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Renal cell carcinomas with t(6;11) chromosome translocation has been classically characterized by the rearrangement of the TFEB gene, located on chromosome 6, and MALAT1 gene, located on chromosome 11. Recently, a few other genes have been described as fusion partners in TFEB rearranged renal cell carcinomas. Although most of TFEB rearranged renal cell carcinomas have an indolent behavior, in the rare cases of advanced metastatic disease targeted therapy and predictive markers remain lacking. In the present study, we collected 13 TFEB rearranged renal cell carcinomas, confirmed by FISH, analyzing their morphology and exploring the novel gene partners. Looking for predictive markers, we have also performed PDL1 immunohistochemical analysis by using four different assays (E1L3N, 22C3, SP142, and SP263). MALAT1 gene rearrangement has been found in ten tumors, five cases showing classical biphasic morphology with “rosettes”, five cases without “rosettes” mimicking other renal cell carcinomas or epithelioid angiomyolipoma/pure epithelioid PEComa. We identified two different partner genes, ACTB and NEAT1, the latter previously unreported and occurring in a tumor with an unusual solid and cystic appearance. In both cases, the “rosettes” were absent. In one case no gene partner was identified. Overall, in 12 of 13 TFEB-rearranged renal cell carcinomas staining for PDL1 SP263 was observed, whereas the other antibodies were less reliable or more difficult to interpret. In conclusion, we described the third case of ACTB-TFEB rearranged renal cell carcinoma and a novel NEAT1-TFEB rearranged renal cell carcinoma, both without the distinctive biphasic morphology typical of t(6;11) renal cell carcinoma. Finally, PDL1 SP263 was constantly expressed in TFEB rearranged renal cell carcinoma with possible clinical benefit which requires further investigations.

MiT family translocation renal cell carcinoma (MiT-RCC) harbors translocations involving the TFE3 or TFEB genes. RCC with TFEB amplification is also identified and is associated with a more aggressive clinical course. Accurate diagnosis of MiT-RCC is crucial for patient management. In this study, we evaluated the performance of the Archer FusionPlex assay for detection of MiT-RCC with TFE3 or TFEB translocations and TFEB amplifications. RNA was extracted from 49 RCC FFPE tissue samples with known TFE3/TFEB status (26 TFE3 FISH positive, 12 TFEB FISH positive, 4 TFEB amplified (1 case both split and amplified), and 8 FISH negative) using the Covaris extraction kit. Target enriched cDNA libraries were prepared using the Archer FusionPlex kit and sequenced on the Illumina NextSeq 550. We demonstrate that the age of the specimen, quality of RNA, and sequencing metrics are important for fusion detection. Fusions were identified in 20 of 21 cases less than 2 years old, and TFE3/TFEB rearrangements were detected in all cases with Fusion QC ≥ 100. The assay identified intrachromosomal inversions in two cases (TFE3-RBM10 and NONO-TFE3), usually difficult to identify by FISH assays. TFEB mRNA expression and the TFEB/TFE3 mRNA expression ratio were significantly higher in RCCs with TFEB fusion and TFEB gene amplification compared to tumors without TFEB fusion or amplification. A cutoff TFEB/TFE3 ratio of 0.5 resulted in 97.3% concordance to FISH results with no false negatives. Our study demonstrates that the FusionPlex assay successfully identifies TFE3 and TFEB fusions including intrachromosomal inversions. Age of the specimen and certain sequencing metrics are important for successful fusion detection. Furthermore, mRNA expression levels may be used for predicting cases harboring TFEB amplification, thereby streamlining testing. This assay enables accurate molecular detection of multiple subtypes of MiT-RCCs in a convenient workflow.

The antiplatelet agent clopidogrel, a prodrug that requires bioactivation through the cytochrome P450 2C19 (CYP2C19) enzyme, is commonly prescribed post‐percutaneous coronary intervention (PCI). Genetic variation in CYP2C19 contributes to individual variability in clopidogrel response, and can lead to adverse cardiovascular events. Incorporating CYP2C19 testing during routine clinical care helps identify high‐risk patients, and provides the opportunity for pharmacotherapeutic interventions in the early post‐PCI period. The Spartan RX CYP2C19 System has emerged as an optimal genotyping assay for use in clinical care due to ease of use, utilization of buccal swabs, and rapid turnaround time. However, workflow constraints related to sample collection and processing, storage, time, and personnel were encountered when integrating testing into clinical care. To improve clinical workflow and successfully implement CYP2C19 genotyping at our institution, we validated the Spartan RX System to return genotype utilizing blood samples. Our Molecular Diagnostic Laboratory tested 26 known reference materials and both blood and buccal swab samples from 23 patients and volunteers using the Spartan RX Assay. Genotype results were 100% concordant between DNA from blood and buccal swabs for all patients or volunteers, and consistent with expected results for the 26 reference materials. For reproducibility, three samples were tested in at least four separate runs, with all resulting genotypes in agreement between runs. Post‐validation, the laboratory began offering CYP2C19 testing during clinical care. DNA extracted from blood can serve as a genomic DNA source for the Spartan RX Assay. Alteration of the methodology allowed for clinical implementation to support genotype‐guided therapy.

