Combining copy number, methylation markers, and mutations as a panel for endometrial cancer detection via intravaginal tampon collection.

OBJECTIVE: We aimed to assess whether endometrial cancer (EC) can be detected in shed DNA collected with vaginal tampon by analyzing copy number, methylation markers, and mutations. METHODS: Tampons were collected prior to hysterectomy from 38 EC patients and 28 women with benign indications. Extracted tampon DNA underwent the following: 1) low-coverage whole genome sequencing (LC-WGS) to assess copy number, 2) pyrosequencing to measure percent promotor methylation of HOXA9, RASSF1, and CDH13 and 3) next generation sequencing (NGS) to identify mutations in 19 genes associated with EC identified through The Cancer Genome Atlas. Sensitivity and specificity for each test and test combinations were calculated. RESULTS: Methylation analysis yielded the highest specificities but lowest sensitivities (37-40% sensitivity; 100% specificity for HOXA9, RASSF1 and HTR1B) while mutation analysis had improved sensitivity (50% sensitivity; 83% specificity). Only one "false positive" result for copy number variants was identified among women with benign surgical indications, which was based on detection of copy number changes, and associated with a leiomyosarcoma that was only recognized at hysterectomy. Considering any of the 3 biomarker classes as a positive, resulted in a sensitivity of 92% and specificity of 86%. Mutation analysis did not add sensitivity to the combination of analysis of copy number and methylation. CONCLUSIONS: This study demonstrates a proof-of-principle for non-invasive yet precise detection of endometrial cancer. We propose that with improved biomarker testing, it may be possible to develop a clinically useful test for detecting EC.

Mutational Profile in Vulvar, Vaginal, and Urethral Melanomas: Review of 37 Cases With Focus on Primary Tumor Site.

Melanomas of female genital tract are rare tumors with poor prognosis. While BRAF-V600E is the most common pathogenic mutation seen in cutaneous sun-exposed melanomas, mucosal and anogenital melanomas usually lack BRAF mutations and instead they harbor KIT alterations. The American Joint Committee on Cancer staging guideline (AJCC eighth edition) recommends using cutaneous melanoma guidelines for vulvar melanoma staging and does not provide any recommendations for vaginal melanoma staging. The aim of this study is to investigate the mutational status of invasive melanomas arising from different anatomic sites in lower female genital tract (vulvar hair-bearing skin, glabrous skin, vagina and urethra) in a group of 37 patients. Tumors were analyzed using a DNA targeted next-generation sequencing panel covering the 21 most common genes and mutation hotspots in melanomas. The most common genetic alterations in invasive melanomas of lower female genital tract are KIT (32%), TP53 (22%), and NF1 (19%). Overall 66% (21/32) of cases showed a pathogenic alteration in at least one of the MAPK pathway genes. No statistical significance seen between different primary tumor sites and the frequency of the oncogenic mutations, nor were any significant differences found by mutation status. Only one case of urethral melanoma showed a BRAF non-V600E mutation (D594G). Our results suggest a similar molecular pathogenesis and overall survival in melanomas arising from lower female genital tract, irrespective of their exact location in the urogenital area. Future classifications of melanoma should consider grouping vulvar melanomas with mucosal rather than cutaneous melanomas.

Analysis of Cell-Free DNA to Assess Risk of Tumoremia Following Endoscopic Ultrasound Fine-Needle Aspiration of Pancreatic Adenocarcinomas.

BACKGROUND & AIMS: Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. METHODS: We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. RESULTS: Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. CONCLUSION: In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing.

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

Genomic rearrangements in sporadic lymphangioleiomyomatosis: an evolving genetic story.

Sporadic lymphangioleiomyomatosis is a progressive pulmonary cystic disease resulting from the infiltration of smooth muscle-like lymphangioleiomyomatosis cells into the lung. The migratory/metastasizing properties of the lymphangioleiomyomatosis cell together with the presence of somatic mutations, primarily in the tuberous sclerosis complex gene (TSC2), lead many to consider this a low-grade malignancy. As malignant tumors characteristically accumulate somatic structural variations, which have not been well studied in sporadic lymphangioleiomyomatosis, we utilized mate pair sequencing to define structural variations within laser capture microdissected enriched lymphangioleiomyomatosis cell populations from five sporadic lymphangioleiomyomatosis patients. Lymphangioleiomyomatosis cells were confirmed in each tissue by hematoxylin eosin stain review and by HMB-45 immunohistochemistry in four cases. A mutation panel demonstrated characteristic TSC2 driver mutations in three cases. Genomic profiles demonstrated normal diploid coverage across all chromosomes, with no aneuploidy or detectable gains/losses of whole chromosomal arms typical of neoplastic diseases. However, somatic rearrangements and smaller deletions were validated in the two cases which lacked TSC2 driver mutations. Most significantly, one of these sporadic lymphangioleiomyomatosis cases contained two different size deletions encompassing the entire TSC1 locus. The detection of a homozygous deletion of TSC1 driving a predicted case of sporadic lymphangioleiomyomatosis, consistent with the common two-hit TSC2 mutation model, has never been reported for sporadic lymphangioleiomyomatosis. However, while no evidence of the hereditary tuberous sclerosis complex disease was reported for this patient, the potential for mosaicism and sub-clinical phenotype cannot be ruled out. Nevertheless, this study demonstrates that somatic structural rearrangements are present in lymphangioleiomyomatosis disease and provides a novel method of genomic characterization of sporadic lymphangioleiomyomatosis cells, aiding in defining cases with no detected mutations by conventional methodologies. These structural rearrangements could represent additional pathogenic mechanisms in sporadic lymphangioleiomyomatosis disease, potentially affecting response to therapy and adding to the complex genetic story of this rare disease.

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.

Molecular cytology genotyping of primary and metastatic GI stromal tumors by using a custom two-gene targeted next-generation sequencing panel with therapeutic intent.

BACKGROUND AND AIMS: In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. METHODS: DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. RESULTS: Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. CONCLUSIONS: Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens.

Characterization of endoscopic ultrasound fine-needle aspiration cytology by targeted next-generation sequencing and theranostic potential.

Determination of tumor genetic architecture based on tissue analysis yields important information on signaling pathways involved in cancer pathogenesis and plays a growing role in choosing the optimal medical management of malignancies. Specifically, the advent of next-generation sequencing has led to a rapidly evolving era of relatively inexpensive, high-throughput DNA sequencing of tumors. One such example is multiplexed tumor genotyping (ie, panel testing) of more than 2800 mutations across 50 commonly mutated cancer-associated genes. This resulting mutational landscape shows medically actionable pathogenic alterations to optimize antitumor therapy. We recently assessed the performance and outcome of targeted next-generation sequencing with archived endoscopic ultrasound fine-needle aspirates across a broad range of primary and metastatic sites with encouraging accuracy. As a result, endoscopic ultrasound has the potential to move from a test for diagnosis or confirmation of malignancy, to one in which it could facilitate the personalization of cancer-directed therapy.

Kinase genotype analysis of gastric gastrointestinal stromal tumor cytology samples using targeted next-generation sequencing.

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Frequency of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathway pathogenic alterations in EUS-FNA sampled malignant lymph nodes in rectal cancer with theranostic potential.

BACKGROUND: Targeted next-generation sequencing has the potential to stratify a tumor by molecular subtype and aid the development of a biomarker profile for prognostic risk stratification and theranostic potential. OBJECTIVE: To assess the frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens. DESIGN: Multigene molecular profiling of archived malignant EUS-FNA lymph node cytology specimens using the Ion Ampliseq Cancer Hotspot Panel v2, which targets at least 2855 possible mutations within 50 cancer-associated genes. SETTING: Single tertiary referral center. PATIENTS: Sporadic, treatment naive, locally advanced primary rectal cancer by EUS-FNA (n = 76) who subsequently completed neoadjuvant therapy with on-site oncologic surgery. MAIN OUTCOME MEASUREMENTS: The frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens by the mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling pathways, by KRAS or NRAS wild-type lymph node status, by extramesenteric lymph node status, and by a complete pathologic response status. RESULTS: Eleven patients (14.5%) were 50-gene panel wild-type. Sixty-five patients had 139 pathogenic alterations (2 [1-3] per patient) in 13 of 50 evaluated genes. The following represent a spectrum of identified alterations: TP53 (n = 52; 68.4%), APC (n = 36; 47.4%), KRAS (n = 22; 28.9%), FBXW7 (n = 8; 10.5%), NRAS (n = 6; 7.9%), PIK3CA (n = 4; 5.3%), SMAD4 (n = 3; 3.9%), and BRAF (n = 3; 3.9%). Pathogenic alterations were identified in the MAPK and PI3K signaling pathways in 41% and 5% of patients, respectively. LIMITATIONS: Findings were limited to a 50 cancer-associated gene analysis. CONCLUSIONS: Molecular EUS lymph node assessments using cancer "hotspot" panels can identify pathogenic alteration frequency and distribution and have theranostic potential for individualized patient care.

Detection of endometrial cancer via molecular analysis of DNA collected with vaginal tampons.

OBJECTIVE: We demonstrate the feasibility of detecting EC by combining minimally-invasive specimen collection techniques with sensitive molecular testing. METHODS: Prior to hysterectomy for EC or benign indications, women collected vaginal pool samples with intravaginal tampons and underwent endometrial brushing. Specimens underwent pyrosequencing for DNA methylation of genes reported to be hypermethylated in gynecologic cancers and recently identified markers discovered by profiling over 200 ECs. Methylation was evaluated individually across CpGs and averaged across genes. Differences between EC and benign endometrium (BE) were assessed using two-sample t-tests and area under the curve (AUC). RESULTS: Thirty-eight ECs and 28 BEs were included. We evaluated 97 CpGs within 12 genes, including previously reported markers (RASSF1, HSP2A, HOXA9, CDH13, HAAO, and GTF2A1) and those identified in discovery work (ASCL2, HTR1B, NPY, HS3ST2, MME, ADCYAP1, and additional CDH13 CpG sites). Mean methylation was higher in tampon specimens from EC v. BE for 9 of 12 genes (ADCYAP1, ASCL2, CDH13, HS3ST2, HTR1B, MME, HAAO, HOXA9, and RASSF1) (all p<0.05). Among these genes, relative hypermethylation was observed in EC v. BE across CpGs. Endometrial brush and tampon results were similar. Within tampon specimens, AUC was highest for HTR1B (0.82), RASSF1 (0.75), and HOXA9 (0.74). This is the first report of HOXA9 hypermethylation in EC. CONCLUSION: DNA hypermethylation in EC tissues can also be identified in vaginal pool DNA collected via intravaginal tampon. Identification of additional EC biomarkers and refined collection methods are needed to develop an early detection tool for EC.

The impact of concurrent temozolomide with adjuvant radiation and IDH mutation status among patients with anaplastic astrocytoma.

This study assesses the controversial role of temozolomide (TMZ) concurrent with adjuvant radiation (RT) in patients with anaplastic astrocytoma (AA). The impact of isocitrate dehydrogenase (IDH) status on therapy and outcomes is also examined. All adult patients diagnosed with AA from 2001 to 2011 and treated with standard doses of adjuvant RT were identified retrospectively for clinical data extraction. IDH status was determined by IDH1-R132H immunostain and sequencing for other mutations in IDH1/IDH2. Cumulative survival probabilities were estimated using the Kaplan-Meier method. Cox proportional hazards regression models were fit for univariable/multivariable analyses. 136 patients had received concurrent TMZ while 29 had not. Of these, IDH status was determined on 114 and 27 patients, respectively. On univariable analysis, improved five-year survival was independently associated with concurrent TMZ (46.2 vs. 29.3%, p = 0.02) and IDH mutation (78.9 vs. 22.0%, p < 0.001). IDH mutation was additionally associated with a greater likelihood of extensive resection possibly secondary to a more favorable tumor location. Gross total/subtotal resections also led to improved survival when compared to biopsy alone on univariable analysis. On multivariable analysis, the association with five-year survival persisted for both concurrent TMZ and IDH mutation, but not with extent of surgery. Both IDH mutation and concurrent TMZ are associated with improved five-year survival in patients with AA who are receiving adjuvant RT. Secondarily, the association between five-year survival and extent of resection is lost on multivariable analysis. This suggests a possible association between IDH mutation, tumor location and consequent resectability.

Evaluating User Experience and DNA Yield from Self-Collection Devices.

BACKGROUND: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal. METHODS: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability. RESULTS: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices. CONCLUSIONS: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

Molecular diagnostics, personalized medicine, and the evolving role of the cytotechnologist: an institutional experience.

The role of cytotechnologists has focused primarily on the microscopic examination of cytologic specimens for diagnosing disease. Cytotechnologists currently evaluate a wide assortment of both gynecological and nongynecological cytology specimens. However, the Pap test remains the primary test for most cytology laboratories. Recently, human papillomavirus testing and newer cervical cancer screening guidelines have reduced the number of Pap tests, resulting in some anxiety and concern among the cytology community. However, as Pap test volumes continue to decrease, molecular oncology and ancillary testing volumes continue to increase with the advent of new biomarkers and associated personalized therapies. This change in clinical practice has resulted in evolving roles for many cytotechnologists. Cytotechnologists have skills based not only in morphology but also in understanding concepts of disease including neoplasia. These skills allow cytotechnologists to excel in many other types of laboratory testing. This article discusses how the roles of the cytotechnologist have recently expanded at our institution to include involvement in DNA ploidy analysis, quantitative immunohistochemistry, fluorescence in situ hybridization, circulating tumor cells, and molecular oncology testing. Lastly, this article discusses how these newer roles benefit both the cytotechnologist and the clinical laboratory.

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.

Fluorescence in situ hybridization testing algorithm improves lung cancer detection in bronchial brushing specimens.

RATIONALE: Bronchoscopically collected cytology specimens are commonly used to obtain a diagnosis of cancer in patients with pulmonary lesions. However, the sensitivity of cytology is suboptimal, especially for peripheral lesions less than 2 cm in diameter. OBJECTIVES: We assessed the performance of a testing algorithm using cytology and fluorescence in situ hybridization (FISH) as part of clinical practice. METHODS: Bronchial brushing specimens (n = 343) were obtained from patients undergoing bronchoscopy for indeterminate pulmonary lesions. Routine cytology was performed and specimens without a positive diagnosis (n = 294) were analyzed by FISH, using residual brushing material. Pathology-confirmed lung cancer or clinical/radiographic evidence of disease was considered diagnostic of malignancy. MEASUREMENTS AND MAIN RESULTS: Routine cytology had a sensitivity and specificity of 41% (23 of 56) and 100% (45 of 45) for central lesions and 20% (26 of 133) and 100% (109 of 109) for peripheral nodules, respectively. FISH detected an additional 32% of lung cancers (18 central and 43 peripheral) not detectable by cytology alone, while producing false positive diagnoses in 22% (10 of 45) and 6% (6 of 109) benign central and peripheral lesions, respectively. In peripheral nodules, FISH detected (relative to routine cytology) an additional 44% (15 of 34) and 28% (25 of 91) of lung cancers less than 2 cm and 2 cm or more in size, respectively. A positive FISH result had a likelihood ratio of 1.45 and 5.87 for central and peripheral lesions and 3.44 and 15.38 for peripheral nodules less than 2 cm and 2 cm or more in size, respectively. CONCLUSIONS: FISH testing significantly increases the detection of lung cancer over routine cytology alone. It is especially useful for peripheral nodules.

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.

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair.

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

LPCAT1-TERT fusions are uniquely recurrent in epithelioid trophoblastic tumors and positively regulate cell growth.

Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD.