Utility of methylated DNA markers for the diagnosis of malignant biliary strictures.

BACKGROUND AND AIMS: Early identification of malignant biliary strictures (MBSs) is challenging, with up to 20% classified as indeterminants after preliminary testing and tissue sampling with endoscopic retrograde cholangiopancreatography. We aimed to evaluate the use of methylated DNA markers (MDMs) from biliary brushings to enhance MBS detection in a prospective cohort. APPROACH: Candidate MDMs were evaluated for their utility in MBS diagnosis through a series of discovery and validation phases. DNA was extracted from biliary brushing samples, quantified, bisulfite-converted, and then subjected to methylation-specific droplet digital polymerase chain reaction.  Patients were considered to have no malignancy if the sampling was negative and there was no evidence of malignancy after 1 year or definitive negative surgical histopathology. RESULTS: Fourteen candidate MDMs were evaluated in the discovery phase, with top-performing and new markers evaluated in the technical validation phase. The top 4 MDMs were TWIST1, HOXA1, VSTM2B, and CLEC11A, which individually achieved AUC values of 0.82, 0.81, 0.83, and 0.78, respectively, with sensitivities of 59.4%, 53.1%, 62.5%, and 50.0%, respectively, at high specificities for malignancy of 95.2%-95.3% for the final biologic validation phase. When combined as a panel, the AUC was 0.86, achieving 73.4% sensitivity and 92.9% specificity, which outperformed cytology and fluorescence in situ hybridization (FISH). CONCLUSIONS: The selected MDMs demonstrated improved performance characteristics for the detection of MBS compared to cytology and FISH. Therefore, MDMs should be considered viable candidates for inclusion in diagnostic testing algorithms.

Spinal cord high-grade infiltrating gliomas in adults: clinico-pathological and molecular evaluation.

Primary high-grade infiltrating gliomas of the spinal cord are rare, with prior series including limited numbers of cases and reporting poor outcomes. Additionally, the molecular profile of high-grade infiltrating gliomas of the spinal cord has not been well characterized. We identified 13 adult patients whose surgery had been performed at our institution over a 26-year-period. Radiologically, nine cases harbored regions of post-contrast enhancement. Existing slides were reviewed, and when sufficient tissue was available, immunohistochemical stains (IDH1-R132H, H3-K27M, H3K27-me3, ATRX, p53 and BRAF-V600E), and a targeted 150-gene neuro-oncology next-generation sequencing panel were performed. The 13 patients included 11 men and 2 women with a median age of 38 years (range = 18-69). Histologically, all were consistent with an infiltrating astrocytoma corresponding to 2016 WHO grades III (n = 5) and IV (n = 8). By immunohistochemistry, six cases were positive for H3K27M, all showing concomitant loss of H3K27-me3. Next-generation sequencing was successfully performed in ten cases. Next-generation sequencing studies were successfully performed in four of the cases positive for H3K27M by immunohistochemistry, and all were confirmed as H3F3A K27M-mutant. Additional recurrent mutations identified included those of TERT promoter (n = 3), TP53 (n = 5), PPM1D (n = 3), NF1 (n = 3), ATRX (n = 2), and PIK3CA (n = 2). No HIST1H3B, HIST1H3C, IDH1, IDH2, or BRAF mutations were detected. Ten patients have died since first surgery, with a median survival of 13 months and 1 year of 46%. Median survival was 48.5 months for H3K27M-positive cases, compared to 1 month for those with TERT promoter mutation and 77 months for those harboring neither (p = 0.019). Median survival for cases with TP53 mutations was 11.5 months and for those with PPM1D mutations was 84 months. Our findings suggest that high-grade infiltrating gliomas of the spinal cord in adults represent a heterogeneous group of tumors, with variable outcomes possibly related to their molecular profiles.

A Multicenter Study of a Fluorescence In Situ Hybridization Probe Set for Diagnosing High-Grade Dysplasia and Adenocarcinoma in Barrett's Esophagus.