ABSTRACT Objectives To compare the effects of two methods of formalin-fixed paraffin-embedded (FFPE) tissue harvesting on DNA quality and next-generation sequencing (NGS) quality metrics. Methods DNA integrity number (DIN) and NGS quality metrics resulting from DNA extraction and sequencing of 199 sequential samples harvested via the Pinpoint Slide DNA Isolation System and the punch method were compared. Results DNA extracted from FFPE tissue punches had higher DIN than that extracted from Pinpoint samples (mean ± SD, 6.18 ± 0.83 vs 5.09 ± 0.91; P < .0001), indicating less degradation. Lower DIN correlated with lower-quality metrics of NGS, that is lower percentage of unique on-target reads, average depth of coverage, and percentage of positions with coverage depth greater than or equal to 100×, 400×, and 1,000×. Conclusions Our study demonstrated methods to harvest tissue from FFPE blocks may affect quality of DNA, which in turn has an effect on other NGS quality metrics.

Angiomyolipomas (AMLs) are triphasic tumors (smooth muscle, vascular and adipocytic components) with myomelanocytic differentiation, arising most commonly in the kidneys, which can show predominant epithelioid morphology and fat-predominant or fat-poor variants. Fat-predominant AMLs can show areas of hypercellularity and lipoblast-like cells, and these features can mimic well-differentiated liposarcoma (WDLS). To date, only one documented metastatic epithelioid AML showed unequivocal MDM2 amplification by fluorescence in situ hybridization. We describe our findings in a series of 35 AMLs including epithelioid, fat-poor, and fat-predominant variants, following interrogation of the MDM2 locus by FISH and CISH assays. MDM2 amplification was detected in 1 fat-predominant AML. Our findings demonstrate that rare MDM2 amplifications can occur in AMLs. We favor that this finding likely represents a “molecular bystander” event since these tumors are mainly driven by aberrations in the TSC1/TSC2 genes. Nevertheless, the presence of MDM2 amplification in a fat-predominant AML could present a potential diagnostic pitfall, particularly when confronted with the differential diagnosis of fat-predominant AML and WDLS in limited material from the retroperitoneum.

Background Human papillomavirus (HPV) genotype testing has limited utility to identify human immunodeficiency virus‐infected (HIV+) women's risk for developing cervical cancer (CC) due to high positivity rate of high‐risk (HR) HPVs. We investigated the accuracy of HPV testing in isolation/in combination with CD4 and HIV viral load (VL) to identify HIV+ women at risk for developing CC. Methods Study consisted of 344 HIV+ women on combination antiretroviral therapy (cART), tested for cervical cytology/HPV using the Cobas test and had data on absolute CD4 count and VL measurements. We calculated the positive predictive value (PPV) and negative predictive value (NPV) of HPV testing, pre‐, post‐cART, and current CD4 and VL in isolation and in combinations to identify those with or free of higher than atypical squamous cells of unknown significance (ASCUS+) or low‐grade intraepithelial lesions (LSIL+). Results HPV test in combination with pre‐/post‐cART or current CD4 counts and VL had higher PPVs compared to HPV test alone for identifying ASCUS+ or LSIL+. PPV of HPV‐CD4 combinations yielded higher PPVs compared to HPV‐VL combinations. The NPVs with pre‐, post‐cART, or current CD4 count and VL in isolation or in combinations were comparable to that of HPV test alone. Conclusions Our results provide a more accurate tool for managing HIV+ women by combining Cobas HPV with CD4 and VL, especially those who had an undesirable pre‐cART CD4 and VL status. Our results also indicate the usefulness of CD4 and VL measurements to identify those at lower risk in the absence of HPV testing. The purpose of the study was to investigate the accuracy of HPV genotype testing in isolation and in combination with CD4 and HIV viral load (VL) for the identification of women infected with HIV at risk for developing cervical cancer. Our results indicated the importance of knowing CD4 and VL status in addition to HPV infection status in identifying women infected with HIV who are at risk for developing CC.

Therapy-related myeloid neoplasms (t-MN) have a common origin in prior cytotoxic therapy and/or radiation. These neoplasms include therapy-related acute myeloid leukemia, myelodysplastic syndrome (t-MDS), and myelodysplastic/myeloproliferative neoplasms (t-MDS/MPN). Myeloid sarcoma (MS), on the other hand, is a rare disease manifesting as an extramedullary collection of immature cells of myeloid lineage. Rarer still is therapy-related MS (t-MS), which has not been adequately studied due to its rarity and its lack of recognition as a unique entity among other t-MN. Here, we report what is to our knowledge the first case series of t-MS, with the aim of investigating and establishing salient clinicopathological and molecular features of this entity.

Using variants at LOD for NGS PT is a crucial aspect of ensuring the accuracy and reliability of NGS assays because it allows participating laboratories to assess the sensitivity of their NGS LDT by challenging them to identify variants present at low allelic fractions. Because clinical samples are frequently small and contain variants at low allelic frequencies, testing against PT samples with low VAF helps laboratories identify potential issues with sensitivity, assay performance, and data analysis, allowing for the improvement of processes and procedures. Because a major goal of the SPOT/Dx pilot study was to determine the performance of NGS LDTs, it was designed to include challenging variants with low VAF, similar to CAP PT programs.5 However, the design of the SPOT/Dx pilot study differed from conventional PT programs in several ways, and as a result its conclusions represent an underestimation of the actual performance of the participating laboratories.4 For example, the reference materials in the SPOT/Dx pilot were designed with VAFs hovering near the LOD of NGS LDTs used by most of the participating laboratories. [...]the performance of NGS LDTs was underestimated, leading to erroneous impressions regarding the quality and accuracy of these LDTs. Laboratories frequently encounter situations where they must analyze new or emerging substances, employ customized testing methods, or cater to specific patient groups not covered by existing PT programs. [...]the introduction of innovative technologies and testing approaches may not always align with the availability