Cell-Free DNA Genomic Profiling and Its Clinical Implementation in Advanced Prostate Cancer.

Most men with prostate cancer (PCa), despite potentially curable localized disease at initial diagnosis, progress to metastatic disease. Despite numerous treatment options, choosing the optimal treatment for individual patients remains challenging. Biomarkers guiding treatment sequences in an advanced setting are lacking. To estimate the diagnostic potential of liquid biopsies in guiding personalized treatment of PCa, we evaluated the utility of a custom-targeted next-generation sequencing (NGS) panel based on the AmpliSeq HD Technology. Ultra-deep sequencing on plasma circulating free DNA (cfDNA) samples of 40 metastatic castration-resistant PCa (mCRPC) and 28 metastatic hormone-naive PCa (mCSPC) was performed. CfDNA somatic mutations were detected in 48/68 (71%) patients. Of those 68 patients, 42 had matched tumor and cfDNA samples. In 21/42 (50%) patients, mutations from the primary tumor tissue were detected in the plasma cfDNA. In 7/42 (17%) patients, mutations found in the primary tumor were not detected in the cfDNA. Mutations from primary tumors were detected in all tested mCRPC patients (17/17), but only in 4/11 with mCSPC. AR amplifications were detected in 12/39 (31%) mCRPC patients. These results indicate that our targeted NGS approach has high sensitivity and specificity for detecting clinically relevant mutations in PCa

RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC

Molecular pathology in colorectal cancer: from early detection to treatment predictions.

Background: In the precision medicine era, the increasing request of clinical relevant biomarkers to improve the patients management lead to the need of most biological source. To address this issue, also if tissue represents the gold standard for the assessment of clinical relevant biomarkers mutational status, some alternative approaches, based on the analysis of circulating free DNA (cfDNA) extracted from “liquid biopsies”, are under evaluation. The aim of this thesis was to investigate the role of the molecular pathology in colorectal cancer from the “early detection” to treatment predictions. In particular, it has been investigated the role of the liquid biopsy as a screening tool for Colorectal Cancer (CRC) in comparison of Fetal Immunochemical Test (FIT) but also as a monitoring tool in patients with metastatic colorectal cancer to predict the possibility of relapse. Moreover, in order to the treatment approach for CRC patients, it was evaluated the feasibility to perform the Idylla™ Assay to select the wilde-type K-N-RAS patients to the treatment with the monoclonal antibodies that target and inhibit the EGFR gene. Methods: In relation to CRC patients, employing the analytical validated Real Time PCR-based ColoScape assay Kit, mutations in the APC, KRAS, BRAF and CTNNB1 genes were assessed on 52 prospectively collected whole-blood samples obtained from FIT+ patients enrolled in the CRC screening program of ASL NAPOLI 3 SUD, using colonoscopy as confirmation. It was, also, performed the NGS analysis of the cfDNA samples isolated from mCRC patients and their matching metastatic tissue using the SiRe® panel, which covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) to monitor the possibility of relapse. Moreover, it was assessed the K-NRAS mutational status by using a fully automated Real Time PCR, the Idylla™ System, as first-level screening method to quickly address the CRC patients to the best personalized treatment. Results: The application of Real Time PCR based ColoScape assay Kit as a screening tool for CRC patients, the assay's sensitivity for advanced adenomas was 53.8% and the specificity was 92.3%. The Positive Predictive Value was 70.0% and negative predictive value was 85.7%. Of note, four of the six positive cases missed by ColoScape had a less than suboptimal DNA input. Had they been ruled out as inadequate, sensitivity would have increased from 53.8% to 69%. Regarding the NGS analysis of the cfDNA samples isolated from mCRC patients the molecular analysis was successfully performed in matched tissue specimen for a preliminary set (n=15) of enrolled patients. In n=3 cases, gDNA was not available for molecular analysis. In details, tissue sample analysis showed at least a molecular alteration in n=8/12 (66,6%) cases. In order to evaluate the possibility to use the Idylla™ Assay to address the CRC patients to best target therapy n=450 mCRC patients and a total of 457 samples were tested. In particular, for 5 patients were analyzed also the metastatic tissue and for 2 patients we analyzed two synchronous cancers. A total of n= 200/457 (43,8%) samples gave a WT result, otherwise n=257/457 (56,2%) samples showed a mutation in the clinical relevant genes in patients with colorectal cancer. In particular n=213/257 (82,9%) were KRAS positive, n= 20/257 (7,8%) were NRAS positive and n=24/257 (9,3%) were positive in BRAF gene. Conclusions: In CRC patients setting, ColoScape is a promising tool for screening program aims to evaluate the triage of FIT+ patients and the cfDNA analysis, using the Sire® panel, has proven to be a valid monitoring tool to follow up the mCRC patients. Moreover, the Idylla ™ results combined with the treatment collected data from the oncologists could represent the best strategy to address wild type patients to a targeted therapy. The collected data obtained with this analysis could be useful to improve the treatment approach in the clinical practice setting

Next generation sequencing identifies novel potential actionable mutations for grade I meningioma treatment

Meningiomas are common brain tumors that arise from the meningeal membranes that envelope the brain and spinal cord. The World Health Organization classifies these tumors into three histopathological grades. Because of tumor recurrence, treating meningiomas may be challenging even in well- differentiated grade I (GI) neoplasms. Indeed, around 5% of completely resected GI meningiomas relapse within 5 years. Therefore, identifying driver mutations in GI meningiomas through next generation sequencing (NGS) assays is paramount. The aim of this study was to validate the use of the 50-gene AmpliSeq Hotspot Cancer Panel v2 to identify the mutational status of 23 GI meningioma, namely, 12 non recurrent and 11 recurrent. In 18 out of the 23 GI meningiomas analyzed, we identified at least one gene mutation (78.2%). The most frequently mutated genes were c-kit (39.1%), ATM (26.1%), TP53 (26.1%), EGFR (26.1%), STK11 (21.7%), NRAS (17.4%), SMAD4 (13%), FGFR3 (13%), and PTPN11 (13%); less frequent mutations were SMARCB1 (8.7%), FLT3 (8.7%), KRAS (8.7%), FBWX7 (8.7%), ABL1 (8.7%), ERBB2 (8.7%), IDH1 (8.7%), BRAF (8.7%), MET (8.7%), HRAS (4.3%), RB1 (4.3%), CTNNB1 (4.3%), PIK3CA (4.3%), VHL (4.3%), KDR (4.3%), APC (4.3%), NOTCH1 (4.3%), JAK3 (4.3%), and SRC (4.3%). To our knowledge, mutations in all of these genes, except for TP53, STK11, SMARCB1, PIK3CA, VHL, and BRAF, have never been described before in meningiomas. Hence, these findings demonstrate the viability of NGS to detect new genetic alterations in GI meningiomas. Equally important, this technology enabled us to detect possible novel actionable mutations not previously associated with GI and for which selective inhibitors already exis

Evaluation of a fully closed real time PCR platform for the detection of SARS-CoV-2 in nasopharyngeal swabs: a pilot study

To date, reverse transcriptase PCR (RT-PCR) on nasopharyngeal swabs is the 'gold standard' approach for the diagnosis of COVID-19. The need to develop easy to use, rapid, robust and with minimal hands-on time approaches are warranted. In this setting, the Idylla SARS-CoV-2 Test may be a valuable option. The aim of our study is to evaluate the analytical and clinical performance of this assay on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up

Analysis of Programmed Death-Ligand 1 Expression, Stromal Tumor-Infiltrating Lymphocytes, and Mismatch Repair Deficiency in Invasive Micropapillary Carcinoma of the Breast

Introduction: Invasive micropapillary carcinoma (IMPC) of the breast is a rare and aggressive subtype of invasive ductal carcinomas, associated with poor prognosis and without a well-established treatment. Programmed death-ligand 1 (PD-L1) expression, high tumor-infiltrating lymphocytes (TILs), and microsatellite instability have recently been linked to susceptibility to immunotherapies against PD-1/PD-L1 axis. No exhaustive data is available on the status of these predictive markers in IMPCs of the breast. The aim of our study is to analyze PD-L1 expression, stromal TIL (sTIL), and mismatch repair (MMR) gene status in IMPCs of the breast, to extend the therapeutic possibilities of these rare aggressive tumors. Materials and Methods: Thirty-seven cases of IMPCs diagnosed in two European institutions between 2003 and 2017 with detailed clinical and pathologic data were analyzed. sTILs were assessed in hematoxylin and eosin-stained sections. MMR deficiency was tested by either immunohistochemistry (IHC) for MMR proteins (MLH1, MSH2, MSH6, and PMS2) or capillary electrophoresis for microsatellite instability using a standardized panel of five loci (Bat25, Bat26, D2S123, D5S346, and D17S250). For PD-L1, expression in both tumor cells (TCs) and immune cells (ICs) was determined using the antibody clone SP263. Results: The median sTILs was 3% (mean: 6%, range: 0–40). Thirty-one cases (84%) showed ≤10% of sTILs and only one case had 40% of sTILs. Higher median TILs were more frequently observed in lymph node metastases. PD-L1 expression (≥1%) was observed in 4 (11%) and 14 (38%) cases in TCs and ICs, respectively. None of the tumors showed PD-L1 expression in >1% of TCs. Only three cases showed expression in >10% of ICs. All cases were microsatellite stable by either IHC or polymerase chain reaction analyses. Conclusions: IMPCs of the breast are microsatellite-stable and immune desert tumors with low PD-L1 expression, thus arguing against the use of immune-checkpoint inhibitors in these patients. Active immunotherapy strategies attempting to stimulate self-immune system to attack tumor are needed

Next generation sequencing in cytology

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high-quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre-analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre-analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field

Is the Idylla EGFR Mutation Assay feasible on archival stained cytological smears? A pilot study

AIM: The rapid and fully automated Idylla EGFR Mutation Assay has been specifically designed to process formalin-fixed, paraffin-embedded sections without requiring preliminary DNA extraction. This study evaluates whether this approach can also process archival smears from patients with non-small cell lung cancer (NSCLC) by scraping the stained cellular material directly into the cartridge. METHODS: The study was divided into two parts. In the first part, we carried out Idylla EGFR Mutation Assay on archival stained smears from 39 patients with NSCLC. Among these, 14 cases harboured a mutation in either exon 19 (n=11) or exon 21 (n=3), previously detected on DNA extracts by fragment length and TaqMan assays. In the second part, we evaluated whether de-staining of the smears could reduce background fluorescence. RESULTS: The Idylla EGFR Mutation Assay confirmed the presence of EGFR mutation in 11 instances (78.6%). However, concordance was higher for exon 19 deletions (10/11) than for exon 21 p.L858R assessments. Raw data showed a high background fluorescence in channel 2, where the EGFR exon 21 p.L858R mutation was detected. This interference, due to dye residues from the original staining, was partially reduced by de-staining the cytological material. CONCLUSIONS: Our data, although preliminary, show that the Idylla EGFR Mutation Assay can reliably process most archival smears without requiring preliminary DNA extraction. Results may be further improved by de-staining the cellular material before insertion into the cartridge

Next Generation Sequencing in Cytopathology: Focus on Non-Small Cell Lung Cancer

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants