Fibroepithelial Neoplasm with Hybrid Features of Benign Phyllodes Tumor, Juvenile Papillomatosis, and Juvenile Fibroadenoma: A Case Report.

Phyllodes tumor is an uncommon breast fibroepithelial neoplasm mainly found in middle-aged patients, presenting a morphologic continuum from benign to malignant. Juvenile papillomatosis represents a rare benign proliferative breast tumor primarily affecting young individuals and carries a potential elevated risk of subsequent breast cancer development. Juvenile fibroadenoma is a well-circumscribed biphasic neoplasm that often occurs in adolescent girls, characterized by a pericanalicular growth pattern with usual-type epithelial hyperplasia and gynaecomastia-like micropapillary proliferation. Herein, we present an unusual example of a 26-year-old woman with a left breast outer lower quadrant palpable mass. Ultrasonography identified a 5.9 cm lobulated hypoechoic solid mass with scattered small cysts. The preoperative biopsy initially diagnosed a fibroepithelial lesion, considering giant cellular fibroadenoma and phyllodes tumor in the differential. Subsequent complete excision revealed areas of benign phyllodes tumor features closely admixed with distinctive elements such as prominent multiple cysts exhibiting apocrine and papillary apocrine metaplasia, duct papillomatosis, and duct stasis characteristic of juvenile papillomatosis, and hyperplastic ductal epithelium with micropapillary projections demonstrating a pericanalicular growth pattern indicative of juvenile fibroadenoma. The diagnosis was conclusively established as a fibroepithelial lesion with combined features of benign phyllodes tumor, juvenile papillomatosis, and juvenile fibroadenoma. Further investigation uncovered a family history of breast cancer. Molecular analysis revealed a pattern of unique and overlapping mutations within these distinct histopathological areas. This unusual presentation with hybrid features within a single tumor is described for the first time in the literature along with the molecular signature of the individual components.

Platinum Sensitivity in IDH1/2 Mutated Intrahepatic Cholangiocarcinoma: Not All "BRCAness" Is Created Equal.

Preclinical data suggest that IDH1/2 mutations result in defective homologous recombination repair (HRR). We hypothesized that patients with IDH1/2mt intrahepatic cholangiocarcinoma (IHCC) would benefit more from 1 L platinum chemotherapy than patients with wildtype (WT) tumors. We performed a multicenter retrospective study of 81 patients with unresectable IHCC treated with 1 L platinum with a primary endpoint of clinical benefit rate (CBR). Patients with IDH1/2mt tumors had a similar CBR and objective response rate compared to those with IDH WT disease (59 versus 54%; p = 0.803), suggesting that a relationship between platinum sensitivity and HRR gene defects may be specific to tumor context.

Characteristics Associated With Disparities Among Older Adults in Coronavirus Disease 2019 Outcomes in an Academic Health Care System.

BACKGROUND: An improved understanding of the coronavirus disease 2019 (COVID-19) pandemic is needed to identify predictors of outcomes among older adults with COVID-19. OBJECTIVE: The objective of this study was to examine patient and health system factors predictive of in-hospital mortality, intensive care unit (ICU) admission, and readmission among patients with COVID-19. DESIGN, SETTING, AND PARTICIPANTS: A cohort study of patients aged 18 years and older with COVID-19 discharged from 5 New York hospitals within the Mount Sinai Health System (March 1, 2020-June 30, 2020). MEASURES: Patient-level characteristics (age, sex, race/ethnicity, comorbidities/serious illness, transfer from skilled nursing facility, severe acute respiratory syndrome coronavirus 2 viral load, Sequential Organ Failure Assessment score, treatments); hospital characteristics. OUTCOMES: All-cause in-hospital mortality; ICU admission; 30-day readmission. RESULTS: Among 7556 subjects, mean age 61.1 (62.0) years; 1556 (20.6%) died, 949 (12.6%) had an ICU admission, and 227 (9.1%) had a 30-day readmission. Increased age [aged 55-64: odds ratio (OR), 3.28; 95% confidence interval (CI), 2.41-4.46; aged 65-74: OR, 4.67; 95% CI, 3.43-6.35; aged 75-84: OR, 10.73; 95% CI, 7.77-14.81; aged 85 y and older: OR, 20.57; 95% CI, 14.46-29.25] and comorbidities (OR, 1.11; 95% CI, 1.16, 2.13) were independent risk factors for in-hospital mortality. Yet older adults (aged 55-64 y: OR, 0.56; 95% CI, 0.40-0.77; aged 65-74: OR, 0.46; 95% CI, 0.33-0.65; aged 75-84: OR, 0.27; 95% CI, 0.18-0.40; aged above 85 y: OR, 0.21; 95% CI, 0.13-0.34) and those with Medicaid (OR, 0.74; 95% CI, 0.56-0.99) were less likely to be admitted to the ICU. Race/ethnicity, crowding, population density, and health system census were not associated with study outcomes. CONCLUSIONS: Increased age was the single greatest independent risk factor for mortality. Comorbidities and serious illness were independently associated with mortality. Understanding these risk factors can guide medical decision-making for older adults with COVID-19. Older adults and those admitted from a skilled nursing facility were half as likely to be admitted to the ICU. This finding requires further investigation to understand how age and treatment preferences factored into resource allocation.