BACKGROUND AND AIMS: Preliminary single-institution data suggest that fluorescence in situ hybridization (FISH) may be useful for detecting high-grade dysplasia (HGD) and esophageal adenocarcinoma (EA) in patients with Barrett's esophagus (BE). This multicenter study aims to validate the measurement of polysomy (gain of at least two loci) by FISH as a way to discriminate degrees of dysplasia in BE specimens. METHODS: Tissue specimens were collected from four different hospitals and read by both the local pathology department ("Site diagnosis") and a single central pathologist ("Review diagnosis") at a separate institution. The specimens then underwent FISH analysis using probes 8q24 (MYC), 9p21 (CDKN2A), 17q12 (ERBB2), and 20q13 (ZNF217) for comparison. A total of 46 non-BE, 42 non-dysplastic specialized intestinal metaplasia (SIM), 23 indefinite-grade dysplasia (IGD), 10 low-grade dysplasia (LGD), 29 HGD, and 42 EA specimens were analyzed. RESULTS: We found that polysomy, as detected by FISH, was the predominant chromosomal abnormality present as dysplasia increased. Polysomy was also the best predictor for the presence of dysplasia or EA when comparing its area under the curve to that of other FISH abnormalities. We observed that if at least 10% of cells had polysomy within a specimen, the FISH probe was able to differentiate between EA/HGD and the remaining pathologies with a sensitivity of 80% and a specificity of 88%. CONCLUSIONS: This study demonstrates that using FISH to determine the percentage of cells with polysomy can accurately and objectively aid in the diagnosis of HGD/EA in BE specimens.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

BACKGROUND & AIMS: Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. METHODS: We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. RESULTS: The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011). CONCLUSIONS: We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.

FGFR1 and FGFR2 in fibrolamellar carcinoma.

AIMS: Fibrolamellar carcinoma is characterized by a recurrent DNAJB1-PRKACA chimeric transcript. The functional properties of the fusion are unknown, but are believed to include PRKACA up-regulation. PRKCA is a subunit of protein kinase A. The downstream targets of protein kinase A are unknown, but may include interactions with fibroblast growth factor receptor (FGFR) pathways. In addition, inhibitors for FGFR proteins have been developed recently. METHODS AND RESULTS: Nineteen histologically confirmed fibrolamellar carcinomas were studied. All showed the characteristic DNAJB1-PRKACA transcript by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry for FGFR1 was negative in 19 of 19 cases using a monoclonal antibody, while a polyclonal antibody showed no expression (n = 11) or weak and focal expression (n = 8). RNAin-situ hybridization was 2+ in two cases, 1+ in four cases and negative in four cases. FGFR1 fluorescence in-situ hybridization (FISH) revealed polysomy of chromosome 8 in 17 of 19 cases. Break-apart FISH for FGFR2 was negative for rearrangements in 12 of 12 informative cases. CONCLUSIONS: Fibrolamellar carcinomas show polysomy of chromosome 8 and the FGFR1 locus, and only modest mRNA expression and weak or absent expression at the protein level. FGFR2 rearrangement was not detected. These data reduce the likelihood that FGFR inhibitors will be effective in the treatment of most fibrolamellar carcinomas.

Fluorescence in situ hybridization compared with conventional cytology for the diagnosis of malignant biliary tract strictures in Asian patients.

BACKGROUND AND AIMS: Fluorescence in situ hybridization (FISH) has improved the diagnostic performance of cytology for the evaluation of malignant biliary strictures in the United States and Europe. The utility of FISH for the diagnosis of biliary strictures in Asia is currently unknown. We aimed to compare the sensitivity of FISH and conventional cytology for the diagnosis of malignant biliary strictures in Thai patients. METHODS: A prospective study was performed at 2 university hospitals between 2010 and 2013. Patients being evaluated for malignant-appearing biliary strictures were included (N = 99). Bile duct brushings were collected and assessed by cytology and FISH. Sensitivities with 95% confidence intervals of cytology and FISH were the main outcome measures. RESULTS: The overall sensitivities of cytology and FISH were 38% and 55%, respectively (P = .001). For those with a diagnosis of cancer based on clinical evidence without biopsy confirmation (n = 44), the sensitivities of cytology and FISH were 43% and 57%, respectively (P = .06). For the 49 patients for whom a cancer diagnosis was confirmed by pathology, FISH had a significantly higher sensitivity than cytology, with a sensitivity of 53% versus 33%, respectively (P = .008). CONCLUSIONS: FISH improves the diagnostic performance of cytology and can be used as a complementary tool to bile duct brushing and biopsy for the evaluation of malignancy in biliary strictures in Asian populations.

Biliary multifocal chromosomal polysomy and cholangiocarcinoma in primary sclerosing cholangitis.

OBJECTIVES: Polysomy detected by fluorescence in situ hybridization (FISH) is associated with cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis (PSC). However, a subset of PSC patients with polysomy do not manifest CCA even after long-term follow-up. It is unknown if patients with chromosomal gains detected by FISH in multiple areas of the biliary tree (i.e., multifocal polysomy, MFP) are more likely to be diagnosed with CCA than patients with unifocal polysomy (UFP). Therefore, our aim is to determine whether patients with MFP are more likely to manifest CCA compared with patients with other chromosomal abnormalities including UFP and other FISH subtypes. METHODS: We performed a retrospective review of PSC patients without a mass lesion who underwent FISH testing at our institution from 1 January 2005 to 1 July 2013. RESULTS: Three-hundred and seventy-one PSC patients were included. Compared with patients with UFP, those with MFP were more likely to have weight loss (32 vs. 9%), suspicious cytology (45 vs. 13%) and develop serial polysomy (91 vs. 35%). MFP was associated with CCA (hazard ratio (HR), 82.42; 95% confidence interval (CI), 24.50-277.31) and was the strongest predictor of cancer diagnosis. Suspicious cytology (HR, 26.31; 95% CI, 8.63-80.24) and UFP (HR, 13.27; 95% CI, 3.32-53.08) were also predictive of CCA. MFP, UFP and suspicious cytology remained associated with CCA in the multivariable model. CONCLUSIONS: Compared with other FISH subtypes, MFP is the strongest predictor of CCA. However, patients with UFP and suspicious cytology (regardless of FISH status) are also at an increased risk for CCA.

Clinical Validation of a Targeted Next-Generation Sequencing Panel for Lymphoid Malignancies.

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin.

MICon Contamination Detection Workflow for Next-Generation Sequencing Laboratories Using Microhaplotype Loci and Supervised Learning.

Innovation in sequencing instrumentation is increasing the per-batch data volumes and decreasing the per-base costs. Multiplexed chemistry protocols after the addition of index tags have further contributed to efficient and cost-effective sequencer utilization. With these pooled processing strategies, however, comes an increased risk of sample contamination. Sample contamination poses a risk of missing critical variants in a patient sample or wrongly reporting variants derived from the contaminant, which are particularly relevant issues in oncology specimen testing in which low variant allele frequencies have clinical relevance. Small custom-targeted next-generation sequencing (NGS) panels yield limited variants and pose challenges in delineating true somatic variants versus contamination calls. A number of popular contamination identification tools have the ability to perform well in whole-genome/exome sequencing data; however, in smaller gene panels, there are fewer variant candidates for the tools to perform accurately. To prevent clinical reporting of potentially contaminated samples in small next-generation sequencing panels, we have developed MICon (Microhaplotype Contamination detection), a novel contamination detection model that uses microhaplotype site variant allele frequencies. In a heterogeneous hold-out test cohort of 210 samples, the model displayed state-of-the-art performance with an area under the receiver-operating characteristic curve of 0.995.

FGFR mRNA Expression in Cholangiocarcinoma and Its Correlation with FGFR2 Fusion Status and Immune Signatures.

