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AimsTargeted next-generation sequencing (NGS) panels, which identify genomic alterations, are the stronghold of molecular oncology laboratories. In spite of technological advances, the quantity and quality of DNA from formalin-fixed paraffin-embedded tissue and paucicellular specimens are barriers to successful sequencing. Here, we describe an NGS assay employing single tube stem-loop inhibition mediated amplification technology that delivers highly accurate results with low input DNA. Rigorous quality metrics, regular monitoring and in-depth validation make the test attractive for clinical laboratories.MethodsThe study used a customised NGS panel, targeting 48 genes across several solid tumour types. Validation, in accordance with guidelines from New York State, sequenced patient samples harbouring 136 known variants, including single-nucleotide variants (SNVs) and indels. Specimen types included formalin-fixed paraffin embedded blocks, core biopsies and cytology material. Neoplastic cellularity of the tumours ranged from 10% to 80%.ResultsThe assay was highly specific and sensitive with excellent accuracy, reproducibility and repeatability/precision. Concordant results for identification of SNVs and indels were obtained from specimens with DNA input of 2–3 ng, tissue with 10% neoplastic cellularity and variant allelic frequencies of 2.5%–3%. Over 99% of the target areas are shown to achieve at least 500X coverage when parsed through two bioinformatics pipelines. With over 2000 clinical specimens analysed, the success of the panel for reporting of results is 95.3%ConclusionsThe advanced technology enables accurate identification of clinically relevant variants with uniformity of coverage and an impressive turn-around-time. The overall workflow and cost-effectiveness provide added value.

Next-generation sequencing is becoming increasingly important for the diagnosis, risk stratification, and management of patients with established or suspected myeloid malignancies. These tests are being incorporated into clinical practice guidelines and many genetic alterations now constitute disease classification criteria. However, the reimbursement for these tests is uncertain. This study analyzed the clinical impact, ordering practices, prior authorization, and reimbursement outcomes of 505 samples from 477 patients sequenced with a 50-gene myeloid next-generation sequencing panel or a 15-gene myeloproliferative neoplasm subpanel. Overall, 98% (496 of 505) of tests provided clinically useful data. Eighty-nine percent of test results, including negative findings, informed or clarified potential diagnoses, 94% of results informed potential prognoses, and 19% of tests identified a potential therapeutic target. Sequencing results helped risk-stratify patients whose bone marrow biopsy specimens were inconclusive for dysplasia, monitor genetic evolution associated with disease progression, and delineate patients with mutation-defined diagnoses. Despite the clinical value, prior authorization from commercial payors or managed government payors was approved for less than half (45%) of requests. Only 51% of all cases were reimbursed, with lack of medical necessity frequently cited as a reason for denial. This study demonstrates the existence of a substantial gap between clinical utility and payor policies on test reimbursement.

•Survey results from 18 academic laboratories reveal differences in molecular testing approaches and practices for hematologic neoplasms.•Shared understanding of the capabilities and limitations of current and future molecular testing will help optimize patient care.

Papillary intralymphatic angioendothelioma (PILA) is an extremely rare vascular tumor and its pathogenesis is unknown. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS) is a heterogeneous group of disorders caused by mosaicism for activating mutations of PIK3CAand characterized by asymmetric overgrowth, skeletal anomalies, skin lesions, and vascular malformations. An association between PILA and PROS has not been known. We report a case of PILA involving the spleen of a young girl with the clinical and molecular diagnosis of PROS. Sequencing of the patient’s germ-line DNA detected a pathogenic PIK3CAvariant c.1357G>A in 10.6% of alleles. Splenectomy revealed a 4-cm tumor composed of ectatic lymphatics with intraluminal papillary projections, consistent with PILA. The tumor cells showed immunohistochemical expression of CD31, CD34, ERG, FLI-1, PROX1, and caldesmon, while D2-40 was negative. The latter may suggest that the tumor derived from an endothelial precursor arrested in the final steps of lymphothelial differentiation, in keeping with the known role of the PIK3CA-governed molecular pathway in the progression of vascular progenitors to mature endothelial cells. The data implicates PIK3CAin the pathogenesis of PILA and broadens the spectrum of phenotypic expressions of PROS.