Primary hemangioblastoma of the kidney with molecular analyses by next generation sequencing: a case report and review of the literature.

BACKGROUND: Hemangioblastoma is an indolent mesenchymal tumor most frequently occurring in the central nervous system (CNS), but can also arise extraneuraxially, as part of Von Hippel-Lindau (VHL) disease or in sporadic tumors. Extraneuraxial hemangioblastomas occur outside the central nervous system. It includes tumors arising from the nervous paraneuraxial structures and visceral organs. Sporadic hemangioblastoma of the kidney, a rare subset of extraneuraxial hemangioblastomas, is an under-recognized renal neoplasm. There have been only 25 cases described to date in the English language literature. We report herein one additional sporadic tumor in a patient without VHL disease. CASE PRESENTATION: A 61 year old male presenting with gross hematuria was found to have a 3.5 cm renal mass at the lateral mid to lower pole of the left kidney on computed tomography urogram. The patient underwent a partial nephrectomy for the mass. Pathological examination showed a well-circumscribed non-encapsulated tumor composed of sheets of large polygonal cells traversed by a rich vascular network. The tumor cells showed clear to eosinophilic cytoplasm and overall bland nuclei. The diagnosis of hemangioblastoma was confirmed by positive immunostaining for alpha-inhibin, S100, neuron-specific enolase, and PAX8. No significant gene mutations, including VHL gene and copy number changes were detected in the tumor using next generation sequencing supporting the diagnosis of sporadic renal hemangioblastoma. CONCLUSION: Sporadic renal hemangioblastoma is a rare subset of extraneuraxial hemangioblastomas. We report one such tumor in a patient without clinical or molecular evidence of VHL disease. The literature was reviewed to better understand the clinical, radiological, pathological, and molecular features of this neoplasm. The majority of renal hemangioblastomas showed positive immunostaining for PAX8, which supports the idea that the immunoprofiles of extraneuraxial hemangioblastomas can vary depending on sites of origin. Diagnosis of renal hemangioblastoma is challenging because of its rarity and overlapping microscopic and immunophenotypic features with other renal tumors, including clear cell renal cell carcinoma. In some cases, molecular or genetic studies may be necessary to obtain an accurate diagnosis. Since renal hemangioblastoma is clinically benign, recognition of this pathological entity is important to avoid unnecessary over-treatment.

Anti-invasive efficacy and survival benefit of the YAP-TEAD inhibitor verteporfin in preclinical glioblastoma models.

BACKGROUND: Glioblastoma (GBM) remains a largely incurable disease as current therapy fails to target the invasive nature of glioma growth in disease progression and recurrence. Here, we use the FDA-approved drug and small molecule Hippo inhibitor Verteporfin (VP) to target YAP-TEAD activity, known to mediate convergent aspects of tumor invasion/metastasis, and assess the drug's efficacy and survival benefit in GBM models. METHODS: Up to 8 low-passage patient-derived GBM cell lines with distinct genomic drivers, including 3 primary/recurrent pairs, were treated with VP or vehicle (VEH) to assess in vitro effects on proliferation, migration, invasion, YAP-TEAD activity, and transcriptomics. Patient-derived orthotopic xenograft (PDX) models were used to assess VP's brain penetrance and effects on tumor burden and survival. RESULTS: VP treatment disturbed YAP/TAZ-TEAD activity; disrupted transcriptome signatures related to invasion, epithelial-to-mesenchymal, and proneural-to-mesenchymal transition, phenocopying TEAD1-knockout effects; and impaired tumor migration/invasion dynamics across primary and recurrent GBM lines. In an aggressive orthotopic PDX GBM model, short-term VP treatment consistently diminished core and infiltrative tumor burden, which was associated with decreased tumor expression of Ki67, nuclear YAP, TEAD1, and TEAD-associated targets EGFR, CDH2, and ITGB1. Finally, long-term VP treatment appeared nontoxic and conferred survival benefit compared to VEH in 2 PDX models: as monotherapy in primary (de novo) GBM and in combination with Temozolomide chemoradiation in recurrent GBM, where VP treatment associated with increased MGMT methylation. CONCLUSIONS: We demonstrate combined anti-invasive and anti-proliferative efficacy for VP with survival benefit in preclinical GBM models, indicating potential therapeutic value of this already FDA-approved drug if repurposed for GBM patients.

Urothelial carcinoma of the graft kidney with molecular analyses: a rare case report.

BACKGROUND: Malignancy after transplantation is a leading cause of death among kidney transplant recipients. However, donor-derived malignancies are rare. We report a case of a high grade papillary urothelial carcinoma arising in a transplanted kidney. CASE PRESENTATION: A 62-year-old female who received a kidney transplantation more than 30 years ago presented with urinary tract infection, acute renal failure, and hydronephrosis of the transplant kidney. Anterograde nephrostogram showed a large filling defect in the lower pole of the transplant kidney and in the proximal 3-4 cm of the ureter. A biopsy from the renal pelvic mass showed a high grade urothelial carcinoma. She underwent an anterior exenteration, resection of both transplant and native kidneys and bilateral pelvic lymph node dissection. Pathologic examination showed a high grade papillary urothelial carcinoma which appeared to arise in the pelvis of the graft kidney, involve the graft ureter and native urinary bladder. The tumor had metastasized to one left obturator lymph node but spared the two native kidneys and ureters. Short tandem repeat (STR) analysis confirmed the tumor to be of donor origin. Next-generation sequencing identified amplification of TERT and loss of CDKN2A/CDKN2B in the primary tumor. CONCLUSION: While it is known that transplant recipients have an increased risk of urothelial carcinoma compared to the general population, the lack of the well-documented risk factors, such as older age at transplantation, BK polyomavirus infection, and prolonged post-transplantation history and dissemination of the tumor in this case shed light on the de novo tumorigenesis of the graft kidney within the host microenvironment. Amplification of Telomerase reverse transcriptase (TERT) and loss of cyclin dependent kinase inhibitor 2A/2B (CDKN2A/CDKN2B) detected in the tumor by next gene sequencing suggests that they may play an important role in this case.

Liquid phase human papillomavirus genotype analysis of aspirated metastatic head and neck squamous cell carcinoma: Fine needle aspiration supernatant is a rich source of tumor DNA that can increase the diagnostic yield.

BACKGROUND: Most patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSC) present with lymph node metastasis. In these patients, fine needle aspiration (FNA) is not only a diagnostic tool, but a means for determining HPV status. HPV status, in turn, is used to determine tumor origin, prognosis, and even guide therapy. Thus, the limited sampling afforded by FNA must be optimized to meet heavy clinical demands. PURPOSE: The purpose of this study was to determine whether the residual supernatant portion of the FNA could serve as a resource for reliable determination of HPV status DESIGN/METHOD: 25 FNAs from 24 patients with metastatic HNSC underwent HPV genotyping of post-centrifuged supernatant fluid from FNA needle rinses. HPV genotyping was performed using two real time PCR-based assays, the two-step LightCycler and the one-step automated cobas HPV tests. HPV status of the supernatant was compared with the paired FNA cell blocks and/or surgical tissue samples. RESULTS: The supernatant was adequate for HPV testing in 24 (96%) of 25 cases. Of these, 14 (56%) were HPV positive and 11 (44%) negative by the LightCycler assay. HPV16 was the most commonly detected genotype (n = 12). When results of supernatant and paired cell block testing were compared, HPV status was concordant in all cases. The LightCycler method was more sensitive than the cobas assay due to its ability to detect an expanded profile of HPV variant genotypes. CONCLUSION: The current standard of practice for patients with HNSC who undergo FNA is to construct a cell block and then discard the supernatant. This supernatant is a rich source of tumor DNA that can be used to detect HPV status. It should not be wasted.