PURPOSE: Selective FGFR inhibitors are effective against cholangiocarcinomas that harbor gene alterations in FGFR2. Clinical trials suggest that expression of wild-type FGFR mRNA can predict sensitivity to FGFR inhibitors, but this biomarker has not been well characterized in cholangiocarcinoma. This study explores the prevalence of FGFR mRNA overexpression in cholangiocarcinoma, its role in predicting sensitivity to FGFR inhibitors, and its association with immune markers. EXPERIMENTAL DESIGN: Tissue microarrays of intrahepatic (ICC) and extrahepatic cholangiocarcinomas (ECC) resected between 2004 and 2015 were used to evaluate FGFR1-4 mRNA expression levels by RNA in situ hybridization (ISH). Expression levels of FGFR2 mRNA were correlated with FGFR2 fusion status and with patient outcomes. Immune markers expression was assessed by IHC and CSF1 and CSF1 receptor expression were examined by RNA ISH. RESULTS: Among 94 patients with resected cholangiocarcinoma, the majority had ICC (77%). FGFR2 fusions were identified in 23% of ICCs and 5% of ECCs. High levels of FGFR mRNA in FGFR2 fusion-negative ICC/ECC were seen for: FGFR1 (ICC/ECC: 15%/0%), FGFR2 (ICC/ECC: 57%/0%), FGFR3 (ICC/ECC: 53%/18%), and FGFR4 (ICC/ECC: 32%/0%). Overall, 62% of fusion-negative cholangiocarcinomas showed high levels of FGFR mRNA. In patients with advanced FGFR2 fusion-positive ICC, high levels of FGFR2 mRNA did not correlate with clinical benefit. FGFR2 fusion-positive tumors showed a paucity of PD-L1 on tumor cells. CONCLUSIONS: FGFR mRNA overexpression occurs frequently in cholangiocarcinoma in the absence of genetic alterations in FGFR. This study identifies a molecular subpopulation in cholangiocarcinoma for which further investigation of FGFR inhibitors is merited outside currently approved indications.

Evaluating the Significance of Pancreatobiliary Fluorescence In Situ Hybridization Polysomy on Prognosis in De Novo Cholangiocarcinoma.

INTRODUCTION: We recently developed a fluorescence in situ hybridization probe set for evaluating suspicious biliary and pancreatic duct strictures (PB-FISH). We aimed to determine whether PB-FISH results in biliary brush cytology specimens are associated with outcomes of patients with cholangiocarcinoma (CCA). METHODS: We performed a retrospective study of patients with CCA tested by PB-FISH from January 2015 to August 2018. CCA was stratified by primary sclerosing cholangitis (PSC) into those with (PSC CCA) or without PSC ( de novo CCA). PB-FISH results were categorized as polysomy (gain of multiple loci), nonpolysomy (single locus gain, single locus gain with 9p21 loss, homozygous 9p21 loss, tetrasomy), and disomy (no abnormalities). Overall survival (OS) was estimated using Kaplan-Meier methods and compared between the PB-FISH results using log-rank tests. Cox models were adjusted for age, sex, CA 19-9, cytology results, source of brushing sample, and treatments. RESULTS: Characteristics of 264 eligible patients (median age 60.4; range 18-92) were comparable for patients with PB-FISH polysomy vs nonpolysomy vs disomy. The median OS was similar between disomy, nonpolysomy, and polysomy in the overall population (22.7 vs 22.7 vs 20.3 months, respectively). For de novo CCA, both polysomy and nonpolysomy were associated with worse OS compared with disomy (polysomy: hazard ratio [HR] = 2.09, 95% confidence interval [CI] = 1.14-3.83; nonpolysomy: HR = 2.4, 95% CI = 0.54-2.46; P = 0.027). For PSC CCA, neither polysomy nor nonpolysomy were significantly associated with worse OS (polysomy: 0.90, 95% CI = 0.47-1.75; nonpolysomy: HR = 1.78, CI = 0.71-4.49; P = 0.27). DISCUSSION: PB-FISH alterations are associated with worse survival in de novo CCA, though statistical significance was lost when adjusting for confounding variables.

Primary sclerosing cholangitis patients with serial polysomy fluorescence in situ hybridization results are at increased risk of cholangiocarcinoma.