Tumour mutational burden (TMB) is used to predict response to immunotherapies. Although several groups have proposed calculation methods for TMB, a clear consensus has not yet emerged. In this study, we explored TMB calculation approaches with a 586-gene cancer panel (1.75 Mb) benchmarked to TMB measured by whole-exome sequencing (WES), using 30 samples across a range of tumour types. We explored variant allelic fraction (VAF) cut-offs of 5% and 10%, population database filtering at 0.001, 0.0001 and 0.000025, as well as different combinations of synonymous, insertion/deletion and intronic (splice site) variants, as well as exclusion of hotspot mutations, and examined the effect on TMB correlation. Good correlation (Spearman, range 0.66–0.78) between WES and panel TMB was seen across all methods evaluated. Each method of TMB calculation evaluated showed good positive per cent agreement and negative per cent agreement using 10 mutations/Mb as a cut-off, suggesting that multiple TMB calculation approaches may yield comparable results.

Resected lesions from the pineal region are rare specimens encountered by surgical pathologists, and their heterogeneity can pose significant diagnostic challenges. Here, we reviewed 221 pineal region lesions resected at New York-Presbyterian Hospital/Columbia University Irving Medical Center from 1994 to 2019 and found the most common entities to be pineal parenchymal tumors (25.3%), glial neoplasms (18.6%), and germ cell tumors (17.6%) in this predominantly adult cohort of patients. Six cases of a rare midline entity usually found exclusively in the fourth ventricle, the rosette-forming glioneuronal tumor, were identified. These tumors exhibit biphasic morphology, with a component resembling pilocytic astrocytoma admixed with variable numbers of small cells forming compact rosettes and perivascular pseudorosettes. Targeted sequencing revealed a 100% co-occurrence of novel and previously described genetic alterations in the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways, suggesting a synergistic role in tumor formation. The most common recurrent mutation, PIK3CA H1047R, was identified in tumor cells forming rosettes and perivascular pseudorosettes. A review of the literature revealed 16 additional cases of rosette-forming glioneuronal tumors in the pineal region. Although rare, this distinctive low-grade tumor warrants consideration in the differential diagnosis of pineal region lesions.

This multi-institutional study was undertaken to evaluate interrater reliability of the 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists guidelines for interpretation and reporting of oncology sequence variants and to assess current practices and perceptions surrounding these guidelines. Fifty-one variants were distributed to 20 participants from 10 institutions for classification using the new guidelines. Agreement was assessed using chance-corrected agreement (Cohen κ). κ was 0.35. To evaluate if data sharing could help resolve disagreements, a summary of variant classifications and additional information about each variant were distributed to all participants. κ improved to 0.7 after the original classifications were revised. Participants were invited to take a web-based survey regarding their perceptions of the guidelines. Only 20% (n= 3) of the survey respondents had prior experience with the guidelines in clinical practice. The main perceived barriers to guideline implementation included the complexity of the guidelines, discordance between clinical actionability and pathobiologic relevance, lack of familiarity with the new classifications, and uncertainty when applying criteria to potential germline variants. This study demonstrates noteworthy discordances between pathologists for variant classification in solid tumors when using the 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists guidelines. These findings highlight potential areas for clarification/refinement before mainstream clinical adoption.

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP).MethodsAltered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay.ResultsThe classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours.ConclusionThis study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Changes in coding and coverage generate an uncertain reimbursement environment for molecular pathology laboratories. We analyzed our experience with two representative molecular oncology tests: a T-cell receptor (TCR) β rearrangement test and a large (467-gene) cancer next-generation sequencing panel, the Columbia Combined Cancer Panel (CCCP). Before 2013, the TCR β test was coded using stacked current procedural terminology codes and subsequently transitioned to a tier 1 code. CCCP was coded using a combination of tier 1 and 2 codes until 2015, when a new Genomic Sequencing Procedure code was adopted. A decrease in reimbursement of 61% was observed for the TCR β test on moving from stacking to tier 1 codes. No initial increase in total rejection rate was observed, but a subsequent increase in rejection rates in 2015 and 2016 was noted. The CCCP test showed a similar decrease (48%) in reimbursement after adoption of the new Genomic Sequencing Procedure code and was accompanied by a sharp increase in rejection rates both on implementation of the new code and over time. Changes in coding can result in substantial decreases in reimbursement. This may be a barrier to patient access because of the high cost of molecular diagnostics. Revisions to the molecular code set will continue. These findings help laboratories and manufacturers prepare for the financial impact and advocate appropriately.

AimsRefractory coeliac disease type II (RCDII), a rare complication of coeliac disease (CD) associated with high morbidity, requires identification of a clonal population of phenotypically aberrant intraepithelial lymphocytes (IELs) for diagnosis. However, data regarding the frequency and significance of clonal T cell receptor (TCR) gene rearrangements (TCR-GRs) in small bowel (SB) biopsies of patients without RCDII are limited.MethodsWe analysed results of TCR-GR analyses performed on SB biopsies at our institution over a 3-year period, which were obtained from eight active CD, 172 CD on gluten-free diet (GFD), 33 RCDI, and three RCDII patients and 14 patients without CD. TCR-GR patterns were divided into clonal, polyclonal and prominent clonal peaks (PCPs) and these patterns were correlated with clinical and pathological features.ResultsClonal TCR-GR products were detected in biopsies from 67% of patients with RCDII, 17% of patients with RCDI and 6% of patients with GFD. PCPs were observed in all disease phases (range 12%–33%). There was no significant difference in the TCR-GR patterns between the non-RCDII disease categories (p=0.39). A higher frequency of surface CD3(−) IELs was noted in cases with clonal TCR-GR, but the PCP pattern did not show associations with any clinical or pathological feature. Persistence of clonal or PCP pattern on repeat biopsy was seen for up to 2 years without evidence of RCDII.ConclusionsClonal TCR-GRs are not infrequent in cases lacking features of RCDII, while PCPs are frequent in all disease phases. TCR-GR results should be assessed in conjunction with immunophenotypic, histological and clinical findings for appropriate diagnosis and classification of RCD.

The staging of multiple pulmonary adenocarcinomas requires the distinction of intrapulmonary metastasis (IPM) from multiple primary lung cancers (MPLCs). This can be challenging in some patients, and the addition of data from oncogenic driver mutations in these tumors may be helpful in this determination.

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATMgene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM(c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

Large cancer panels are being increasingly used in the practice of precision medicine to generate genomic profiles of tumors with the goal of identifying targetable variants and guiding eligibility for clinical trials. To facilitate identification of mutations in a broad range of solid and hematological malignancies, a 467-gene oncology panel (Columbia Combined Cancer Panel) was developed in collaboration with pathologists and oncologists and is currently available and in use for clinical diagnostics. Herein, we share our experience with this testing in an academic medical center. Of 255 submitted specimens, which encompassed a diverse range of tumor types, we were able to successfully sequence 92%. The Columbia Combined Cancer Panel assay led to the detection of a targetable variant in 48.7% of cases. However, although we show good clinical performance and diagnostic yield, third-party reimbursement has been poor. Reimbursement from government and third-party payers using the 81455 Current Procedural Terminology code was at 19.4% of billed costs, and 55% of cases were rejected on first submission. Likely contributing factors to this low level of reimbursement are the delays in valuation of the 81455 Current Procedural Terminology code and in establishing national or local coverage determinations. In the absence of additional demonstrations of clinical utility and improved patient outcomes, we expect the reimbursement environment will continue to limit the availability of this testing more broadly.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KITmutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KITmutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRASmutation, as well as a BRAFmutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1in 20% of cases, which represented the second-most frequently mutated gene after KITin our series. Furthermore, we identify SF3B1mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

