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Background: Sotorasib showed a significant improvement of progression free survival (PFS), safety and quality of life over docetaxel in patients with KRASp.G12C-mutated advanced non-small-cell lung cancer (NSCLC) within the CodeBreak-200 study. Here we report real-world efficacy and tolerability data from NSCLC patients who received sotorasib within the Italian expanded access program (EAP). Methods: Sotorasib (960 mg, orally, once daily) was available on physician request for KRASp.G12C mutant advanced NSCLC patients. Clinical-pathological and molecular data were collected from the Italian ATLAS real-world registry. Patients underwent CT-scan and responses were evaluated by RECIST criteria. Efficacy and tolerability outcomes have been assessed. Results: A total of 196 advanced NSCLC patients were treated across 30 Italian centers. Median age was 69 years old (range 33-86). Most patients were male (61 %), former (49 %) or current smokers (43 %), with ECOG-PS 0/1 (84 %) and adenocarcinoma subtype (90 %). 45 % and 32 % of patients received sotorasib in 2nd and 3rd line, respectively. Overall, response rate was 26 % and the median duration of response was 5.7 months (95 % CI: 4.4-7.0). Median PFS and OS were 5.8 months (95 % CI: 5 - 6.5) and 8.2 months (95 % CI: 6.3 - 9.9). Grade 3-4 TRAEs occurred in 16.5 % of patients, with Grade >= 3 liver enzyme increase and TRAEs-related discontinuation reported in 12 % and 4.6 % of cases. Conclusion: Real-world data from the Italian EAP confirm the tolerability and effectiveness of sotorasib in patients with KRASp.G12C-mutated advanced NSCLC and highlight the value of the national ATLAS network as source of real-world evidence driving the clinical management of NSCLC patients.

Introduction:PIK3CA gene mutations occur in approximately 40% of hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancers (MBCs), electing them to targeted therapy. Testing PIK3CA status is complex due to selection of biological specimen and testing method. Materials & methods: This work investigates real-life experience on PIK3CA testing in HR+/HER2- MBC. Clinical, technical and molecular data on PIK3CA testing were collected from two referral laboratories. Additionally, the results of a nationwide PIK3CA survey involving 116 institutions were assessed. Results: Overall, n = 35 MBCs were PIK3CA-mutated, with mutations mostly occurring in exons 9 (n = 19; 51.4%) and 20 (n = 15; 40.5%). The nationwide survey revealed significant variability across laboratories in terms of sampling methodology, technical assessment and clinical report signing healthcare figures for PIK3CA molecular testing in diagnostic routine practice. Conclusion: This study provides insights into the real-world routine of PIK3CA testing in HR+/HER2- MBC and highlights the need for standardization and networking in predictive pathology.

Molecular biomarker testing is increasingly becoming standard of care for advanced non-small cell lung cancer (NSCLC). Tissue and liquid biopsy-based next-generation sequencing (NGS) is now highly recommended and has become an integral part of the routine management of advanced NSCLC patients. This highly sensitive approach can simultaneously and efficiently detect multiple biomarkers even in scant samples. However full optimization of NGS in clinical practice requires accurate reporting and interpretation of NGS findings. Indeed, as the number of NSCLC biomarkers continues to grow, clinical reporting of NGS data is becoming increasingly complex. In this scenario, achieving standardization, simplification, and improved readability of NGS reports is key to ensuring timely and appropriate treatment decisions. In an effort to address the complexity and lengthy reporting of NGS mutation results, an Italian group of 14 healthcare professionals involved in NSCLC management convened in 2023 to address the content, structure, and ease-of-use of NGS reporting practices and proposed a standard report template for clinical use This article presents the key discussion points addressed by the Italian working group and describes the essential elements of the report template.

Background: The molecular diagnostic and therapeutic pathway of Non-Small Cell Lung Cancer (NSCLC) stands as a successful example of precision medicine. The scarcity of material and the increasing number of biomarkers to be tested have prompted the routine application of next-generation-sequencing (NGS) techniques. Despite its undeniable advantages, NGS involves high costs that may impede its broad adoption in laboratories. This study aims to assess the detailed costs linked to the integration of NGS diagnostics in NSCLC to comprehend their financial impact. Materials and methods: The retrospective analysis encompasses 210 cases of early and advanced stages NSCLC, analyzed with NGS and collected at the IRCCS San Gerardo dei Tintori Foundation (Monza, Italy). Molecular analyses were conducted on FFPE samples, with an hotspot panel capable of detecting DNA and RNA variants in 50 clinically relevant genes. The economic analysis employed a full-cost approach, encompassing direct and indirect costs, overheads, VAT (Value Added Tax). Results: We estimate a comprehensive cost for each sample of €1048.32. This cost represents a crucial investment in terms of NSCLC patients survival, despite constituting only around 1% of the expenses incurred in their molecular diagnostic and therapeutic pathway. Conclusions: The cost comparison between NGS test and the notably higher therapeutic costs highlights that the diagnostic phase is not the limiting economic factor. Developing NGS facilities structured in pathology networks may ensure appropriate technical expertise and efficient workflows.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids’ quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6–8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6–8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Aims: To date, precision medicine has revolutionized the clinical management of Non-Small Cell Lung Cancer (NSCLC). International societies approved a rapidly improved mandatory testing biomarkers panel for the clinical stratification of NSCLC patients, but harmonized procedures are required to optimize the diagnostic workflow. In this context a knowledge-based database (Biomarkers ATLAS, https://biomarkersatlas.com/) was developed by a supervising group of expert pathologists and thoracic oncologists collecting updated clinical and molecular records from about 80 referral Italian institutions. Here, we audit molecular and clinical data from n = 1100 NSCLC patients collected from January 2019 to December 2020. Methods: Clinical and molecular records from NSCLC patients were retrospectively collected from the two coordinating institutions (University of Turin and University of Naples). Molecular biomarkers (KRAS, EGFR, BRAF, ROS1, ALK, RET, NTRK, MET) and clinical data (sex, age, histological type, smoker status, PD-L1 expression, therapy) were collected and harmonized. Results: Clinical and molecular data from 1100 (n = 552 mutated and n = 548 wild-type) NSCLC patients were systematized and annotated in the ATLAS knowledge-database. Molecular records from biomarkers testing were matched with main patients’ clinical variables. Conclusions: Biomarkers ATLAS (https://biomarkersatlas.com/) represents a unique, easily managing, and reliable diagnostic tool aiming to integrate clinical records with molecular alterations of NSCLC patients in the real-word Italian scenario.

The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, − 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

Epidermal growth factor receptor (EGFR) exon 20 insertions occur rarely among different cancer types, with the highest frequency reported among non-small-cell lung cancer (NSCLC) patients, particularly adenocarcinomas (ADCs). Exon 20 insertions fall back in the tyrosine kinase domain, and can be clustered into two principal groups represented by in frame insertions and three to 21 bp (corresponding to 1–7 amino acids) duplications within amino acids 762 and 774. The identification of these alterations is key for an adequate management of NSCLC patients due to the possibility to treat these patients with specific targeted therapies. Next generation sequencing (NGS) technology, able to detect several hotspot gene mutations for different patients simultaneously, is the best detection approach due to its higher sensitivity and specificity compared to other techniques. Here we reviewed the principal biological characteristics, the main detection technologies and treatment options for NSCLC patients harbouring EGFR exon 20 insertions.

Aim: ALK, ROS1, NTRK and RET gene fusions and MET exon 14 skipping alterations represent novel predictive biomarkers for advanced non-small-cell lung cancer (NSCLC). Therefore, testing patients for these genetic variants is crucial for choosing the best selective treatment. Over the last couple of decades, next-generation sequencing (NGS) platforms have emerged as an extremely useful tool for detecting these variants. Materials & methods: In the present study, we report our NGS molecular records produced during a year of diagnostic activity. Results: Overall, our in-house developed NGS workflow successfully analyzed n = 116/131 (88.5%) NSCLC samples. Of these, eight (6.8%) and five (4.3%) out of 116 patients harbored ALK and RET gene rearrangements, respectively: one case harbored ROS1 gene fusion (0.7%). Conclusion: Our results highlight that an RNA-based NGS analysis can reliably detect gene fusion alterations, thereby playing a pivotal role in the management of NSCLC patients.

Constitutional BRCA1/BRCA2 pathogenic or likely pathogenic variants (PVs) are associated with an increased risk for developing breast and ovarian cancers. Current evidence indicates that BRCA1/2 PVs are also associated with pancreatic cancer, and that BRCA2 PVs are associated with prostate cancer risk. The identification of carriers of constitutional PVs in the BRCA1/2 genes allows the implementation of individual and family prevention pathways, through validated screening programs and risk-reducing strategies. According to the relevant and increasing therapeutic predictive implications, the inclusion of BRCA testing in the routine management of patients with breast, ovarian, pancreatic and prostate cancers represent a key requirement to optimize medical or surgical therapeutic and prevention decision-making, and access to specific anticancer therapies. Therefore, accurate patient selection, the use of standardized and harmonized procedures, and adherence to homogeneous testing criteria, are essential elements to implement BRCA testing in clinical practice. This consensus position paper has been developed and approved by a multidisciplinary Expert Panel of 64 professionals on behalf of the AIOM–AIRO–AISP–ANISC–AURO–Fondazione AIOM–SIAPEC/IAP–SIBioC–SICO–SIF–SIGE–SIGU–SIU–SIURO–UROP Italian Scientific Societies, and a patient association (aBRCAdaBRA Onlus). The working group included medical, surgical and radiation oncologists, medical and molecular geneticists, clinical molecular biologists, surgical and molecular pathologists, organ specialists such as gynecologists, gastroenterologists and urologists, and pharmacologists. The manuscript is based on the expert consensus and reports the best available evidence, according to the current eligibility criteria for BRCA testing and counseling, it also harmonizes with current Italian National Guidelines and Clinical Recommendations.

The introduction of next-generation sequencing (NGS) in the molecular diagnostic arma-mentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

Aims: The KWAY project aims to investigate the economic sustainability of the up-front NGS technologies adoption in the analysis of clinically relevant molecular alterations in NSCLC patients. Methods: The diagnostic workflow and the related sustained costs of five Italian referral centers were assessed in four different evolving scenarios were analyzed. For each scenario, two alternative testing strategies were evaluated: the Maximized Standard strategy and the Maximized NGS strategy. Results: For each center, the robustness of obtained results was verified through a deterministic sensitivity analysis, observing the variation of total costs based on a variation of ±20 % of the input parameters and ensuring that results would present a consistent behavior compared to the original ones. Conclusions: our project, highlighted that the adoption of NGS allows to save personnel time dedicated to testing activities and to reduce the overall cost of testing per patient.

Background: Rearranged during transfection (RET) gene fusions are detected in 10–20% of thyroid cancer patients. Recently, RET fusion-positive metastatic thyroid cancers have attracted much attention owing to the FDA approval of two highly selective anti-RET tyrosine kinase inhibitors, namely, selpercatinib, and pralsetinib. Areas covered: This review summarizes the available evidence on the biological and predictive role of RET gene fusions in thyroid carcinoma patients and the latest screening assays currently used to detect these genomic alterations in histological and cytological specimens. Expert opinion: Management of advanced thyroid carcinoma has significantly evolved over the last decade thanks to the approval of three multikinase inhibitors, i.e. sorafenib, lenvatinib, cabozantinib, and of two selective RET-tyrosine inhibitors, i.e. selpercatinib and pralsetinib. In this setting, the detection of RET-fusions in advanced thyroid cancer specimens through the use of next-generation sequencing has become a commonly used strategy in clinical practice to select the best treatment options.

Mucoepidermoid carcinoma (MEC) of the breast is an extremely rare salivary gland-type tumor characterized by epidermoid, basaloid, intermediate, and/or mucinous cells arranged in solid and cystic patterns. Despite their triple-negative phenotype, breast MECs are generally considered low-risk malignancies but their biology is largely unexplored; therefore, guidelines for clinical management are lacking. Here, we sought to characterize the molecular landscape of breast MECs. Thirteen cases were histologically reviewed, characterized for tumor-infiltrating lymphocytes (TILs), and were subjected to immunohistochemistry for programmed death-ligand 1 (PD-L1, clone 22C3), EGFR, and amphiregulin (AREG). Rearrangements in MAML2 and EWSR1 were investigated by fluorescent in situ hybridization. Targeted next-generation sequencing of 161 genes was performed on eight cases. Most MECs had low histological grade (n = 10, 77%), with the presence of TILs (n = 9/12; 75%) and PD-L1 combined positive score ranging from 10 to 20 (n = 4/6; 67%). All cases showed EGFR and AREG overexpression and were fusion negative. Enrichment of genetic alterations was observed in PI3K/AKT/mTOR and cell cycle regulation pathways, while only one case harbored TP53 mutations. This is the first study providing extensive molecular data on breast MECs and the largest collection of cases available to date in the literature. Breast MECs lack TP53 mutations found in high-grade forms of triple-negative breast cancers and MAML2 or EWSR1 rearrangements pathognomonic of salivary MECs. Triple-negativity and PD-L1 positivity suggest a window of opportunity for immunotherapy in these patients. The EGFR/AREG axis activation, coupled with the mutational patterns in PI3K/AKT/mTOR and cell cycle pathways warrants caution in considering MECs as low-risk neoplasms.

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.