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

Design and validation of a next generation sequencing custom panel for the pre-surgical risk stratification of indeterminate thyroid fine needle aspirations

Background: Thyroid carcinoma is the most common malignant neoplasm of the endocrine system, with a growing incidence peak in the world (1). Thyroid tumors generally consist of benign lesions that can affect one lobe or both; however, only a small fraction of them are classified as malignant (7-15%). The correct classification of thyroid tumors represents a crucial point for the stratification of patients with thyroid cancer in terms of prognosis and therapy. Currently, aspiration of the fine needle of the thyroid (FNA) is the most reliable and economic diagnostic tool to evaluate the morphological characteristics of the thyroid neoplasm, but very scarce nucleic acids are available to perform molecular tests. Methods: A commercially available test based on real-time PCR (RT-PCR) technology was tested on a series of indeterminate thyroid FNAs. In addition, a customized NGS gene panel was designed and tested on a different set of retrospective FNA samples. Results: the RT-PCR assay, which evaluates the genomic alterations of BRAF, N-H-KRAS, RET / PTC and PAX8 / PPARG, was chosen for subsequent clinical validation on a prospective series of n = 1172 thyroid FNA. In addition, 76 samples were tested to validate the gene panel. The custum gene panel allows to widen the reference reference range. In fact, with it it is possible to test 15 important genes for the stratification and evaluation of ROM in thyroid nodules. Conclusions: Our preliminary data show that 7 RT-qPCR genes and a customized NGS panel are feasible tests that should be implemented in routine diagnostics in order to avoid surgical treatment for low-risk nodules. The significant difference in post-test ROM between MT-pos and MT-neg FNA confirmed the high positive predictive value of BRAFV600E and BRAF type mutations compared to RAS-like genomic alterations. Furthermore, the preliminary results of the customized NGS panel have been satisfactory enough to expect its actual future adoption on our routine clinical samples

Multiple predictive biomarker testing in melanoma: another challenge for the optimal approach on cytological samples

Background: The management of cutaneous melanoma has dramatically changed in recent years thanks to the development of tyrosine kinase and immune-checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important to identify patients potentially eligible for these treatments. One reliable approach for molecular evaluation of metastatic melanoma is fine-needle cytology (FNC). To examine the utility of this approach for PD-L1 assessment, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas previously tested for BRAF and NRAS mutations. Methods: We retrieved from our internal archives a series of FNC samples of metastatic melanoma subjected to molecular testing on residual CB material or dedicated needle rinse, between January 2016 and July 2022. Briefly, RT-PCR was adopted to assess BRAF and NRAS status, and SP263 assay was employed to assess PD-L1 expression level. Results: Overall n=19 cases were selected. Of these, 11 (57,9%) cases revealed a BRAF exon 15 p.V600E mutation, one case (5,3%) revealed NRAS mutation, and seven cases (36,8%) showed no mutations. Regarding PD-L1 assessment, 16/19 (84,2%) cases were deemed adequate, containing at least 100 viable cells. Conclusions: We highlighted the feasibility of PD-L1 assessment on residual CB material from metastatic melanomas previously tested for BRAF and NRAS mutations. Moreover, we pointed out that FNC needle rinses may be an alternative sources of nucleic acids for molecular testing, preserving CB material for ICC evaluation

Molecular Testing of Thyroid Fine-Needle Aspiration: Local Issues and Solutions. An Interventional Cytopathologist Perspective

Molecular testing has acquired a relevant role for diagnostic and prognostic stratification of indeterminate thyroid nodules. Besides the available commercial solutions marketed in the United States, various local testing strategies have been developed in the last decade. In this setting, the modern interventional cytopathologist, the physician who performs the both aspirate and the morphologic interpretation plays a key role in the correct handling of fine-needle aspiration (FNA) samples not only for microscopy but also for molecular techniques. This review summarizes experiences with local approaches to the molecular testing of thyroid FNA, highlighting the role of the modern interventional cytopathologist

Fine-Needle Aspiration Is Suitable for Breast Cancer BRCA Molecular Assessment: A Case Report

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status

Multiplex digital colour-coded barcode technology on RNA extracted from routine cytological samples of patients with non-small cell lung cancer: pilot study

In the advanced stages of non-small cell lung cancer (NSCLC), molecular testing is often performed on archival cytological smears. The nCounter system (NanoString Technologies) is a new promising multiplex digital colour-coded barcode technology. However, its feasibility to evaluate the RNA expression of clinical relevant biomarkers on routine cytological smears is still uncertain. To this end, RNA was extracted from 12 NSCLC routine stained cytological smears, and nCounter analysis performed by using a 48-gene panel. Overall, 11/12 (92%) of the smears were adequate for the secondary analysis, fulfilling the quality check parameter analysis of nSolver software. This pilot study shows that RNA nCounter analysis is feasible on routine cytological smears preparing the field for the implementation of this technology in the routine setting

Liquid Biopsy Analysis in Clinical Practice: Focus on Lung Cancer

Lung cancer is the leading cause of cancer death worldwide. Despite the emergence of highly effective targeted therapies, up to 30% of advanced stage non-small cell lung cancer (NSCLC) patients do not undergo tissue molecular testing because of scarce tissue availability. Liquid biopsy, on the other hand, offers these patients a valuable opportunity to receive the best treatment options in a timely manner. Indeed, besides being much faster and less invasive than conventional tissue-based analysis, it can also yield specific information about the genetic make-up and evolution of patients’ tumors. However, several issues, including lack of standardized protocols for sample collection, processing, and interpretation, still need to be addressed before liquid biopsy can be fully incorporated into routine oncology practice. Here, we reviewed the most important challenges hindering the implementation of liquid biopsy in oncology practice, as well as the great advantages of this approach for the treatment of NSCLC patients

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

Predictive molecular pathology in the time of COVID-19

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