Transient expansion of TP53 mutated clones in polycythemia vera patients treated with idasanutlin.

Activation of the P53 pathway through inhibition of MDM2 using nutlins has shown clinical promise in the treatment of solid tumors and hematologic malignancies. There is concern, however, that nutlin therapy might stimulate the emergence or expansion of TP53-mutated subclones. We recently published the results of a phase 1 trial of idasanutlin in patients with polycythemia vera (PV) that revealed tolerability and clinical activity. Here, we present data indicating that idasanutlin therapy is associated with expansion of TP53 mutant subclones. End-of-study sequencing of patients found that 5 patients in this trial harbored 12 TP53 mutations; however, only 1 patient had been previously identified as having a TP53 mutation at baseline. To identify the origin of these mutations, further analysis of raw sequencing data of baseline samples was performed and revealed that a subset of these mutations was present at baseline and expanded during treatment with idasanutlin. Follow-up samples were obtained from 4 of 5 patients in this cohort, and we observed that after cessation of idasanutlin, the variant allele frequency (VAF) of 8 of 9 TP53 mutations decreased. Furthermore, disease progression to myelofibrosis or myeloproliferative neoplasm blast phase was not observed in any of these patients after 19- to 32-month observation. These data suggest that idasanutlin treatment may promote transient TP53 mutant clonal expansion. A larger study geared toward high-resolution detection of low VAF mutations is required to explore whether patients acquire de novo TP53 mutations after idasanutlin therapy.

Detection of EGFR T790M Mutation by Droplet Digital Polymerase Chain Reaction in Lung Carcinoma Cytology Samples.

CONTEXT.­: Advanced-stage non-small cell lung carcinoma patients on EGFR-targeted tyrosine kinase inhibitors frequently present with an acquired EGFR T790M resistance mutation. Early detection using a high-sensitivity assay is critical to allow patients to switch to third-generation tyrosine kinase inhibitors. The detection of EGFR T790M mutation is often challenging because of low tumor fraction in posttreatment specimens. Because a large fraction of non-small cell lung carcinoma patients are given a diagnosis by cytology, evaluating a high-sensitivity technique for EGFR T790M detection in these specimens is essential. OBJECTIVE.­: To evaluate a high-sensitivity droplet digital polymerase chain reaction (ddPCR) assay for EGFR T790M using different cytologic specimen preparations. DESIGN.­: A total of 42 cytology samples, including smears and cell block preparation, were evaluated for EGFR T790M using ddPCR. The results of the mutation assay were compared to the patient's known EGFR T790M mutation status. RESULTS.­: The ddPCR assay successfully determined the EGFR T790M mutation status in 36 of 42 samples (86%), including samples with low tumor fraction (≤20%). In 4 cases the results of the ddPCR assay could not be compared because the mutation status was unknown at the time of collection of the cytology sample. There was 1 false-positive result, with borderline positivity, and 1 false-negative result. Overall sensitivity and specificity of the ddPCR assay were 93% and 96%, respectively. CONCLUSIONS.­: Our results indicate that EGFR T790M ddPCR is a highly sensitive and specific mutational assay that can be used reliably in cytologic specimens, including samples with low tumor fraction.

Non-invasive genotyping of metastatic colorectal cancer using circulating cell free DNA.