OBJECTIVES: A polysomy fluorescence in situ hybridization (FISH) result in a pancreatobiliary brushing from a patient with primary sclerosing cholangitis (PSC) is very worrisome for carcinoma. However, treatment is not recommended unless verified by corroborative evidence of malignancy because of less than perfect test specificity in this population. The aim of this study was to evaluate the clinical outcome of PSC patients with serial polysomy FISH results. METHODS: Patients with PSC underwent endoscopic retrograde cholangiopancreatography with brushings when clinically indicated per standard practice. Brushings were evaluated by routine cytology and FISH. Retrospective review identified patients with a polysomy FISH result without definitive imaging or pathological evidence of malignancy at the time of the first polysomy, who underwent follow-up examinations including subsequent FISH testing (n=30). Patient records were reviewed to determine clinical outcome. RESULTS: In all, 9 of 13 patients (69%) with a subsequent polysomy result (i.e., serial polysomy) were diagnosed with cholangiocarcinoma (CCA) compared with 3 of 17 patients (18%) with subsequent non-polysomy results (P=0.008). There was a significant difference in time to a diagnosis of CCA between PSC patients with serial polysomy compared with those with subsequent non-polysomy (P=0.01). In four patients with serial polysomy results, imaging/pathological evidence of CCA was not found until 1-2.7 years after the initial polysomy FISH result. CONCLUSIONS: FISH may detect polysomic cells in pancreatobiliary brushings before other pathological or imaging techniques identify CCA. Patients with serial polysomy FISH results are at higher risk for having CCA than those with subsequent non-polysomy FISH results.

Long-term outcomes of positive fluorescence in situ hybridization tests in primary sclerosing cholangitis.

UNLABELLED: Patients with primary sclerosing cholangitis (PSC) are at increased risk for developing cholangiocarcinoma (CCA). Fluorescence in situ hybridization (FISH) is a cytological test designed to enhance early CCA diagnosis. The long-term outcome of PSC patients with a positive FISH test (polysomy, trisomy/tetrasomy) are unclear. All PSC patients with at least one FISH test were identified and defined to have CCA if they had a positive tissue biopsy, positive cytology, or evidence of cancer in the explant after liver transplantation. A total of 235 PSC patients had at least one FISH test performed, and 56 patients had CCA on histopathology (n = 35) or cytology (n = 21). Overall, 120 of 235 (51%) of PSC patients tested for FISH were positive, but only one third of these positive patients had CCA. Sensitivity and specificity for FISH polysomy were 46% and 88%, and for trisomy/tetrasomy they were 25% and 67%, respectively. Survival analysis showed that patients with FISH polysomy had an outcome similar to patients with CCA; whereas FISH trisomy/tetrasomy patients had an outcome similar to patients with negative FISH tests. The FISH polysomy patients without cancer compared with those with CCA had lower serum bilirubin, lower carbohydrate antigen 19-9 (CA 19-9), lower Mayo risk score, and lower occurrence of dominant strictures. CONCLUSION: In PSC patients, the presence of a dominant stricture plus FISH polysomy has a specificity of 88% for CCA. Patients with FISH showing trisomy or tetrasomy have a similar outcome to patients with negative FISH. FISH testing should be used selectively in patients with other signs indicating CCA and not as a screening tool in all PSC patients undergoing endoscopic retrograde cholangiopancreatography (ERCP).

Molecular Genetic Landscape of Sclerosing Pneumocytomas.

OBJECTIVES: Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. METHODS: To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). RESULTS: Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. CONCLUSIONS: Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.

SeekFusion - A Clinically Validated Fusion Transcript Detection Pipeline for PCR-Based Next-Generation Sequencing of RNA.

Detecting gene fusions involving driver oncogenes is pivotal in clinical diagnosis and treatment of cancer patients. Recent developments in next-generation sequencing (NGS) technologies have enabled improved assays for bioinformatics-based gene fusions detection. In clinical applications, where a small number of fusions are clinically actionable, targeted polymerase chain reaction (PCR)-based NGS chemistries, such as the QIAseq RNAscan assay, aim to improve accuracy compared to standard RNA sequencing. Existing informatics methods for gene fusion detection in NGS-based RNA sequencing assays traditionally use a transcriptome-based spliced alignment approach or a de-novo assembly approach. Transcriptome-based spliced alignment methods face challenges with short read mapping yielding low quality alignments. De-novo assembly-based methods yield longer contigs from short reads that can be more sensitive for genomic rearrangements, but face performance and scalability challenges. Consequently, there exists a need for a method to efficiently and accurately detect fusions in targeted PCR-based NGS chemistries. We describe SeekFusion, a highly accurate and computationally efficient pipeline enabling identification of gene fusions from PCR-based NGS chemistries. Utilizing biological samples processed with the QIAseq RNAscan assay and in-silico simulated data we demonstrate that SeekFusion gene fusion detection accuracy outperforms popular existing methods such as STAR-Fusion, TOPHAT-Fusion and JAFFA-hybrid. We also present results from 4,484 patient samples tested for neurological tumors and sarcoma, encompassing details on some novel fusions identified.

DNA Methylation Markers for Detection of Cholangiocarcinoma: Discovery, Validation, and Clinical Testing in Biliary Brushings and Plasma.

Cholangiocarcinoma (CCA) has poor prognosis due to late-stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced-representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin-fixed, paraffin-embedded CCA tumors and adjacent hepatobiliary control tissues using methylation-specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95-1.0). In the clinical pilot on plasma, a cross-validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81-0.95]) but not for primary sclerosing cholangitis-associated eCCA (AUC, 0.54 [0.35-0.73]). Conclusion: Next-generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

Ki67: a time-varying biomarker of risk of breast cancer in atypical hyperplasia.

Uncontrolled proliferation is a defining feature of the malignant phenotype. Ki67 is a marker for proliferating cells and is overexpressed in many breast cancers. Atypical hyperplasia is a premalignant lesion of the breast (relative risk approximately 4.0). Here, we asked if Ki67 expression could stratify risk in women with atypia. Ki67 expression was assessed immunohistochemically by digital image analysis in archival sections from 192 women with atypia diagnosed at the Mayo Clinic 1/1/67-12/31/91. Risk factor and follow-up data were obtained via study questionnaire and medical records. Observed breast cancer events were compared to population expected rates (Iowa SEER) using standardized incidence ratios (SIRs). We examined two endpoints: risk of breast cancer within 10 years and after 10 years of atypia biopsy. Thirty-two (16.7%) of the 192 women developed breast cancer over a median of 14.6 years. Thirty percent (58) of the atypias had >or=2% cells staining for Ki67. In these women, the risk of breast cancer within 10 years after atypia was increased (SIR 4.42 [2.21-8.84]) but not in those with <2% staining. Specifically, the cumulative incidence for breast cancer at 10 years was 14% in the high Ki67 vs. 3% in the low Ki67 group. Conversely, after 10 years, risk in the low Ki67 group rose significantly (SIR 5.69 [3.63-8.92]) vs. no further increased risk in the high Ki67 group (SIR 0.78 [0.11-5.55]). Ki67 appears to be a time-varying biomarker of risk of breast cancer in women with atypical hyperplasia.

Secondary renal neoplasia following chemotherapy or radiation in pediatric patients.

Renal neoplasia occurring as a second malignancy following childhood cancer has been most closely associated with neuroblastoma and Wilms tumor. While some cases have been associated with a genetic predisposition, nearly all are thought to result from "late effects" of therapy-related toxicity that involves chemotherapy or radiation. It is unclear if these tumors are enriched for specific molecular or morphologic characteristics. A query of our institutional nephrectomy registry of 8295 patients for renal neoplasia occurring post-treatment for childhood cancer revealed 6 patients with Wilms tumor, 4 with neuroblastoma, and 1 with acute lymphoblastic leukemia (ALL). Three additional cases of MiT family translocation renal cell carcinoma (RCC), from 2 patients, following chemotherapy for neuroblastoma and systemic lupus erythematosus and another of clear cell RCC post-ALL were included. The most common tumor type was clear cell RCC: 9/19 cases (47.4%), followed by metanephric adenoma and MiT family translocation RCC (3/19, 15.8%). There were no characteristic features to indicate a unique renal neoplasia subtype. Potential syndromic renal neoplasia occurred in 2 patients, metanephric adenomas and oncocytoma in a patient with hyperparathyroidism-jaw tumor syndrome post-treatment of Wilms tumor and a fumarate hydratase-deficient RCC in a patient post-treatment for ALL. The mean age at diagnosis of childhood neoplasia or treatment with chemotherapy or radiation was 4.7 years, and the average time to subsequent renal neoplasia was 31 years. Five (of 14) patients developed metastatic RCC, and there were 2 RCC-related deaths. These results indicate the need for extended clinical follow-up of these patients.