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Whenever tissue sample is not available, non-small cell lung cancer (NSCLC) biomarker testing is performed with liquid biopsy. The Kirsten rat sarcoma viral oncogene homolog (KRAS) p.G12C mutation is a novel target in patients with NSCLC. In this study, 33 NSCLC frozen plasma samples, previously characterised for KRAS mutational status by next generation sequencing (NGS), were processed by the fully automated Idylla KRAS assay. In 30/33 cases, archival matched cell-free DNA (cfDNA) was also directly pipetted in the cartridge. Overall, 30/33 plasma and 28/30 cfDNA samples yielded valid results. In 29/30 of KRAS p.G12C mutant plasma samples and 26/28 of cfDNA, Idylla confirmed the NGS results. In conclusion, the Idylla NSCLC KRAS liquid biopsy assay may represent a reliable tool to assess KRAS p.G12C mutation.
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Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Ácidos Nucleicos Libres de Células/genética , Análisis Mutacional de ADN/métodos , Humanos , Biopsia Líquida , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIMS: In thyroid cytopathology, the undetermined diagnostic categories still pose diagnostic challenges. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid fine-needle aspiration (FNA) specimens, access to such technology can be difficult because of its prohibitive cost and lack of reimbursement in countries with universal health coverage. To overcome these issues, we developed and validated a novel custom NGS panel, Nexthyro, specifically designed to target 264 clinically relevant mutations involved in thyroid tumourigenesis. Moreover, in this study, we compared its analytical performance with that of our previous molecular testing strategy. METHODS: The panel, which includes 15 genes (BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter), was validated with a cell-line derived reference standard and 72 FNA archival samples previously tested with the 7-gene test. RESULTS: Nexthyro yielded 100% specificity and detected mutant alleles at levels as low as 2%. Moreover, in 5/72 (7%) FNAs, it detected more clinically relevant mutations in BRAF and RAS genes compared with the 7-gene test. Nexthyro also revealed better postsequencing metrics than the previously adopted commercial 'generic' NGS panel. CONCLUSION: Our comparative analysis indicates that Nexthyro is a reliable NGS panel. The study also implies that a custom-based solution for routine thyroid FNA is sustainable at the local level, allowing patients with undetermined thyroid nodules affordable access to NGS.
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Neoplasias de la Tiroides , Nódulo Tiroideo , Biopsia con Aguja Fina , ARN Helicasas DEAD-box/genética , ADN , Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Ribonucleasa III/genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Nódulo Tiroideo/diagnóstico , Nódulo Tiroideo/genética , Nódulo Tiroideo/patologíaRESUMEN
AIM: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow. METHODS: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols. RESULTS: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants. CONCLUSION: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.
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Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Adhesión en ParafinaRESUMEN
V-Raf murine sarcoma viral oncogene homolog B (BRAF) gene mutations have recently been approved to select advanced stages non-small cell lung cancer (NSCLC) patients for tyrosine kinase inhibitors treatments. In this setting, liquid biopsy may represent a valuable option for BRAF mutational testing in patients without tissue availability. Here, we reviewed 196 plasma based liquid biopsies analysed by an in-house developed next generation sequencing panel, termed SiRe. On the overall, 6 (3.1%) out of 196 BRAF mutated cases were identified, with an overall median allelic frequency of 3.4%. Exon 15 p.V600E was the most common detected mutation (2/6, 33.3%). Our data highlighted that the SiRe panel is a robust tool for BRAF mutation assessment on circulating tumour DNA. Further investigation is required to develop a diagnostic algorithm to harmonise BRAF testing on tissue and blood in advanced stages NSCLC patients.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas B-raf/genética , Anciano , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/genética , Exones/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos , Análisis de Secuencia de ADNRESUMEN
Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Fusión Génica/genética , Neoplasias Pulmonares/genética , Oncología Médica/métodos , Oncología Médica/tendencias , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.
