we generated genome-wide H3K36me3 profiles from four cytoreductive nephrectomies and SETD2 isogenic renal cell carcinoma (RCC) cell lines using chromatin immunoprecipitation coupled with high-throughput DNA sequencing and RNA sequencing. SETD2 loss of methyltransferase activity leads to regional alterations of H3K36me3 associated with aberrant RNA splicing in a SETD2 mutant RCC and SETD2 knockout cell line. These data suggest that during progression of ccRCC, a decline in H3K36me3 is observed in distant metastases, and regional H3K36me3 alterations influence alternative splicing in ccRCC.

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gene panels for hereditary breast and ovarian cancer risk assessment are gaining acceptance, even though the clinical utility of these panels is not yet fully defined. Technical questions remain, however, about the performance and clinical interpretation of gene panels in comparison with traditional tests. We tested 1105 individuals using a 29-gene next-generation sequencing panel and observed 100% analytical concordance with traditional and reference data on >750 comparable variants. These 750 variants included technically challenging classes of sequence and copy number variation that together represent a significant fraction (13.4%) of the pathogenic variants observed. For BRCA1 and BRCA2, we also compared variant interpretations in traditional reports to those produced using only non-proprietary resources and following criteria based on recent (2015) guidelines. We observed 99.8% net report concordance, albeit with a slightly higher variant of uncertain significance rate. In 4.5% of BRCA-negative cases, we uncovered pathogenic variants in other genes, which appear clinically relevant. Previously unseen variants requiring interpretation accumulated rapidly, even after 1000 individuals had been tested. We conclude that next-generation sequencing panel testing can provide results highly comparable to traditional testing and can uncover potentially actionable findings that may be otherwise missed. Challenges remain for the broad adoption of panel tests, some of which will be addressed by the accumulation of large public databases of annotated clinical variants., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity., (©2014 American Association for Cancer Research.)

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage and easily-manipulated non-pathogenic viruses as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylated affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of Vascular Endothelial Growth Factor, a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage fluid.

Purpose: Translocation renal cell carcinoma (tRCC) is a rare subtype of kidney cancer involving the TFEB/TFE3 genes. We aimed to investigate the genomic and epigenetic features of this entity., Experimental Design: Cytogenomic analysis was conducted with 250K single-nucleotide polymorphism microarrays on 16 tumor specimens and four cell lines. LINE-1 methylation, a surrogate marker of DNA methylation, was conducted on 27 cases using pyrosequencing., Results: tRCC showed cytogenomic heterogeneity, with 31.2% and 18.7% of cases presenting similarities with clear-cell and papillary RCC profiles, respectively. The most common alteration was a 17q gain in seven tumors (44%), followed by a 9p loss in six cases (37%). Less frequent were losses of 3p and 17p in five cases (31%) each. Patients with 17q gain were older (P=0.0006), displayed more genetic alterations (P<0.003), and had a worse outcome (P=0.002) than patients without it. Analysis comparing gene-expression profiling of a subset of tumors bearing 17q gain and those without suggest large-scale dosage effects and TP53 haploinsufficiency without any somatic TP53 mutation identified. Cell line-based cytogenetic studies revealed that 17q gain can be related to isochromosome 17 and/or to multiple translocations occurring around 17q breakpoints. Finally, LINE-1 methylation was lower in tRCC tumors from adults compared with tumors from young patients (71.1% vs. 76.7%; P=0.02)., Conclusions: Our results reveal genomic heterogeneity of tRCC with similarities to other renal tumor subtypes and raise important questions about the role of TFEB/TFE3 translocations and other chromosomal imbalances in tRCC biology., (©2013 AACR.)

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

Metachronous bilateral renal cell carcinomas (RCCs) are rare but well known. We present a case of metachronous bilateral RCCs with a ureter orifice metastasis, for which the pathological diagnosis was confirmed with single nucleotide polymorphism microarray (SNP-M) and gene expression assay (GEA). A 53-year-old man presented with a right ureteral obstruction. A cystoscopy showed a large pedunculated tumor emanating from the right ureteral orifice, consistent with a drop metastasis, which was biopsied. He had a history of a clear cell RCC (ccRCC) 1.5 years prior and a right renal pelvic mass found 8 months later. Histologically, the biopsied right ureteral tumor demonstrated sheets of poorly differentiated cancer cells composed of a mixture of spindled and focal clear cell components. The main differential diagnosis was metastatic RCC versus urothelial carcinoma, but the immunohistochemical profile was not contributory. SNP-M revealed a genomic profile consistent with a metastatic ccRCC with loss of chromosome 3p. GEA showed a gene expression pattern consistent with kidney origin. The cytogenomic array also identified chromosome copy number patterns that were shared between both kidney tumors. This finding suggests that both tumors had a common origin, and thus, the metachronous ccRCC in the contralateral kidney represents a metastasis., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Renal cell carcinoma (RCC) is an important contributor to cancer-specific mortality worldwide. Targeted agents that inhibit key subtype-specific signaling pathways have improved survival times and have recently become part of the standard of care for this disease. Accurately diagnosing and classifying RCC on the basis of tumor histology is thus critical. RCC has been traditionally divided into clear-cell and non-clear-cell categories, with papillary RCC forming the most common subtype of non-clear-cell RCC. Renal neoplasms with overlapping histologies, such as tumors with mixed clear-cell and papillary features and hybrid renal oncocytic tumors, are increasingly seen in contemporary practice and present a diagnostic challenge with important therapeutic implications. In this review, we discuss the histologic, immunohisto-chemical, cytogenetic, and clinicopathologic aspects of these differential diagnoses and illustrate how the classification of RCC has evolved to integrate both the tumor's microscopic appearance and its molecular fingerprint.

Juxtaglomerular cell tumors (JGCTs) are rare tumors characterized by renin synthesis, hyperaldosteronism and hypertension. A curious immunohistochemical overlap between JGCT and gastrointestinal stromal tumor (GIST) including the expression of vimentin, CD34, CD117, α-smooth muscle actin was previously reported, prompting us to further investigate JGCT and its phenotypic and molecular genetic characteristics. Virtual karyotyping showed gain of chromosomes 3, 4, 10, 13, 17 and 18 in one JGCT, and fluorescence in situ hybridization (FISH) study confirmed this multiple gain pattern. Additionally, loss of chromosome 9 was observed in four of six cases analyzed with FISH. A whole genome expression analysis revealed 415 up-regulated (including renin, and CD117) and 325 down-regulated genes between the 2 cases. The study confirmed earlier reports on the gain of chromosomes 4 and 10, and provided further evidence of up-regulation of the genes located on these 2 chromosomes. For the first time our study indicated the importance of the loss of chromosome 9 and loss of expression of several tumor suppressor genes located on this chromosome as possible pathogenetic events important in development of JGCT., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Loss-of-function defects in DNA mismatch repair (MMR), which manifest as high levels of microsatellite instability (MSI), occur in approximately 15% of all colorectal carcinomas (CRCs). This molecular subset of CRC characterizes patients with better stage-specific prognoses who experience no benefit from 5-fluorouracil chemotherapy. Most MMR-deficient (dMMR) CRCs are sporadic, but 15% to 20% are due to inherited predisposition (Lynch syndrome). High penetrance of CRCs in germline MMR gene mutation carriers emphasizes the importance of accurate diagnosis of Lynch syndrome carriers. Family-based (Amsterdam), patient/family-based (Bethesda), morphology-based, microsatellite-based, and IHC-based screening criteria do not individually detect all germline mutation carriers. These limitations support the use of multiple concurrent tests and the screening of all patients with newly diagnosed CRC. This approach is resource intensive but would increase detection of inherited and de novo germline mutations to guide family screening. Although CRC prognosis and prediction of 5-fluorouracil response are similar in both the Lynch and sporadic dMMR subgroups, these subgroups differ significantly with regard to the implications for family members. We recommend that new CRCs should be classified into sporadic MMR-proficient, sporadic dMMR, or Lynch dMMR subgroups. The concurrent use of MSI testing, MMR protein IHC, and BRAF c.1799T>A mutation analysis would detect almost all dMMR CRCs, would classify 94% of all new CRCs into these MMR subgroups, and would guide secondary molecular testing of the remainder., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Seven percent of renal cell carcinoma (RCC) cases are diagnosed as "unclassified" RCC by morphology. Genetic profiling of RCCs helps define renal tumor subtypes, especially in cases where morphologic diagnosis is inconclusive. This report describes a patient with synchronous clear cell RCC (ccRCC) and a tubulocystic renal carcinoma (TCRC) in the same kidney, and discusses the pathologic features and genetic profile of both tumors. A 67 year-old male underwent CT scans for an unrelated medical event. Two incidental renal lesions were found and ultimately removed by radical nephrectomy. The smaller lesion had multiple small cystic spaces lined by hobnail cells with high nuclear grade separated by fibrous stroma. This morphology and the expression of proximal (CD10, AMACR) and distal tubule cell (CK19) markers by immunohistochemistry supported the diagnosis of TCRC. The larger lesion was a typical ccRCC, with Fuhrman's nuclear grade 3 and confined to the kidney. Molecular characterization of both neoplasms using virtual karyotyping was performed to assess relatedness of these tumors. Low grade areas (Fuhrman grade 2) of the ccRCC showed loss of 3p and gains in chromosomes 5 and 7, whereas oncocytic areas displayed additional gain of 2p and loss of 10q; the high grade areas (Fuhrman grade 3) showed several additional imbalances. In contrast, the TCRC demonstrated a distinct profile with gains of chromosomes 8 and 17 and loss of 9. In conclusion, ccRCC and TCRC show distinct genomic copy number profiles and chromosomal imbalances in TCRC might be implicated in the pathogenesis of this tumor. Second, the presence of a ccRCC with varying degrees of differentiation exemplifies the sequence of chromosomal imbalances acquired during tumor progression., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1790525735655283.

