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• Genomic studies were reviewed for renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, and the translocation RCC involving TFE3 and TFEB rearrangements, as well as benign oncocytoma, which together comprise about 95% of all renal cell neoplasia. • This evidence-based review provides a validated role for assessment of CNAs in renal cell neoplasia in the clinical setting. • It can assist in renal cell neoplasm diagnosis and sub-classification within subtypes that is integral to the management of patients. Renal cell neoplasia are heterogeneous with diverse histology, genetic alterations, and clinical behavior that are diagnosed mostly on morphologic features. The Renal Cell Neoplasia Workgroup of the Cancer Genomics Consortium systematically evaluated peer-reviewed literature on genomic studies of renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, and the translocation RCC involving TFE3, TFEB and MITF rearrangements, as well as benign oncocytoma, which together comprise about 95% of all renal cell neoplasia. The Workgroup curated recurrent copy number alterations (CNAs), copy-neutral loss-of-heterozygosity (cnLOH), rearrangements, and mutations, found in each subtype and assigned clinical relevance according to established criteria. In clear cell RCC, loss of 3p has a disease-initiating role and most likely also in progression with mutations detected in VHL and other genes mapped to this arm, and loss of 9p and/or 14q has well-substantiated prognostic utility. Gain of chromosomes 7 and 17 are hallmark CNAs of papillary RCC, but patterns of other CNAs as detected by chromosomal microarray analysis (CMA) afford sub-classification into Type 1 and 2 with prognostic value, and for further sub-stratification of Type 2. Inherent chromosome loss in chromophobe RCC as detected by CMA is useful for distinguishing the eosinophilic variant from benign oncocytoma which in contrast exhibits few CNAs or rearranged CCND1 , but share mitochondrial DNA mutations. In morphologically atypical RCCs, rearrangement of TFE3 and TFEB should be considered in the differential diagnosis, portending an aggressive RCC subtype. Overall, this evidence-based review provides a validated role for assessment of CNAs in renal cell neoplasia in the clinical setting to assist in renal cell neoplasm diagnosis and sub-classification within subtypes that is integral to the management of patients, from small incidentally found renal masses to larger surgically resected specimens, and simultaneously identify the presence of key alterations portending outcome in malignant RCC subtypes. [ABSTRACT FROM AUTHOR]

Molecular testing to detect clonal T-cell populations is commonly applied to dermatologic specimens to support cutaneous T-cell lymphoma diagnosis. With clinical implementation of TRG and TRB clonality by NGS using Lymphotrack (Invivoscribe) sequenced on the Miseq (Illumina), increased specimen numbers have been submitted for testing. For 66 specimens (50 patients) received to date, high insufficiency rates were found for TRG (30%) and TRB (56%). Thus, to improve testing efficiency and effect better specimen triage, we examined tissue features, DNA size by fragment analysis, library concentration, reads and overall call. Nine specimens either had inadequate DNA for library preparation or amplified fragments <300bp in size. Another 9 supported 300bp fragment amplification, 11 400bp, and 37 600bp which yielded acceptable reads for interpretation (>50,000) for TRG (33%, 82%, 78%) and TRB (0%, 45%, 54%) respectively. This was not unexpected given the larger size of the TRB amplicon compared to TRG. For the latter 48 specimens, estimated biopsy lymphocyte content was available for 34, and exhibited a weak correlation with TRG and TRB library concentrations (R2=0.26, 0.29). Only 64% of punch biopsies (9/14) but 85% (29/34) of shave biopsies achieved acceptable reads, and 40% and 66% respectively for TRB. For all specimens, 50% of punch biopsies were deemed insufficient and 19% of shave biopsies. Thus biopsy type, ability to support 400bp amplification, and amplicon size impacted the overall diagnostic yield of TCR clonality testing by NGS on dermatologic specimens. Diagnostic advantages of TCR clonality assessed by NGS will be discussed for specific cases. [ABSTRACT FROM AUTHOR]

Technical barriers, panel complexity and bioinformatics are major challenges for the clinical implementation of large NGS panels. For optimization of the 500+ gene Oncomine comprehensive plus targeted amplicon based NGS assay to detect SNVs, Indels, CNVs, fusions and TMB, 40ng DNA and RNA from 115 specimens from different tumor types (tumor cellularity ≥10%) were used for library preparation and sequenced on the Ion S5XL sequencer. Ion Reporter 5.18 (IR) and Alissa custom classification trees were optimized for known variants. CDKN2A, PTEN and TERT genes were underperformers. SNVs/Indels demonstrated 95% concordance with OCAv3 panel (n=71) sequenced comparator specimens, and 94% and 56% for SNVs and Indels respectively compared to a hybrid capture 324 gene panel (n=22). Discordant SNVs had poor coverage (<100x) and low VAF (<3%), while 76% discrepant Indel calls were in homopolymer region of length ≥5. After augmentation of the CNV IR baseline with 44 tumor and normal specimens, concordance for 85 CNVs was 92% for known CNV ≥6 copies, 83% for ≥5-6, and 46% for ≥4-5, and 100% for loss. Fusions were detected with 100% concordance. 22 specimens with prior TMB score (14 high (≥10) and 8 low) demonstrated 86% concordance. TMB discrepant specimens (range 11-25) showed low score due to missed variants in homopolymer region. In the current optimization study, challenges faced in clinical implementation included lower confidence for Indel calling in homopolymer length >4, CNV gain 4-5 and borderline TMB. [ABSTRACT FROM AUTHOR]

For therapeutic purpose, tumor molecular profiling (MP) of both DNA and RNA is standard-of-care in lung cancer patients. With increased use of fine needle aspiration (FNA) to assist in diagnosis, MP can be challenging given the limited cell block material. At our institution, only 40% of 309 FNA with lung cancer diagnosis over 2 years were successful for DNA and RNA MP as a send out, compared with 90% of surgically-resected specimens. To improve the diagnostic yield, we salvaged left over FNA washes (FW, 30-40mL) from the Cytology Laboratory within a median of 10 days from collection. For 211 FW with a malignant diagnosis, DNA/RNA extraction from 4 mL FW was performed using QIAamp ccfDNA kit (QIAGEN), where 97% and 83% yielded adequate levels for MP of DNA and RNA respectively. MP of DNA only (48) or DNA/RNA (35) workflows were performed using a 50-gene Hotspot, OCAV3, or OCAplus NGS panels (ThermoFisher). For 67 cases with a concurrent or other profiled specimen, 94% concordance for SNVs/indels and 77% for CNVs detection was observed, and expected EML4-ALK and FGFR3-TACC fusions detected. Discordance of 21 variants calls in 13 FW was due to low VAF, low coverage, or failure of CNV QC metrics. Importantly, 7 relevant variants and fusions were detected in 4 specimens previously reported as insufficient and one with prior DNA results only. Thus, the use of FW as a source of nucleic acids afforded increased diagnostic yield of FNAs for MP in lung cancer and timely targeted therapeutic intervention. [ABSTRACT FROM AUTHOR]

Chromosomal copy number alterations (CNAs) and structural rearrangements (SR) are a key part of risk stratification and management of patients with MM. We developed a clinical paired tumor/normal WES/WTS assay that detects CNAs at one-copy difference from focal to whole genome, and gene fusion at 20% and 10% limit of detection, respectively. We performed WES/WTS analysis utilizing DNA/RNA extracted from CD138+ enriched cells and buccal swab from 92 MM patients. Plasma cell content was not available for most cases. We compared the detection of CNAs, and SRs with other techniques currently in use including FISH, arrays and molecular analysis. Molecular alterations (MA) were detected in 82/92(89.1%) cases, whereas 65/92(70.7%) cases had CNAs. In cases with CNAs (indicating sufficient tumor content), deletions of (1p32), (1p12), or both were detected in 7(10.8%), 12(18.5%), and 5(7.7%) cases, respectively. Gain of (1q22), and hyperdiploidy were detected in 19(29.2%) and 32(49.2%), respectively. Deletions of 13q, 14q, 16q, and 17p(TP53) were detected in 32(49.2%), 14(21.6%), 16(24.6%), and 13(20%) cases, respectively. In cases with MA, 10/82(12.2%), and 3/82(3.7%) cases had TP53 mono- and bi-allelic inactivation, respectively. Translocations (4;14)(p16;q32)(IGH::NSD2/FGFR3) and t(14;16)(q32;q23)(IGH::MAF) were detected in 11/82(13.4%), and 3/82(3.7%) cases with MA, respectively. Overexpression of CCND1 (research use only) was observed in 8/82(9.8%) cases. In this series, our WES/WTS detected abnormalities similarly to what has been reported by techniques currently needed in combination such as FISH, arrays or molecular analysis. By incorporating RNA expression analysis, WES/WTS may reliably detect all clinically-relevant alterations in MM. [ABSTRACT FROM AUTHOR]