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Reparación de la Incompatibilidad de ADN , Neoplasias del Sistema Digestivo/genética , Neoplasias de los Genitales Femeninos/genética , Inestabilidad de Microsatélites , Neoplasias de la Próstata/genética , Biomarcadores de Tumor/análisis , Enzimas Reparadoras del ADN/análisis , Neoplasias del Sistema Digestivo/enzimología , Neoplasias del Sistema Digestivo/patología , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Neoplasias de los Genitales Femeninos/enzimología , Neoplasias de los Genitales Femeninos/patología , Humanos , Inmunohistoquímica , Italia , Masculino , Técnicas Analíticas Microfluídicas , Técnicas de Diagnóstico Molecular , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Reproducibilidad de los Resultados , Estudios Retrospectivos , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
Liquid biopsy, which allows the isolation of circulating cell-free (ccf) DNA from blood, is an emerging noninvasive tool widely used in oncology for diagnostic and prognosis purposes. Previous data have shown that serum cfDNA discriminates idiopathic pulmonary fibrosis (IPF) from other interstitial lung diseases. Our study aimed to measure plasma levels of ccfDNA in 59 consecutive therapy-naive and clinically stable IPF patients. The single nucleotide polymorphism (SNP) of the MUC5B gene promoter (rs35705950), associated with increased susceptibility of developing IPF, has been sought in plasma cfDNA and genomic DNA for comparison. Thirty-five age- and sex-matched healthy volunteers were recruited as the control group. Our results show that concentrations of small-size ccfDNA fragments were significantly higher in IPF patients than in controls and inversely correlated with lung function deterioration. Moreover, the median level of 104 ng/mL allowed discriminating patients with mild disease from those more advanced. The rs35705950 polymorphism was found in 11.8% of IPF patients and 8% of controls, with no differences. Complete concordance between ccfDNA and genomic DNA was detected in all control samples, while four out of seven IPF cases (57%) carrying the rs35705950 polymorphism were discordant from genomic DNA (7% of total IPF). Liquid biopsy is a suitable tool with optimistic expectations of application in the field of IPF. In analogy with cancer biology, finding some discrepancies between ccfDNA and genomic DNA in IPF patients suggests that the former may convey specific genetic information present in the primary site of the disease.
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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.
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Citodiagnóstico/métodos , Técnicas Citológicas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologíaRESUMEN
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.
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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.
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AIMS: In the time of COVID-19, predictive molecular pathology laboratories must still timely select oncological patients for targeted treatments. However, the need to respect social distancing measures may delay results generated by laboratory-developed tests based on sequential steps a long hands-on time. Laboratory workflows should now be simplified. METHODS: The organisation of the University of Naples Federico II predictive pathology laboratory was assessed before (March-April 2019) and during (March-April 2020) the Italian lockdown. RESULTS: The number of patients undergoing single or multiple biomarker testing was similar in 2019 (n=43) and in 2020 (n=45). Considering adequate samples for molecular testing, before the outbreak, next-generation sequencing was mostly used (35/42, 83.3%). Testing six genes had a reagent cost of 98/patient. Conversely, in 2020, almost all cases (38/41, 92.7%) were analysed by automated testing. This latter had for any single assay/gene a significant reagent cost (95-136) and a faster mean turnaround time (5.3 vs 7.9 working days). CONCLUSION: In the times of coronavirus, laboratory fully automated platforms simplify predictive molecular testing. Laboratory staff may be more safely and cost-effectively managed.
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Biomarcadores/análisis , COVID-19/diagnóstico , Brotes de Enfermedades , Oncología Médica , Patología Molecular , SARS-CoV-2/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Automatización de Laboratorios , COVID-19/epidemiología , COVID-19/virología , Femenino , Humanos , Italia/epidemiología , Laboratorios , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Valor Predictivo de las Pruebas , Factores de Tiempo , Flujo de Trabajo , Adulto JovenRESUMEN
BACKGROUND: Immune-checkpoint inhibitors (ICIs) represent an important treatment option for patients who have advanced stage non-small cell lung cancer (NSCLC). Currently, evaluation of the expression level of programmed death-ligand 1 (PD-L1) has proven highly successful as a positive predictive biomarker for ICIs. In addition to PD-L1, other promising predictive biomarkers are emerging, including high tumor mutational burden (TMB-H). However, measuring TMB-H remains challenging for several reasons, among which is the difficulty in obtaining adequate tissue material from NSCLC patients. There are no data in the current literature regarding the possibility of adopting cell blocks (CBs) for TMB evaluation; therefore, our goal was to evaluate the feasibility of analyzing TMB on CBs. METHODS: For evaluation of differences in run metric parameters, 8 pairs of histological and CB samples from patients with NSCLC were analyzed using the Oncomine Tumor Mutational Load Assay on Ion Torrent S5 GS next-generation sequencing (NGS) platform. RESULTS: Most CBs (6/8, 75.0%) were successfully analyzed by adopting the broad NGS panel approach. CBs provided results similar to those obtained on histological matched specimens in terms of median total reads (7207048.80 vs 7558817.80), median mapped reads (7075753.83 vs 7513822.00), median read lengths (115.50 vs. 113.00), median percentage of reads on-target (97.49% vs. 98.45%), median average reads per amplicon (454.67 vs 476.14), and median uniformity of amplicon coverage (83.52% vs 84.13%). CONCLUSION: In this pilot study, we demonstrated the technical feasibility of assessing TMB on CBs.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Técnicas Citológicas/métodos , Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/genética , Mutación , Anciano , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/patología , Masculino , Proyectos Piloto , Pronóstico , Estudios RetrospectivosRESUMEN
PURPOSE: Nowadays, the clinical management of thyroid nodules needs to be multi-disciplinary. In particular, the crosstalk between endocrinologists and cytopathologists is key. When FNAs are properly requested by endocrinologists for nodules characterised by relevant clinical and ultrasound features, cytopathologists play a pivotal role in the diagnostic work-up. Conversely, improper FNA requests can lead to questionable diagnostic efficiency. Recently, recommendations to delay all non-urgent diagnostic procedures, such as thyroid FNAs, to contain the spread of COVID-19 infection, have made the interplay between endocrinologists and cytopathologists even more essential. The objective of this study was to assess the impact of COVID-19 pandemic on our practice by evaluating the total number of FNAs performed and the distribution of the Bethesda Categories before, during, and after the lockdown. METHODS: We analysed the FNA trends before (1st January 2019 to March 13th 2020), during (March 14th to May 15th), and after (May 16th to July 7th) the lockdown. RESULTS: Although the total number of weekly FNAs dropped from 62.1 to 23.1, our referring endocrinologists managed to prioritise patients with high-risk nodules. In fact, in the post-lockdown, the weekly proportion of benign diagnoses dropped on average by 12% and that of high-risk diagnoses increased by 6%. CONCLUSIONS: The lesson we have learned so far from this pandemic is that by applying safety protocols to avoid contagion and by increasing the threshold for FNA requests for thyroid nodules, we can continue to guarantee our services to high-risk patients even in times of a health crisis.