Circulating cell-free DNA (ccfDNA) in plasma provides an easily accessible source of circulating tumor DNA (ctDNA) for detecting actionable genomic alterations that can be used to guide colorectal cancer (CRC) treatment and surveillance. The goal of this study was to test the feasibility of using a traditional amplicon-based next-generation sequencing (NGS) on Ion Torrent platform to detect low-frequency alleles in ctDNA and compare it with a digital NGS assay specifically designed to detect low-frequency variants (as low as 0.1%) to provide evidence for the standard care of CRC. The study cohort consisted of 48 CRC patients for whom matched samples of formalin-fixed, paraffin-embedded tumor tissue, plasma, and peripheral blood mononuclear cells were available. DNA samples from different sources were sequenced on different platforms using commercial protocols. Our results demonstrate that the ccfDNA sequencing with the traditional NGS can be reliably used in an integrated workflow to detect low-frequency somatic variants in CRC. We found a high degree of concordance between traditional NGS and digital NGS in profiling mutant alleles in ccfDNA. These findings suggest that the traditional NGS is a viable alternative to digital sequencing of ccfDNA at allele frequency above 1%. ccfDNA sequencing can not only provide real-time monitoring of CRC, but also lay the basis for its application as a clinical diagnostic test to guide personalized therapy.

Rational "Error Elimination" Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies.

The emergence of highly sensitive molecular diagnostic approaches, such as droplet digital PCR, has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytic strategies that integrate molecular barcodes have rarely been applied to clinical settings. In this study, we evaluated the parameters that are crucial in the use of molecular barcodes in next-generation sequencing for genotyping clinical specimens from patients with hematologic malignancies. The uniform incorporation of molecular barcodes into DNA templates through PCR was found to be crucial, and the extent of uniformity was governed by multiple interdependent variables. An error elimination strategy was developed for removing sequencing background errors by using molecular barcode sequence information as an alternative to the conventional error correction approach. This approach was successfully used to identify mutations with frequencies as low as 0.15%, and the clonal heterogeneity of hematologic malignancies was revealed. These findings have implications for elucidating heterogeneity and temporal and spatial clonal evolution, evaluating response to therapy, and monitoring relapse in patients with hematologic malignancies.

Centrifuged supernatants from FNA provide a liquid biopsy option for clinical next-generation sequencing of thyroid nodules.

BACKGROUND: Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid-based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from thyroid FNA samples (n = 156) were evaluated. A 50-gene next-generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. RESULTS: All 156 samples yielded adequate DNA (median, 135 ng; range, 11-3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow-up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n = 8) showed 100% concordance. CONCLUSIONS: This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management.

Targeted next-generation sequencing of endometrial cancer and matched circulating tumor DNA: identification of plasma-based, tumor-associated mutations in early stage patients.

There is currently no blood-based marker in routine use for endometrial cancer patients. Such a marker could potentially be used for early detection, but it could also help to track tumor recurrence following hysterectomy. This is important, as extra-vaginal recurrence of endometrial endometrioid adenocarcinoma is usually incurable. This proof-of-principle study was designed to determine if tumor-associated mutations could be detected in cell-free DNA from the peripheral blood of early and late stage endometrial endometrioid carcinoma patients. Approximately 90% of endometrioid carcinomas have at least one mutation in the genes CTNNB1, KRAS, PTEN, or PIK3CA. Using a custom panel targeting 30 hotspot amplicons in these four genes, next-generation sequencing was performed on cell-free DNA extracted from plasma obtained from a peripheral blood draw at the time of hysterectomy and the matching tumor DNA from 48 patients with endometrioid endometrial carcinomas. At least one mutation in the tumor was detected in 45/48 (94%) of patients. Fifteen of 45 patients (33%) had a mutation in the plasma that matched a mutation in the tumor. These same mutations were not detected in the matched negative control buffy coat. Presence of a plasma mutation was significantly associated with advanced stage at hysterectomy, deep myometrial invasion, lymphatic/vascular invasion, and primary tumor size. Detecting a plasma-based mutation was independent of the amount of cell-free DNA isolated from the plasma. Overall, 18% of early stage patients had a mutation detected in the plasma. These results demonstrate that mutations in genes relevant to endometrial cancer can be identified in the peripheral blood of patients at the time of surgery. Future studies can help to determine the post-operative time course of mutation clearance from the peripheral blood and if mutation re-emergence is predictive of recurrence.

Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors.