An epidemic caused by an outbreak of monkeypox (mpox) in May 2022 rapidly spread internationally, requiring an urgent response from the clinical diagnostics community. A detailed description of the clinical validation and implementation of a laboratory-developed real-time PCR test for detecting nonvariola Orthopoxvirus-specific DNA based on the newly designed RealStar Zoonotic Orthopoxvirus assay is presented. The validation was performed using an accuracy panel (n = 97) comprising skin lesion swabs in universal transport media and from mpox virus genomic DNA spiked into pooled mpox virus–negative remnant universal transport media of lesion specimens submitted for routine clinical testing in the NewYork-Presbyterian Hospital clinical laboratory system. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to three different reference tests. Analytical sensitivity with 95% detection probability was 126 copies/mL, and analytical specificity, clinical sensitivity, and clinical specificity were 100%. In summary, the RealStar Zoonotic Orthopoxvirus assay provides a sensitive and reliable method for routine diagnosis of mpox infections.

Thymidylate synthase expression is known to be higher in squamous cell carcinoma than in adenocarcinoma of the lung. It is thought that this is the reason for the poor efficacy of pemetrexed in squamous cell carcinoma. However, there is limited data on thymidylate synthase expression in adenosquamous carcinoma, a distinct subtype of lung cancer containing both squamous and glandular differentiation. Furthermore, molecular alterations like epidermal growth factor receptor and Kirsten rat sarcoma 2 viral oncogene homolog mutations, which are seen in adenocarcinomas, are not well understood in mixed histology tumors such as adenosquamous carcinoma. In our study, we sought to better characterize adenosquamous tumors of the lung. Using immunohistochemistry to evaluate thymidylate synthase protein levels, we found that the expression of thymidylate synthase in these mixed tumors roughly parallel that of squamous cell carcinoma, instead of falling in between squamous cell and adenocarcinoma. Of note, in adenosquamous samples, the expression of thymidylate synthase was more closely correlated within the two components than would be expected by random chance alone. Also, we had a relatively high rate of epidermal growth factor receptor (11%) and Kirsten rat sarcoma 2 viral oncogene homolog (33%) mutations in these specimens, with the mutations showing convergence in both the glandular and squamous components upon microdissection. Our results indicate that adenosquamous carcinomas are not simple mixtures of their two histological components; they rather behave as their own entity, and it is important to further understand their behavior. Given the similarity of thymidylate synthase expression between squamous cell and adenosquamous carcinoma, and that thymidylate synthase is the main target of pemetrexed, we extrapolate that pemetrexed may also have inferior clinical activity in adenosquamous carcinoma.

Thymidylate synthaseexpression is known to be higher in squamous cell carcinoma than in adenocarcinoma of the lung. It is thought that this is the reason for the poor efficacy of pemetrexed in squamous cell carcinoma. However, there is limited data on thymidylate synthaseexpression in adenosquamous carcinoma, a distinct subtype of lung cancer containing both squamous and glandular differentiation. Furthermore, molecular alterations like epidermal growth factor receptor and Kirsten rat sarcoma 2 viral oncogene homolog mutations, which are seen in adenocarcinomas, are not well understood in mixed histology tumors such as adenosquamous carcinoma. In our study, we sought to better characterize adenosquamous tumors of the lung. Using immunohistochemistry to evaluate thymidylate synthaseprotein levels, we found that the expression of thymidylate synthasein these mixed tumors roughly parallel that of squamous cell carcinoma, instead of falling in between squamous cell and adenocarcinoma. Of note, in adenosquamous samples, the expression of thymidylate synthasewas more closely correlated within the two components than would be expected by random chance alone. Also, we had a relatively high rate of epidermal growth factor receptor (11%) and Kirsten rat sarcoma 2 viral oncogene homolog (33%) mutations in these specimens, with the mutations showing convergence in both the glandular and squamous components upon microdissection. Our results indicate that adenosquamous carcinomas are not simple mixtures of their two histological components; they rather behave as their own entity, and it is important to further understand their behavior. Given the similarity of thymidylate synthaseexpression between squamous cell and adenosquamous carcinoma, and that thymidylate synthaseis the main target of pemetrexed, we extrapolate that pemetrexed may also have inferior clinical activity in adenosquamous carcinoma.

Collagenous sprue is associated with high morbidity; however, the etiology of this disorder is unclear. Data regarding the pathological and clinical manifestations of patients with collagenous sprue are also limited. We, thus, undertook this study to gain insight into the etiology, disease manifestations and outcomes of collagenous sprue. We searched our departmental database (1999–2008) to identify cases of collagenous sprue and to obtain clinical and laboratory data. Small bowel histology, including thickness of subepithelial collagen, intra-epithelial lymphocyte phenotype and results of T-cell clonality assays were evaluated. Nineteen patients (15 women, 4 men, age 22–80 years, mean 57 years) were identified. Seventeen (89%) had celiac disease and two had unclassified sprue; 9 of 17 (53%) celiac disease patients had refractory disease; 5 of 15 (33%) lacked diarrhea (atypical presentation), including 2 of 6 (33%) with active (untreated) celiac disease and 3 of 9 (33%) with refractory celiac disease. Autoimmune disorders were seen in 12 of 19 (63%) patients and microscopic colitis (n=7), lymphocytic gastritis (n=2) or collagenous gastritis (n=2) were seen in nine patients. Subepithelial collagen thickness was mildly (n=6), moderately (n=10), or markedly (n=3) increased and villous atrophy was total (n=13) or subtotal (n=6). Phenotypically aberrant intraepithelial lymphocytes were not detected in any case. Polymerase chain reaction analysis showed a dominant T-cell clone in the only patient with refractory celiac disease type II. Histological improvement occurred in 7 of 11 (64%) patients. Overall, 8 of 19 (42%) responded to gluten-free diet, including 2 of 9 (22%) with refractory celiac disease and 10 responded to immunomodulatory therapy, including 6 of 9 (67%) with refractory celiac disease. Only one patient died from complications of refractory celiac disease. No patient developed lymphoma. The vast majority of our patients with collagenous sprue had celiac disease. Although, many patients required immunomodulatory therapy for symptom control, a subset responded to gluten-free diet alone. In our experience, collagenous sprue patients had relatively good clinical outcomes.

Cell-lineage-specific transcription factors are a group of regulatory proteins expressed in embryonic, differentiated, or neoplastic cells of the same lineage and represent a valuable repertoire of tissue-specific markers for the diagnosis of human tumors. Together with PAX2, PAX8 is a nephric-lineage transcription factor and is required for the establishment of renal-lineage cells and the formation of the kidney. In contrast to PAX2, little is known about the expression of PAX8 in adult kidney and renal tumors. In this study, we used immunohistochemistry to investigate the expression of PAX8 in adult human kidney and renal epithelial tumors. We report here that PAX8 was detected in renal epithelial cells in all segments of renal tubules from the proximal tubules to the renal papillae and in the parietal cells of Bowman's capsule in the adult kidney. PAX8 was also present in 98% of clear cell renal cell carcinomas (RCCs), 90% of papillary RCCs, and 95% of oncocytomas, similar to PAX2. In addition, PAX8 was found in 82% of chromophobe RCCs, 71% of sarcomatoid components of RCCs, and 100% (2/2) of renal medullary carcinomas. Overall, PAX8 was detected in 85% of metastatic renal tumors. Interestingly, expression of PAX8 was noted in some urothelial cells in the renal pelvis and ureters and ∼23% of urothelial carcinomas of the renal pelvis, but not in the urothelium or urothelial carcinomas of the urinary bladder; this probably underlines the different embryonic origins of urothelial cells in the upper and lower urinary tracts. As shown in this study, PAX8 is widely expressed in normal and neoplastic renal tissues. PAX8 may be a useful additional marker for renal epithelial tumors; however, its specificity and sensitivity await further investigation.

Cell-lineage-specific transcription factors are a group of regulatory proteins expressed in embryonic, differentiated, or neoplastic cells of the same lineage and represent a valuable repertoire of tissue-specific markers for the diagnosis of human tumors. Together with PAX2, PAX8is a nephric-lineage transcription factor and is required for the establishment of renal-lineage cells and the formation of the kidney. In contrast to PAX2, little is known about the expression of PAX8in adult kidney and renal tumors. In this study, we used immunohistochemistry to investigate the expression of PAX8in adult human kidney and renal epithelial tumors. We report here that PAX8was detected in renal epithelial cells in all segments of renal tubules from the proximal tubules to the renal papillae and in the parietal cells of Bowman's capsule in the adult kidney. PAX8was also present in 98% of clear cell renal cell carcinomas (RCCs), 90% of papillary RCCs, and 95% of oncocytomas, similar to PAX2. In addition, PAX8was found in 82% of chromophobe RCCs, 71% of sarcomatoid components of RCCs, and 100% (2/2) of renal medullary carcinomas. Overall, PAX8was detected in 85% of metastatic renal tumors. Interestingly, expression of PAX8was noted in some urothelial cells in the renal pelvis and ureters and ∼23% of urothelial carcinomas of the renal pelvis, but not in the urothelium or urothelial carcinomas of the urinary bladder; this probably underlines the different embryonic origins of urothelial cells in the upper and lower urinary tracts. As shown in this study, PAX8is widely expressed in normal and neoplastic renal tissues. PAX8may be a useful additional marker for renal epithelial tumors; however, its specificity and sensitivity await further investigation.

Hispanic-Americans are the fastest growing minority group in the United States. Many studies have compared prostate cancer treatment outcomes between black and white men, but few such studies have been done with Hispanic men. We compared clinical and pathological features as well as the treatment failure rate of radical prostatectomy in contemporaneously treated groups of Hispanic and white men with prostate cancer.

Hispanic-Americans are the most rapidly growing population in the United States. Although many studies have assessed differences in pathological stage at radical prostatectomy between white and black American men, to our knowledge none has assessed it in Hispanic men. We compared pathological stage at radical prostatectomy in contemporaneous groups of Hispanic and white American men.

Most forms of interstitial nephritis are cell mediated and lack tubulointerstitial immune deposits. These forms include allergic, infectious, and idiopathic interstitial nephritis. Immune complex deposits in the tubular basement membranes and interstitium most commonly are encountered in conjunction with glomerular diseases. Predominantly tubulointerstitial immune deposits without significant glomerular involvement can occur in Sjögren's syndrome and in a small subset of lupus nephritis. We report eight unusual cases of tubulointerstitial nephritis with massive tubulointerstitial immune deposits occurring in adults with hypocomplementemia and no evidence of systemic lupus erythematosus or Sjögren's disease. Most patients were older men. The renal biopsy specimens manifested a spectrum of changes ranging from tubulointerstitial nephritis to atypical lymphoid hyperplasia to changes suggestive of marginal zone B-cell lymphoma. Chronic local antigenic stimulation may predispose to lymphoma in these cases, analogous to what is postulated to occur in cases of mucosa-associated lymphoid tissue (MALT) lymphomas in extranodal sites, such as salivary gland, stomach, and thyroid. The preferential tubulointerstitial immune deposition and significant interstitial plasma cell component suggest pathomechanisms that involve local immune complex formation.

The ability to identify inviduals who are predisposed to specific malignant tumors is a promising molecular diagnostic byproduct of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2 of Ashkenazi Jews carry recurrent germline mutations in either the BRCA1 or BRCA2 genes that may predispose these individuals to the development of breast and ovarian cancer. We have developed a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutant alleles, which can be used to identify the BRCA1 185delAG and the BRCA2 6174delT mutations. The same assay can also be used to verify the loss of heterozygosity in a tumor sample arising in an individual with a germline mutation. The four steps described in this report (PCR amplification, heteroduplex formation, acrylamide gel electrophoresis, and ethidium bromide staining/UV-fluorescence photography) can be readily and reproducibly performed in the course of a single day, making this a useful method for the routine identification of these mutations.

P53 and p21WAF1/CIP1(p21) immunostaining was performed on 92 non-Hodgkin's lymphomas (NHLs), and the staining pattern correlated with the presence or absence of p53 hot spot mutations as detected by PCR-SSCP of exons 5–8 and direct sequencing. Twenty-nine of 92 lymphomas overexpressed p53, and 17 overexpressed p21. Of the p53 overexpressing lymphomas, 14 also overexpressed p21, and none of these 14 harbored a detectable hot spot mutation. However, mutations were detected in 13 (87) of 15 p53 overexpressing, p21 negative lymphomas. One of the 63 p53-negative lymphomas harbored a detectable hot spot mutation, and it was also negative for p21. These results demonstrate that among NHLs that overexpress p53 protein, those which also show p21 overexpression do not harbor p53 hot spot mutations, and furthermore, provide evidence that the transactivating function of p53 is retained. On the other hand, p53 overexpression in NHLs that lack p21 expression is usually indicative of p53 gene mutation.

To determine whether programmed myocyte cell death is a major component of the differential growth adaptation of the right and left ventricle during development, the formation of DNA strand breaks in myocyte nuclei was identified and quantitated in hearts of rats at the end of gestation and at 1, 5, 11, and 21 days after birth. Incorporation of BrdU in left and right ventricular myocytes was also evaluated. Moreover, the expression of bcl-2 in myocytes was determined. Programmed myocyte cell death was absent in the fetal heart but affected the myocardium postnatally. This phenomenon was no longer detectable at 21 days. DNA strand breaks in nonmyocyte nuclei were present at all time intervals. Quantitatively, 10.4, 6.1, and 2.5 myocyte nuclei/10,000 nuclei exhibited DNA degradation at 1 day in the right ventricle, interventricular septum, and left ventricle, respectively. Corresponding values at 5 days were 3.7, 3.5, and 2.0 myocyte nuclei/10,000 nuclei. At 11 days, programmed cell death involved 1.2, 1.5, and 0.53 myocyte nuclei/10,000 nuclei in these three regions of the heart. The 4.2-fold, 1.9-fold, and 2.3-fold greater magnitude of programmed cell death in the right ventricle at 1, 5, and 11 days was statistically significant. In contrast, BrdU incorporation in myocytes decreased in a comparable manner in the left and right ventricles with maturation. Importantly, bcl-2 mRNA levels were high in fetal myocytes, decreased markedly at 1 and 5 days, and progressively increased at 11 and 21 days. The expression of bcl-2 was lower in right than in left ventricular myocytes. In conclusion, programmed myocyte cell death is inversely related to bcl-2 expression and affects the right ventricle more than the left ventricle during postnatal development. This phenomenon may be crucial in the modulation of the number of myocytes in the two ventricles during the transition from the fetal to the adult circulatory system.

Mitochondrial DNA (mtDNA) depletion syndromes are a group of autosomal recessive disorders associated with a spectrum of clinical diseases, which include progressive external ophthalmoplegia (PEO). They are caused by variants in nuclear DNA (nDNA) encoded genes, and the gene that encodes for mtDNA polymerase gamma (POLG) is commonly involved. A splice-site mutation in POLG, c.3104+3A > T, was previously identified in three families with findings of PEO, and studies demonstrated this variant to result in skipping of exon 19. Here, we report a 57-year-old female who presented with ophthalmoplegia, ptosis, muscle weakness, and exercise intolerance with a subsequent muscle biopsy demonstrating mitochondrial myopathy on histopathologic evaluation and multiple mtDNA deletions by southern blot analysis. Whole-exome sequencing identified the previously characterized c. 3104+3A > T splice-site mutation in compound heterozygosity with a novel frameshift variant, p.Gly23Serfs∗236 (c.67_88del). mtDNA copy number analysis performed on the patient’s muscle showed mtDNA depletion, as expected in a patient with biallelic pathogenic mutations in POLG. This is the first reported case with POLG p.Gly23Serfs∗236, discovered in a patient presenting with features of PEO.

The 2018 updated molecular testing guidelines for patients with advanced lung cancer incorporated ALKimmunohistochemistry (IHC) analysis as an equivalent to fluorescence in situ hybridization (FISH) method recommended in 2013. Nevertheless, no specific recommendation for alternative methods was proposed owing to insufficient data. The aim of this study was to compare the results of ALKIHC, FISH, RNA next-generation sequencing (NGS), and RNA in situ hybridization (ISH) with available clinical data.

Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations.

No abstract.

Background:Plasmablastic lymphoma (PBL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma (B-NHL) that typically occurs in immunocompromised individuals, especially those with HIV infection or patients receiving immunosuppressive therapy for autoimmune diseases or organ allografts. Cytogenetic and gene expression profile analyses of PBL have provided some biological insights. However, the molecular and genetic alterations underlying PBL are not known. In this study, we assessed the pathologic features and spectrum of genetic mutations in a series of post-transplant PBL (PT-PBL) in order to identify molecular pathways potentially contributing to disease pathogenesis.

No relevant conflicts of interest to declare.

Hairy cell leukemia (HCL) is a rare type of B-cell non Hodgkin lymphoma (B-NHL), which is characterized by neoplastic cells exhibiting slender cytoplasmic projections and an activated phenotype. Unlike other B-NHL, HCL lacks characteristic recurrent chromosome abnormalities and its etiology remains elusive. Recently, the BRAF V600E mutation was described to occur at a high frequency in HCL in contrast to other B-NHL, suggesting that the BRAF V600E mutation could represent a disease defining lesion and a possible therapeutic target. In this study, we determined the prevalence of the BRAF V600E mutation in a large series of low and intermediate grade B-NHL and assessed the relationship of this mutation, if any, with microsatellite instability (MSI).DNA was extracted from various low and intermediate grade B-NHL (n=100), as defined by the 2008 World Health Organization Classification, which comprised HCL (n=10 from 6 patients), chronic lymphocytic leukemia/small lymphocytic lymphoma (n=22), mantle cell lymphoma (n=19), marginal zone lymphoma (n=22), follicular lymphoma (n=20) and lymphoplasmacytic lymphoma (n=7). Percentage of neoplastic cells was assessed by flow cytometry (n=99) or immunohistochemistry (n=1). Presence of BRAF V600E mutation was determined using a real time PCR assay using allele-specific hydrolysis (“Taqman”) probes. The detection limit of the assay was determined by diluting DNA extracted from a fresh HCL sample with normal high molecular weight DNA extracted by the same method. MSI analysis was performed using a multiplex reaction for 5 quasi monomorphic mononucleotide repeat markers (BAT-25, BAT-26, MONO-27, NR-21 and NR-24) and two highly polymorphic penta nucleotide markers (Penta C and Penta D) as sample identifiers. In the absence of normal DNA for comparison, a sample was considered MSI-H if greater than two markers showed an altered pattern, indeterminate if only two showed an alteration and MSS if no marker showed an altered allele.The ubiquitous occurrence and high specificity of the BRAF V600E mutation for HCL was confirmed in our series of B-NHL. The HCL samples consisted of 10 specimens from 6 patients and were the initial diagnostic specimen in 2 patients. The percentage of neoplastic cells in these samples ranged from 1% to 78.4% (median 18.2%). The BRAF V600E mutation was detected in 7 of 10 (70%) samples of HCL from 5 of 6 (83%) patients. The proportion of hairy cells was ≤5% in samples where the mutation was not detected. All 90 of the other low and intermediate grade B-NHL (>50% tumor burden) were negative for the V600E mutation. Compared to the reported sensitivity of approximately 30% tumor cells for detecting the BRAF V600E mutation by Sanger sequencing, our real time PCR assay allowed detection of the mutation in samples containing ≥9.8% tumor cells. Analysis of the HCL cases for MSI revealed that all cases had a microsatellite stable (MSS) phenotype.The BRAF V600E mutation appears specific for HCL among low and intermediate grade B-NHL and is not associated with microsatellite instability. These characteristics thus warrant inclusion in disease definition. Further refinements of a realtime PCR based approach for detecting the BRAF V600E mutation might be of utility in diagnosis and disease monitoring, as the neoplastic cellular yield is often limited in HCL due to underlying myelofibrosis.No relevant conflicts of interest to declare.

The article presents a case study of Diamond Blackfan anemia (DBA) and the use of next-generation sequencing (NGS) to determine the causative mutation of the disease in a 42-day-old female infant. Physical examination of the infant showed no compensatory reticulocytosis, a congenital anemia related to bone marrow failure, is discussed. It mentions the cause of a met1-to-val (M1V) substitution that removes the start codon.