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COVID-19/epidemiología , Accesibilidad a los Servicios de Salud , Pandemias , Cuarentena , Nódulo Tiroideo/diagnóstico , Adulto , Anciano , Actitud Frente a la Salud , Biopsia con Aguja Fina/estadística & datos numéricos , Biopsia con Aguja Fina/tendencias , Control de Enfermedades Transmisibles/métodos , Control de Enfermedades Transmisibles/organización & administración , Control de Enfermedades Transmisibles/normas , Femenino , Adhesión a Directriz/normas , Adhesión a Directriz/estadística & datos numéricos , Adhesión a Directriz/tendencias , Prioridades en Salud/normas , Prioridades en Salud/estadística & datos numéricos , Prioridades en Salud/tendencias , Accesibilidad a los Servicios de Salud/normas , Accesibilidad a los Servicios de Salud/tendencias , Historia del Siglo XXI , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Pautas de la Práctica en Medicina/normas , Pautas de la Práctica en Medicina/estadística & datos numéricos , Pautas de la Práctica en Medicina/tendencias , Cuarentena/organización & administración , Cuarentena/normas , Derivación y Consulta/estadística & datos numéricos , Derivación y Consulta/tendencias , Glándula Tiroides/patología , Nódulo Tiroideo/epidemiología , Factores de Tiempo , Ultrasonografía IntervencionalRESUMEN
Gain-of-function of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) is one of the most frequent oncogenic mutations in numerous cancers, including thyroid papillary carcinoma, melanoma, colon, and lung carcinomas, and to a lesser extent, ovarian and glioblastoma multiforme. This mutation aberrantly activates the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, thereby eliciting metastatic processes. The relevance of BRAF mutations stems from its prognostic value and, equally important, from its relevant therapeutic utility as an actionable target for personalized treatment. Here, we discuss the double facets of BRAF. In particular, we argue the need to implement diagnostic molecular algorithms that are able to detect this biomarker in order to streamline and refine diagnostic and therapeutic decisions.
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Proteínas Proto-Oncogénicas B-raf/genética , Biomarcadores de Tumor/genética , Humanos , Mutación/genética , Neoplasias/genética , Transducción de Señal/genéticaRESUMEN
In advanced stage non-small cell lung cancer (NSCLC) patients, Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) testing may soon acquire a predictive significance to select patients for AMG510 treatment. Since tissue samples are not always available, liquid biopsy may represent a viable option for KRAS testing. Here, we review the last three years clinical practice performed on 194 plasma based liquid biopsies by next generation sequencing (NGS) SiRe® panel. In particular, 36 (18.6%) KRAS mutated cases were identified, with an overall median allelic frequency of 5.0% (ranging between 0.2% and 46.8%). No concomitant mutations were observed in the other NSCLC clinical relevant genes included in the SiRe® panel, such as epidermal growth factor receptor (EGFR) and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Exon 2 p.G12C was the most common detected mutation (13/36, 36.1%). In conclusion, our data update and confirm that SiRe® NGS panel represents a robust analytical tool to assess KRAS mutational status on circulating tumor DNA. Further investigation is required to design more cost-effective diagnostic algorithms to harmonize clinical relevant biomarker testing on tissue and blood in advanced stage NSCLC clinical practice.
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Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.
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BACKGROUND: Thyroid fine-needle aspirates (FNAs) with undetermined morphology can be outsourced to centralized laboratories for comprehensive molecular profiling. When a local, rapid screening rules out easily detectable BRAF and NRAS mutations outsourcing is minimized, leading to cost savings. The fully automated Idylla technology, that does not require trained staff, is an emerging option. However, Idylla platform has only been validated to process formalin fixed paraffin embedded (FFPE) sections. Here we investigate whether also the FNA needle rinse could be genotyped by the same cytopathologist who performs the FNA, a procedure that can be termed rapid on site molecular evaluation (ROME). METHODS: To validate this approach, the Idylla BRAF and NRAS Test was performed on the rinses from 25 simulated (bench-top) FNAs, in a first part of the study. Genotyping data were compared with those obtained on matched histological FFPE blocks. The second part of the study was carried out on 25 prospectively collected routine FNAs to assess the performance of the Idylla BRAF and NRAS assay against a gold standard real time polymerase chain reaction method. RESULTS: Idylla NRAS-BRAF Mutation Test was performed on needle rinse as well as histological FFPE blocks. A sensitivity of 88.9%, a specificity of 100.0% were obtained comparing the Idylla NRAS-BRAF Mutation Test on needle rinse to the reference method. CONCLUSIONS: The FNA needle rinse can be directly genotyped. This obviates the need of cell block preparation, making possible a rapid combined morphological and molecular evaluation. Since DNA extraction is no longer necessary, the cytopathologist can perform ROME him/herself.
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GTP Fosfohidrolasas/genética , Proteínas de la Membrana/genética , Técnicas de Diagnóstico Molecular , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Glándula Tiroides , Neoplasias de la Tiroides , Biopsia con Aguja Fina , Análisis Mutacional de ADN , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Glándula Tiroides/enzimología , Glándula Tiroides/patología , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
BACKGROUND: Thyroid fine-needle aspiration (FNA) is a reliable and cost-effective diagnostic tool for establishing the nature of thyroid nodules, although up to 30% of FNAs are still classified as "indeterminate." Molecular testing of FNAs could improve preoperative diagnosis, thereby reducing unnecessary surgery. In this multicenter prospective study the authors investigated, using a 7-gene assay, the distribution and diagnostic impact of BRAF, RAS, RET/PTC, and PAX8/PPARg, the most frequent genomic alterations occurring during thyroid oncogenesis. METHODS: In total, of 1172 routine FNAs from 7 centers in southern Italy were classified according to the Bethesda System for Reporting Thyroid Cytopathology. Each specimen was tested, and molecular data were compared with available histology or cytologic follow-up. RESULTS: In particular, for atypia of undetermined significance/follicular lesion of undetermined significance cases, the 7-gene test confirmed the high positive predictive value of BRAFV600E and BRAF-like mutations (80%) and the moderate positive predictive value of RAS-like alterations (32.4%), suggesting different surgical management, depending on the type of mutation. The rate of mutation-positive FNAs was strictly related to the risk of malignancy of each diagnostic class, supporting the identification of prognostically relevant diagnostic categories. CONCLUSIONS: The 7-gene panel test improves the preoperative risk stratification of indeterminate thyroid FNAs, especially when considering the biologic significance of the different types of mutations. Moreover, the rate of mutation-positive FNAs is related to the risk of malignancy of each diagnostic class.
Asunto(s)
Biomarcadores de Tumor/análisis , Toma de Decisiones Clínicas/métodos , Pruebas Genéticas/métodos , Glándula Tiroides/patología , Nódulo Tiroideo/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biopsia con Aguja Fina , Carcinogénesis/genética , Niño , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Selección de Paciente , Cuidados Preoperatorios/métodos , Pronóstico , Estudios Prospectivos , Medición de Riesgo/métodos , Nódulo Tiroideo/genética , Nódulo Tiroideo/patología , Nódulo Tiroideo/cirugía , Tiroidectomía , Adulto JovenRESUMEN
AIMS: Following the development for liquid biopsies of the SiRe next-generation sequencing (NGS) panel that covers 568 clinical relevant mutations in EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRa genes, in this current study, we apply this small NGS panel on tissue samples of lung cancer. METHODS: A total of 322 specimens were prospectively tested. Technical parameters were analysed on both cytological and histological samples. In a subset of 75 samples, the EGFR SiRe results were compared with those generated by the European Community (CE)-IVD EGFR assay on Idylla platform. Clinical outcomes of 11 patients treated, on the basis of SiRe results, were also evaluated. RESULTS: Only 28 (8.7%) specimens failed to produce a library; out of the 294 remaining samples, a total of 168 somatic mutations were found. In nearly all instances (74/75-99%), the EGFR SiRe results were confirmed by Idylla. In general, SiRe analytical parameters were excellent. However, histological and cytological specimens differed in relation to average reads for sample, mean number of mapped reads, median read length and average reads for amplicon. Treatment outcome evaluation in 11 patients showed a partial response in 82 % (9/11) patients with a median progression-free survival of 340 days. CONCLUSIONS: The small gene panel SiRe is a clinically relevant tool useful to widespread the adoption of NGS in predictive molecular pathology laboratories.