A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton. A subset of these samples with DNA availability was tested by ddPCR and UltraSEEK MassARRAY for mutation detection in 5 genes (IDH1, PIK3CA, KRAS, BRAF, and NRAS). Sixty one of 104 expected tissue mutations and 6 additional mutations not present in the tissue were detected in cfDNA. ddPCR and MassARRAY showed 83% and 77% concordance with NGS for mutation detection with 100% and 79% sensitivity, respectively. The median OS of patients with lower cfDNA yield (74 vs 50 months; P < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; p < 0.04) was significantly longer than in patients with higher cfDNA yield and positive for mutations. A limit-of-detection of 0.1% was demonstrated for ddPCR and MassARRAY platforms using a serially diluted positive cfDNA sample. The MassARRAY and ddPCR systems enable fast and cost-effective genotyping for a targeted set of mutations and can be used for single gene testing to guide response to chemotherapy or for orthogonal validation of NGS results.

Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling.

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling. In this study, we evaluated the post-centrifuged supernatant from 25 malignant and 10 benign fine needle aspiration needle rinses. The mean and median DNA yields from the supernatants were 445 ng and 176.4 ng (range, 15.1-2958 ng), respectively. Next generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 detected somatic mutations in all 25 malignant samples. No mutations were detected in any of the benign samples tested. When available, mutations detected in the supernatant fluid were compared to the next generation sequencing analysis performed on a prior or concurrent surgical specimen from the same patient and showed 100% concordance. In a subset of cases (n = 19) mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS were successfully confirmed by droplet digital PCR, providing an orthogonal platform for mutation analysis. In summary, in this study we show that post centrifuged supernatants from fine needle aspiration needle rinses can provide a robust substrate for expanded mutation profiling by next generation sequencing, as well as hotspot mutation testing by droplet digital PCR. The ability to detect somatic mutations from otherwise discarded supernatant fluids offers the ability to triage and effectively utilize limited volume fine needle aspiration samples when multiple molecular tests are requested, without the need to re-biopsy for additional tissue samples.

Novel enriched pathways in superficial malignant peripheral nerve sheath tumours and spindle/desmoplastic melanomas.

Superficial malignant peripheral nerve sheath tumour (MPNST) is a rare, soft tissue neoplasm that shares morphological features and some molecular events with spindle/desmoplastic melanoma (SDM). Herein, we sought to identify molecular targets for therapy by using targeted RNA/DNA sequencing and gene expression of key immunological players. DNA and RNA from formalin-fixed paraffin-embedded tissue were extracted and processed. Massive high-throughput deep parallel sequencing was performed with the Oncomine comprehensive panel, enabling detection of relevant single-nucleotide variants, copy number variations, gene fusions and indels for 143 unique genes on the Ion torrent sequencer for clinical trial research programmes. Gene expression analysis was carried out with a customized 770-gene expression panel combining markers for 24 different immune cell types and 30 common cancer antigens, including key checkpoint blockade genes analysed with the Ncounter system. Fifty-one patients (SDM, 16/11; MPNST, 24; male, n = 37; female, n = 16) had sufficient DNA and RNA for testing. NF1 deleterious mutations and/or deep/homozygous deletions were identified in 73% of MPNSTs and 67% of SDMs, with 50% of the mutations involving the RAS-binding domain. Inactivating/deleterious mutations of TSC1/TSC2 were identified in 40% and 41% of MPNSTs and SDMs, respectively. Activating mutations affecting the EGFR-like and the negative regulatory domains of NOTCH1 and KDR (VEGFR2) were identified in 45% and 40% of SDMs and in 30% and 8% of MPNSTs, respectively. Differential gene expression and gene clustering analysis showed significantly perturbed immune pathway components, including nuclear factor-κB (NF-κB), JAK-STAT, and CXCL12-CXCR4, and differentially expressed CD274 and CTLA4, in both SDM and MPNST. Angiogenesis (KDR and NOTCH1) and mammalian target of rapamycin complex (mTORC) pathways offer a rationale for anti-angiogenic and selective mTORC inhibition as treatment strategies for MPNST and SDM. Cytokines and the JAK-STAT, TNF and NF-κB axes were perturbed in both SDM and MPNST. These pathways have been targeted in haematological malignancies and present promising targets for these tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prognostic Significance of Circulating RET M918T Mutated Tumor DNA in Patients With Advanced Medullary Thyroid Carcinoma.

Context: Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. Objective: We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. Design: The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients: Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. Results: RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Conclusions: Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment.