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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. Molecular cytopathology is a rapidly evolving field. Modern molecular cytopathologists play a key role in bridging the gap between conventional microscopy and novel molecular technologies. Cytological samples represent a valid source of high‐quality DNA for next generation sequencing (NGS) analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnosis. [ABSTRACT FROM AUTHOR]

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. Immune‐checkpoint inhibitors (ICIs) are an important treatment option for patients who have advanced stage non–small cell lung cancer, and tumor mutational burden (TMB) is a valid biomarker for ICI administration. Cell blocks may be a suitable starting material for TMB analysis. [ABSTRACT FROM AUTHOR]

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 PDGFR α) 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. [ABSTRACT FROM AUTHOR]

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. The 7‐gene panel test improves the preoperative risk stratification of indeterminate thyroid fine‐needle aspirations, especially when considering the biologic significance of the different types of mutations (ie, BRAF‐like and RAS‐like alterations). Moreover, the rate of mutation‐positive thyroid fine‐needle aspirations is related to the risk of malignancy of each diagnostic class, supporting the establishment, from a molecular standpoint, of prognostically relevant cytologic diagnostic categories. [ABSTRACT FROM AUTHOR]

BACKGROUND: The Bethesda System for Reporting Thyroid Cytopathology category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) includes fine‐needle aspiration (FNA) specimens that cannot straightforwardly be classified as benign or malignant. To determine whether morphological subcategorization based on atypia qualifiers and molecular testing could improve malignancy risk stratification of AUS/FLUS patients, this study assessed the correlation between these qualifiers and the molecular alterations commonly harbored by thyroid neoplasms. METHODS: A total of 162 AUS/FLUS cases were subcategorized by atypia qualifiers (Hürthle cell changes, architectural atypia, and cytologic atypia [CyA]) and were tested for BRAF, N‐H‐KRAS, RET/PTC, and paired box 8 (PAX8)/peroxisome proliferator activated receptor γ (PPARg) mutations. RESULTS: CyA was observed more frequently in mutation‐positive AUS/FLUS (14 of 37 [37.84%]) than mutation‐negative AUS/FLUS (20 of 125 [16.00%]; P < .0084), and it specifically harbored the BRAFV600E point mutation. Malignancy was confirmed in the available follow‐up. Conversely, although RAS was the most frequent mutation identified in AUS/FLUS FNA specimens (26 of 37 cases [70.27%]; P < .0001), it was distributed across various AUS/FLUS subcategories and was not significantly associated with a specific atypia qualifier or malignant outcome according to the available follow‐up. Rearrangements of both RET/PTC (n = 1) and PAX8/PPARg (n = 3) were rarely retrieved in the FNA samples. CONCLUSIONS: BRAF and RAS mutations are associated with different AUS/FLUS qualifiers and hence have different risks of malignancy. Consequently, a hybrid molecular and morphological subcategorization system could improve the malignancy risk stratification of thyroid FNA samples diagnosed as AUS/FLUS. Cancer Cytopathol 2018;126:317‐25. © 2018 American Cancer Society. [ABSTRACT FROM AUTHOR]

Background: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.Methods: Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.Results: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.Conclusions: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice. [ABSTRACT FROM AUTHOR]

The patient referred to the neurosurgeon in October 2015, complaining polyuria, polydipsia and polyphagia, associated with violent headache, unresponsive to anti-inflammatory therapy, vomit and loss of consciousness. In May 2016 the patient underwent surgery, by mean of endoscopic endonasal approach: at this time, due to the hard consistency, the tight adherences of the tumor and the narrow and deep corridor lesion has been only partially removed. Six months thereafter a new MRI disclosed a slight enlargement of tumor (volume increase was 30% more than the prior exam), so that transcranial transcortical-transventricular approach was adopted to remove the lesion. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Gene fusions represent novel predictive biomarkers for advanced Non Small Cell Lung Cancer (NSCLC) patients. In this study, we developed and validated a narrow Next Generation Sequencing gene panel able to cover ALK, ROS1, RET and NTRK gene fusions and MET splicing events in advanced-stage NSCLC patients. Overall, our RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. In addition, It also successfully analyzed 46 (95.8%) out of 48 routine samples previously characterized by conventional non - NGS technology, representing a robust tool for routine setting. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: UM reports personal fees (as speaker bureau or advisor) from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen, and Merck, unrelated to the current work. GT reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, and Bayer, unrelated to the current work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pisapia, Pepe, Iaccarino, Sgariglia, Nacchio, Conticelli, Salatiello, Tufano, Russo, Gragnano, Girolami, Eccher, Malapelle and Troncone.)

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., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jtd.2020.01.19). The series “Improving Outcomes in Lung Cancer Through Early Diagnosis and Smoking Cessation” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare., (2020 Journal of Thoracic Disease. All rights reserved.)

Background: Non small cell lung cancer (NSCLC) is diagnosed in most cases on small tissue samples, such as cytological preparations and histological biopsies; these limited tissue specimens may be not always sufficient for testing epidermal growth factor receptor ( EGFR ) mutations and other relevant predictive biomarkers. Cell-free DNA (cfDNA) can be used as a surrogate for EGFR mutational testing, whenever tissue is unavailable. However, the detection of gene mutations on cfDNA is challenging; in fact, the extremely low concentration of circulating tumor DNA requires the implementation of highly sensitive and validated next generation techniques., Methods: Thus, we have recently validated a novel next generation sequencing (NGS) assay, employing the SiRe ® gene panel to detect on cfDNA mutations of EGFR and KRAS, NRAS, BRAF, cKIT and PDGFR genes. In this current study, we report on a series of NSCLC patients, without available tissue for EGFR testing, who prospectively underwent SiRe ® NGS analysis., Results: The results confirm the high clinical performance, in terms of success rate and mutation detection, of NGS based analysis of cfDNA., Conclusions: SiRe ® NGS panel represent an effective diagnostic tool in cfDNA analysis setting., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Background: Fine-needle aspiration (FNA) cytology is accurate and cost-effective in the evaluation of thyroid nodules. Molecular techniques may contribute to risk stratification in indeterminate cases. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid FNA specimens, thyroid-specific cancer gene panels are not commercially available. Conversely, the Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2), which includes the genes most frequently mutated in thyroid neoplasms, is commercially available and may represent an alternative to thyroid-specific panels. To the authors' knowledge to date, CHPv2 has performed well only on "ideal" cytological samples featuring abundant, high-quality DNA and satisfactory postsequencing metrics. The objective of the current study was to extend NGS to less-than-ideal samples, which represent a large percentage of routine clinical specimens., Methods: A total of 37 thyroid smears were retrospectively analyzed using CHPv2, regardless of any preanalytical and postsequencing metric thresholds. Specifically, the authors evaluated the performance of CHPv2 on the BRAF, NRAS, HRAS, KRAS, and RET genes. Results were verified by pyrosequencing., Results: Of the 37 thyroid FNA specimens, 34 (91.8%) were successfully processed. BRAF, NRAS, and RET somatic variants were detected in 22 of these 34 specimens (64.7%). NGS was found to have a high sensitivity (89.4%), specificity (85.7%), and accuracy (88.4%)., Conclusions: CHPv2 is a valid option for the molecular evaluation of thyroid FNA specimens by NGS. It is interesting to note that this approach is accurate and effective even when applied to routine cytology samples that usually do not have optimal preanalytical and postsequencing requirements. Cancer Cytopathol 2016;124:776-84. © 2016 American Cancer Society., (© 2016 American Cancer Society.)