Background: Body fluid specimens may be the first and only pathologic specimen for clinical evaluation in metastatic cancer cases. The challenge of identifying the tissue of origin in metastatic cancer has led to the emergence of molecular-based assays, such as the microarray-based Pathwork Tissue of Origin gene expression test. The ability to use body fluid specimens in this test would be valuable in providing diagnoses to cancer patients without clearly identifiable primary sites. In the current study, the authors evaluated the Tissue of Origin Test for use with malignant effusion specimens., Methods: A total of 27 metastasis-positive body fluid specimens from different body sites, including pleural, ascites, pericardial, and pelvic wash fluids, were obtained from patients with known diagnoses. Nine specimens from nonmalignant body fluids were included as controls. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and gene expression analysis was performed with the Tissue of Origin Test., Results: Seventeen of 27 metastasis-positive samples were non-necrotic with ≥60% tumor and yielded sufficient RNA. Of these samples, 94.1% (16 of 17) were in agreement with the available diagnosis. Of the 9 negative control samples evaluated, 7 (77.8%) demonstrated microarray expression profiles most similar to lymphoma, which is consistent with the predominance of inflammatory cells in these specimens., Conclusions: The results of the current study demonstrated that FFPE cell blocks from cytologic body fluid specimens yield adequate diagnostic material for the Pathwork test and can be used in the workup of patients with unknown primary tumors., (Copyright © 2011 American Cancer Society.)

Chromosomal abnormalities and gene mutations have become major determinants in the classification of kidney carcinomas. Most renal medullary carcinomas develop in patients with hereditary sickle cell disease, but sporadic cases unassociated with sickle cell disease have also been described, for which underlying genetic abnormality is unknown. We evaluated 3 patients with renal medullary carcinoma (1 patient with sickle cell disease and 2 patients without sickle cell disease) for germ line and somatic mutations in genes commonly involved in pathogenesis of renal carcinomas using denaturing high-performance liquid chromatography and direct sequencing. Chromosomal abnormalities were studied by the conventional cytogenetic and SNP arrays analysis. Expression of hypoxia-inducible factor 1α was examined using immunohistochemistry. Two new mutations in the gene for fumarate hydratase were identified in 1 case of medullary renal carcinoma without sickle cell disease: a germ line mutation in exon 6 (R233H) and an acquired (somatic) mutation in exon 8 (P374S). No fumarate hydratase mutations were identified in the other 2 patients. The second sporadic case of renal medullary carcinoma harbored double somatic mutations in von Hippel-Lindau gene, and renal medullary carcinoma in the patient with sickle cell disease showed von Hippel-Lindau gene promoter methylation (epigenetic silencing). No consistent pattern of chromosomal abnormalities was found between 2 cases tested. All 3 cases showed increased hypoxia-inducible factor 1α expression. Medullary renal carcinomas from patients with or without sickle cell disease show involvement of genes important in hypoxia-induced signaling pathways. Generalized cellular hypoxia (in sickle cell disease) or pseudohypoxia (in tumors with fumarate hydratase and von Hippel-Lindau mutations or epigenetic silencing) may act alone or in concert at the level of medullary tubular epithelium to promote development of this rare type of renal carcinoma, which could then be genetically reclassified as either fumarate hydratase-associated renal carcinomas or high-grade clear cell renal cell carcinomas., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Loss of chromosome 14 has been associated with poor outcomes in clear-cell renal cell carcinoma. Expression of HIFα isoforms has been linked to distinct molecular phenotypes of clear-cell renal cell carcinoma. We hypothesized that chromosome 14 loss could lead to a decrease in HIF1α levels, as its gene (HIF1A) resides in this chromosome. We analyzed 112 archival clear-cell renal cell carcinoma tumor specimens with 250K SNP microarrays. We also evaluated expression of HIFα isoforms by qPCR and immunohistochemistry in a subset of 30 patients. Loss of chromosome 14q was associated with high stage (III-IV, P=0.001), high risk for recurrence (P=0.002, RR 2.78 (1.506-5.153)) and with decreased overall survival (P=0.030) in non-metastatic clear-cell renal cell carcinoma. HIF1α mRNA and protein expression was reduced in specimens with loss of 14q (P=0.014) whereas HIF2α was not. Gain of 8q was associated with decreased overall survival (P<0.0001). Our studies confirm an association between 14q loss and clinical outcome in non-metastatic clear-cell renal cell carcinoma patients and that 8q gain is a candidate prognostic marker for decreased overall survival and appears to further decrease survival in patients with 14q loss. We have also identified that differential expression of HIF1α is associated with 14q loss. Further exploration of 8q gain, 14q loss, MYC, HIF1A and EPAS1 (HIF2α) as molecular markers of tumor behavior and prognosis could aid in personalizing medicine for patients with clear-cell renal cell carcinoma.

Carcinoma arising from Warthin tumor is extremely rare. A 79-year-old man was admitted for a firm, well-defined, 5-cm left infra-auricular mass. Aspiration cytology showed many lymphohistiocytes and oncocytes in a proteinaceous background, compatible with Warthin tumor. A left superficial parotidectomy showed a solid mass around the cyst wall. The tumor cells of the solid area were arranged as infiltrative ducts with a few foci of malignant transformation. Virtual karyotyping disclosed a complex pattern of genetic aberrations with a focal amplification in 12q14-q21.2. This chromosomal region contains the MDM2 (murine double minute) gene, which regulates p53 inactivation. HER2 fluorescence in situ hybridization showed a focal amplification. Subsequently, the patient underwent total parotidectomy and ipsilateral neck dissection for a recurrence. To our knowledge, this is the first case of salivary duct carcinoma arising from Warthin tumor. The essential molecular pathway has not been reported, we presume an important role of MDM2 amplification- P53 inactivation.

Cancer of unknown primary (CUP) is one of the ten most frequently diagnosed cancers in developed countries and accounts for 3-5% of all new cancer cases. For all cancer management, chemotherapeutic regimens and targeted agents have increasingly become primary-site dependent over the last decade, and thus, accurate identification of the primary site is crucial for CUP patients. Histopathologic examination along with immunohistochemical studies and different imaging modalities are most often performed for this purpose, but still a significant number of patients remain with an unidentified primary. Recently, gene-expression profiling assays have emerged as alternative tests for tumor tissue of origin determination. It is expected that molecular determination of the origin in patients with CUP will aid in the determination of management strategies. It is thus possible that personalized treatment of CUP patients based on molecular classification could significantly improve patient outcomes.

Oncocytoma, chromophobe renal cell carcinoma (chRCC), and the eosinophilic variant of clear cell RCC (ccRCC) are morphologically similar tumors with significantly different clinical courses. These renal tumor subtypes show characteristic structural genetic changes; however, the mRNA expression patterns of oncocytoma and chRCC are strikingly similar. MicroRNAs (miRNA) are small RNA molecules that regulate the expression of many genes and have been shown to be useful for tumor classification and identification. The miRNA expression was analyzed from formalin-fixed paraffin-embedded tissue in 5 cases each of oncocytoma, ccRCC, papillary RCC, chRCC, and 4 normal kidney tissues using microarrays. Affymetrix single-nucleotide polymorphism arrays were used to detect chromosomal imbalances in each of the tumors. Eighteen miRNAs were significantly different among the 4 tumor types. The microRNA miR-21, a known oncogenic miRNA, was found to be upregulated in papillary and clear cell carcinomas. Four miRNAs could differentiate oncocytomas from chRCCs and the 3 could differentiate papillary RCC from ccRCC, including miR-126, a known vasculogenic miRNA. Of the 18 differentially expressed miRNAs, only 2 correlated with copy number changes in the chromosomal region harboring these genes. One tumor, originally diagnosed as an oncocytoma by morphology, showed a virtual karyotype and miRNA expression pattern consistent with chromophobe carcinoma. Further investigation of the tumor showed vascular invasion. Our study suggests that miRNA expression can be used to differentiate the common subtypes of renal epithelial neoplasms but further validation is necessary. In addition, the lack of correlation between miRNA expression and virtual karyotype suggests a non-copy-number-related mechanism for miRNA gene expression regulation in renal neoplasia.

Kidney and upper urinary tract cancers account for approximately 54,000 cases every year in the United States, and represent about 3.7% of adult malignancies, with more than 13,000 annual deaths. Classification of renal tumors is typically based on histomorphologic characteristics but, on occasion, morphologic characteristics are not sufficient. Each of the most common histologic subtypes harbors specific recurrent genetic abnormalities, such as deletion of 3p in conventional clear cell carcinoma, trisomy 7 and 17 in papillary renal cell carcinoma, multiple monosomies in chromophobe renal cell carcinoma, and a nearly diploid genome in benign oncocytomas. Knowledge of this information can provide diagnostic support and prognostic refinement in renal epithelial tumors. Identification of the specific subtype of a renal tumor is critical in guiding surveillance for recurrence and the appropriate use of targeted therapies. Cytogenomic arrays are increasingly being used as a clinical tool for genome-wide assessment of copy number and loss of heterozygosity in renal tumors. In addition, the improved understanding of the hereditary causes of renal tumors and their role in sporadic malignancies has led to the development of more effective targeted therapies. This review summarizes the genetic and genomic changes in the most common types of renal epithelial tumors and highlights the clinical implications of these aberrations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Chromosomal imbalances are commonly seen in cancer and inherited genetic diseases. These imbalances may assist in the diagnosis, prognosis, and/or therapeutic management of certain neoplasms. Several methods for detecting chromosomal imbalances, such as, fluorescent in situ hybridization, array comparative genomic hybridization, and single nucleotide polymorphism (SNP) arrays have proven useful in formalin-fixed paraffin-embedded (FFPE) tissues. Here, we report the performance and reproducibility of virtual karyotyping of FFPE tissues with Affymetrix SNP arrays., Methods: Virtual karyotypes from 442 FFPE tumor samples were generated using the Affymetrix GeneChip Mapping 10K Xba 2.0 and/or 250K Nsp SNP mapping arrays. Samples ranged from a few weeks to 17 years in archival storage. Virtual karyotypes were assessed for copy number changes, loss of heterozygosity, and acquired uniparental disomy., Results: Overall, 75.3% of samples produced interpretable virtual karyotypes with the 10K arrays and 76.7% in the 250K arrays. Parameters for the selection of samples for hybridization were determined, which increased the success rate in both platforms to 81.3 and 92.6%, respectively. FFPE virtual karyotypes generated with both 10K Xba 2.0 and 250K Nsp arrays showed 100% concordance in intralaboratory and interlaboratory reproducibility studies. Samples older than 7 years showed decreased performance., Conclusions: SNP arrays are a reliable, reproducible, and robust platform for the virtual karyotyping of FFPE tumor tissues with performance characteristics adequate for clinical application. Parameters that most significantly affected sample performance were sample age and storage conditions.

Specific chromosomal alterations are recognized as important prognostic factors in chronic lymphocytic leukemia (CLL). Array-based karyotyping is gaining acceptance as an alternative to the standard fluorescence in situ hybridization (FISH) panel for detecting these aberrations. This study explores the optimum single nucleotide polymorphism (SNP) array probe density for routine clinical use, presents clinical validation results for the 250K Nsp Affymetrix SNP array, and highlights clinically actionable genetic lesions missed by FISH and conventional cytogenetics. CLL samples were processed on low (10K2.0), medium (250K Nsp), and high (SNP6.0) probe density Affymetrix SNP arrays. Break point definition and detection rates for clinically relevant genetic lesions were compared. The 250K Nsp array was subsequently validated for routine clinical use and demonstrated 98.5% concordance with the standard CLL FISH panel. SNP array karyotyping detected genomic complexity and/or acquired uniparental disomy not detected by the FISH panel. In particular, a region of acquired uniparental disomy on 17p was shown to harbor two mutated copies of TP53 that would have gone undetected by FISH, conventional cytogenetics, or array comparative genomic hybridization. SNP array karyotyping allows genome-wide, high resolution detection of copy number and uniparental disomy at genomic regions with established prognostic significance in CLL, detects lesions missed by FISH, and provides insight into gene dosage at these loci.

Background: Carcinomas of unknown primary (CUP) represent approximately 3%-5% of malignant neoplasms. Identifying the tissue of origin (TOO) in these tumors allows for more specific treatment and improves outcomes. However, primary classification remains a challenge in many cases. We evaluated the ability of a microarray-based gene expression test to identify the TOO in tumor specimens from 21 patients with a diagnosis of CUP., Methods: The Pathwork TOO Test was used to measure gene expression patterns for 1550 genes; these were compared for similarity to patterns from 15 known tissue types., Results: The TOO Test yielded a clear single positive call for the primary site in 16 of 21 (76%) specimens and was indeterminate in 5 (24%). The positive results were consistent with clinicopathologic suggestions in 10 of the 16 cases (62%). In the remaining six cases the positive results were considered plausible based on clinical information. Positive calls included colorectal (5), breast (4), ovarian (3), lung (2), and pancreas (2). The TOO Test ruled out an average of 11 primary tissues in each CUP specimen., Conclusion: The Pathwork TOO Test reduced diagnostic uncertainty in all CUP cases and could be a valuable addition or alternative to current diagnostic methods for classifying uncertain primary cancers.

Clinical workup of metastatic malignancies of unknown origin is an arduous and expensive process, which is reported to be unsuccessful in up to 30% of cases. Global gene expression-based molecular testing may offer accurate classification of metastatic tumors in which a primary site has not been identified. Recently, the US FDA cleared the Pathwork((R)) tissue-of-origin test, which is a gene expression microarray-based test that quantifies the molecular similarity of tumor specimens to 15 known tissue types. A blinded, multicenter validation on poorly differentiated and undifferentiated tumors showed 87.8% sensitivity and 99.4% specificity in frozen tissue samples. The availability of ancillary gene expression-based molecular tests for tissue of origin determination represents a milestone in cancer patient management as part of the personalized medicine revolution.

Approximately 7 % of renal cell tumors are reported to be "unclassified" renal cell carcinoma (RCC) under the current (morphology-based) classification. Genetic lesions characteristic for RCC subtypes can be identified by virtual karyotyping with single nucleotide polymorphisms (SNP) microarrays. In this study, we examined whether virtual karyotypes could be used to better classify a cohort of morphologically challenging/unclassified RCC. Tumor resection specimens from 21 patients were profiled by virtual karyotyping with Affymetrix 10K 2.0 or 250K Nsp SNP mapping arrays and were also evaluated independently by a panel of 7 genito-urinary pathologists. Tumors were classified by the established pattern of genomic imbalances based on a reference cohort of 98 cases with classic morphology and compared with the morphologic diagnosis of the pathologist panel. Virtual karyotyping analysis identified recognized patterns of chromosomal imbalances in all but 1 (16/17 or 94%) cases with successful analysis. Four cases failed owing to low DNA quality. All cases with a panel diagnosis of unclassified RCC and cases in which a majority diagnosis was not reached were classified by their virtual karyotypes. In 1 case, the molecular-based diagnosis was in disagreement with the majority diagnosis. One case with a majority diagnosis of oncocytoma showed a novel genomic pattern not previously identified in the classic morphology cohort. We conclude that virtual karyotypes generated by SNP arrays are a valuable tool for increasing diagnostic accuracy in morphologically challenging or unclassified renal neoplasms. We consider that this technique is a feasible and practical approach for resolving difficult-to-diagnose renal tumors in clinical practice.