Molecular Characterization of Advanced-Stage Melanomas in Clinical Practice Using a Laboratory-Developed Next-Generation Sequencing Panel.

Cutaneous melanoma is one of the most lethal tumors among skin cancers, characterized by complex genetic and molecular alterations that result in uncontrolled cell proliferation and metastatic spread. Next-generation sequencing (NGS) enables the simultaneous examination of numerous genes, making this molecular technique essential for melanoma diagnosis, prognostic stratification, and therapy planning. Herein, we present the experience with our laboratory-designed NGS panel for the routine assessment of advanced-stage melanoma. A total of 260 specimens of advanced-stage melanomas were evaluated utilizing a laboratory-developed multi-gene NGS panel, which allowed the investigation of 229 amplicons in 25 oncogene/oncosuppressor genes. The NGS panel proved to be a reliable tool, failing to produce results in only 1.2% of the samples tested. BRAF and TERT were the two more commonly altered genes in 44.0% and 59.9% of samples, respectively. In 59.3% of the mutated cases, at least two concomitant variants were detected. In eight cases, both primary lesion and metastatic disease were analyzed by NGS. In all specimens (8/8, 100%), a perfect concordance in variants harbored by the primary and recurrence lesions was observed. Finally, this study described the validity of a laboratory-developed multi-gene NGS panel built specifically for advanced-stage melanomas in ordinary clinical practice.

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer.

Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC.

Prognostic Impact of Pathologic Features in Molecular Subgroups of Endometrial Carcinoma.

The molecular characterization of endometrial carcinoma (EC) has recently been included in the ESGO/ESTRO/ESP guidelines. The study aims to evaluate the impact of integrated molecular and pathologic risk stratification in the clinical practice and the relevance of pathologic parameters in predicting prognosis in each EC molecular subgroup. ECs were classified using immunohistochemistry and next-generation sequencing into the four molecular classes: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). According to the WHO algorithm, 219 ECs were subdivided into the following molecular subgroups: 7.8% POLE, 31% MMRd, 21% p53abn, 40.2% NSMP. Molecular classes as well as ESGO/ESTRO/ESP 2020 risk groups were statistically correlated with disease-free survival. Considering the impact of histopathologic features in each molecular class, stage was found to be the strongest prognostic factor in MMRd ECs, whereas in the p53abn subgroup, only lymph node status was associated with recurrent disease. Interestingly, in the NSMP tumor, several histopathologic features were correlated with recurrence: histotype, grade, stage, tumor necrosis, and substantial lymphovascular space invasion. Considering early-stage NSMP ECs, substantial lymphovascular space invasion was the only independent prognostic factor. Our study supports the prognostic importance of EC molecular classification and demonstrated the essential role of histopathologic assessment in patients' management.

Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice.

Introduction: The European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class. Methods: The cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). Results: Immuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between "unknown molecular classification" and "known," the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients. Conclusion: Application of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification.

BRAF V600E-mutated large cell neuroendocrine carcinoma responding to targeted therapy: a case report and review of the literature.

Large cell neuroendocrine carcinoma (LCNEC) is a rare and aggressive high-grade neuroendocrine tumor, commonly arising in the lung or in the gastrointestinal tract, with a frequent proportion of unknown primary origin (20%). In the metastatic setting, platinum-based or fluoropyrimidine-based chemotherapeutic regimens are as considered the first-line treatment, despite the limited duration of response. To date, the prognosis of advanced high-grade neuroendocrine carcinoma remains poor, suggesting the need to explore new treatment strategies in this orphan tumor. The evolving molecular landscape of LCNEC, not yet been completely defined, could explain the heterogeneous response to different chemotherapeutic regimens and suggest that treatment strategy could be driven by molecular features. v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutations, well described in melanoma, thyroid cancer, colon cancer and lung adenocarcinoma, account for approximately 2% of cases in lung LCNEC. Here, we describe the case of a patient with a BRAF V600E-mutated LCNEC of unknown primary origin who partially responded to BRAF/mitogen-activated protein kinase kinase inhibitors after standard treatment. Additionally, BRAF V600E circulating tumor DNA was used to monitor disease response. Thereafter, we reviewed the available literature about the role of targeted therapy in high-grade neuroendocrine neoplasms to provide insight for future research to identify patients with driver oncogenic mutations, who can potentially benefit from target therapy.

Expanding the Spectrum of BRAF Non-V600E Mutations in Thyroid Nodules: Evidence-Based Data from a Tertiary Referral Centre.

The BRAF p.V600E mutation represents the most specific marker for papillary thyroid carcinoma and is potentially related to aggressive behavior and persistent disease. BRAF alterations other than the p.V600E are less common in thyroid carcinoma and represent an alternative mechanism of BRAF activation with unclear clinical significance. The study aims to describe the frequency and clinicopathologic characteristics of BRAF non-V600E mutations in a large cohort (1654 samples) of thyroid lesions characterized by next-generation sequencing. BRAF mutations have been found in 20.3% (337/1654) of thyroid nodules, including classic (p.V600E) mutation in 19.2% (317/1654) of samples and non-V600E variants in 1.1% of cases (19/1654). BRAF non-V600E alterations include 5 cases harboring p.K601E, 2 harboring p.V600K substitutions, 2 with a p.K601G variant, and 10 cases with other BRAF non-V600E alterations. BRAF non-V600E mutations have been reported in one case of follicular adenoma, three cases of conventional papillary thyroid carcinoma, eight cases of follicular variant of papillary carcinomas, one case of columnar cell variant papillary thyroid carcinoma, one case of oncocytic follicular carcinoma, and two bone metastasis of follicular thyroid carcinoma. We confirm that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential. However, in both aggressive cases, the BRAF mutations were concomitant with other molecular alterations, such as TERT promoter mutation.

ADK-VR2, a cell line derived from a treatment-naïve patient with SDC4-ROS1 fusion-positive primarily crizotinib-resistant NSCLC: a novel preclinical model for new drug development of ROS1-rearranged NSCLC.

Background: ROS1 fusions are driver molecular alterations in 1-2% of non-small cell lung cancers (NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods: The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo. Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK-VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions: The ADK-VR2 cell line is a promising NSCLC preclinical model for the study of novel targeted therapies against ROS1 fusions and the mechanisms of resistance to TKI therapies.

Clinical characteristics and treatment outcomes of non-V600 E/K BRAF mutant melanoma patients: a single-institution experience.

The widespread use of more sensitive detection tools, such as next-generation sequencing, has increased the identification of a variety of BRAF mutations other than V600E/K in melanoma patients. However, there is a lack of established data regarding the efficacy of BRAF/MEK inhibitors and immune-checkpoint immune inhibitors (ICI) for these patients. We performed a retrospective study, including all the patients diagnosed with stage III or IV melanoma that were referred to the University Hospital of Bologna from 2011 to 2021, carrying a non-V600E or V600K mutation of BRAF and who were started on systemic treatment. We found 14 patients with stage III or IV melanoma harboring the following BRAF mutations: V600R, V600_K601delinsE, K601E, p.T599_V600insT, L597V, G466R, S467L, and A598T. Of note, G466R and A598T BRAF mutations have never been previously reported in melanoma. Four patients received combined BRAF/MEK inhibitors, two patients BRAF inhibitor monotherapy, and six patients were treated with ICI for advanced melanoma; four patients received adjuvant treatment with nivolumab. Given the few cases and the absence of randomized clinical trials, it is important to report clinical experiences, which can guide physicians in the treatment of melanomas harboring rare BRAF mutations.

Therapeutic Outcomes and Clinical Features of Advanced Non-Small Cell Lung Cancer Carrying KRAS Mutations: A Multicenter Real-life Retrospective Study.

INTRODUCTION: Targeting Kirsten Rat Sarcoma (KRAS) has been deemed impossible for long time, but new drugs have recently demonstrated promising results. Evidence on the outcome of KRAS-mutant advanced-NSCLC treated with new standard regimens are still scarce. Thus, we aimed at assessing the incidence and clinical impact of KRAS mutations in a real-life population of advanced-NSCLC, exploring the prognostic significance of distinct alterations. MATERIALS AND METHODS: The present multicenter retrospective study, conducted by 5 Italian Centers from January 2018 to February 2020, involved 297 advanced KRAS mutant NSCLC. Complete clinico-pathological data were evaluated. RESULTS: Out of 297 patients, 130 carried KRAS_G12C mutation, while 167 presented with mutations other than G12C. Within KRAS_non-G12C group, 73%, 16.8% and 8.9% harboured G12X, codon 13 and Q61H alterations, respectively. No significant differences in survival outcome and treatment response were documented according to KRAS_G12C versus non-G12C, nor KRAS_G12C versus G12X versus other mutations. On univariate analysis ECOG PS, number and sites of metastatic lesions and PD-L1 status significantly impacted on survival. A clear trend towards worse prognosis was apparent in chemotherapy-treated patients, while immunotherapy-based regimens were associated to prolonged survival. Investigating the outcome of PD-L1 ≥ 50% population, we did not detect any significant difference between KRAS_G12C and non-G12C subsets. CONCLUSION: Here, we report on real-life data from a large retrospective cohort of advanced NSCLC harbouring KRAS alterations, with particular attention to G12C mutation. Our study offers useful clues on survival outcome, therapeutic response and clinico-pathological correlations in KRAS-mutant setting, especially in the upcoming era of KRAS G12C targeting therapy.

Correlation of molecular alterations with pathological features in hepatocellular carcinoma: Literature review and experience of an Italian center.

Hepatocellular carcinoma (HCC) represents the primary carcinoma of the liver and the fourth leading cause of cancer-related deaths. The World Health Organization estimates an increase in cases in the coming years. The risk factors of HCC are multiple, and the incidence in different countries is closely related to the different risk factors to which the population is exposed. The molecular mechanisms that drive HCC tumorigenesis are extremely complex, but understanding this multistep process is essential for the identification of diagnostic, prognostic, and therapeutic markers. The development of multigenic next-generation sequencing panels through the parallel analysis of multiple markers can provide a landscape of the genomic status of the tumor. Considering the literature and our preliminary data based on 36 HCCs, the most frequently altered genes in HCCs are TERT, CTNNB1, and TP53. Over the years, many groups have attempted to classify HCCs on a molecular basis, but a univocal classification has never been achieved. Nevertheless, statistically significant correlations have been found in HCCs between the molecular signature and morphologic features, and this leads us to think that it would be desirable to integrate the approach between anatomic pathology and molecular laboratories.

Multicenter Observational Study on Metastatic Non-Small Cell Lung Cancer Harboring BRAF Mutations: Focus on Clinical Characteristics and Treatment Outcome of V600E and Non-V600E Subgroups.

INTRODUCTION: BRAF mutation involved 2-4% of lung adenocarcinoma. Differences in clinicopathologic features and patient outcome exist between V600E and non-V600E BRAF mutated NSCLC. Thus, we sought to assess the frequency and clinical relevance of BRAF mutations in a real-life population of advanced-NSCLC, investigating the potential prognostic significance of distinct genetic alterations. MATERIALS AND METHODS: The present multicenter Italian retrospective study involved advanced BRAF mutant NSCLC. Complete clinicopathologic data were evaluated for BRAF V600E and non-V600E patients. RESULTS: A total of 44 BRAFmut NSCLC patients were included (V600E, n = 23; non-V600E, n = 21). No significant differences in survival outcome and treatment response were documented, according to V600E vs. non-V600E mutations, although a trend towards prolonged PFS was observed in the V600E subgroup (median PFS = 11.3 vs. 6.0 months in non-V600E). In the overall population, ECOG PS and age significantly impacted on OS, while bone lesions were associated with shorter PFS. Compared to immunotherapy, first-line chemotherapy was associated with longer OS in the overall population, and especially in the BRAF V600E subtype. CONCLUSIONS: Here, we report on real-life data from a retrospective cohort of advanced-NSCLC harboring BRAF alterations. Our study offers relevant clues on survival outcome, therapeutic response, and clinicopathologic correlations of BRAF-mutant NSCLC.

Sarcomatoid change in adenocarcinoma arising in adulthood congenital pulmonary airway malformation.

Congenital pulmonary airway malformations (CPAM) are rare conditions generally diagnosed in childhood and possibly harboring malignant tumor growths. We describe a unique case of pleomorphic carcinoma in a longstanding type 1 CPAM diagnosed by wedge resection. The patient underwent completion left lower lobectomy and lymphadenectomy, but cancer recurred in nodal station #7 six months later. Clinicians should keep in mind that CPAM may hide radiologically undetectable malignancy in a relevant rate of cases, then requiring surgery in all patients. While MIA is the most common histology in type 1 CPAM, sarcomatoid change has herein been demonstrated.

Unexpected Widespread Bone Metastases from a BRAF K601N Mutated Follicular Thyroid Carcinoma within a Previously Resected Multinodular Goiter.

Follicular thyroid carcinoma (FTC) represents the second most common malignant thyroid neoplasm after papillary carcinoma (PTC). FTC is characterized by the tendency to metastasize to distant sites such as bone and lung. In the last 20 years, the understanding of the molecular pathology of thyroid tumors has greatly improved. Uncommon BRAF non-V600E mutations have been identified and are generally believed to associate with follicular patterned tumors of low malignant potential, particularly non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTPs) (i.e., non-invasive encapsulated follicular variant PTC). We here report for the first time widespread bone metastases from a BRAF K601N mutated follicular tumor.

Broad spectrum mutational analysis of chromophobe renal cell carcinoma using next-generation sequencing.

BACKGROUND: Chromophobe renal cell carcinoma (ChRCC) is a rare subtype of non-clear cell renal cell carcinoma. Due to its rarity, its molecular characterization as well as therapeutic targets are still not fully understood. METHODS: We performed a next-generation sequencing analysis using the platform Ion PGM System on 20 retrospectively collected ChRCC cases with the aim of identify molecular biomarkers with potential prognostic value or that could have therapeutic implications. RESULTS: We identified mutation onTP53, SMARCB1, RB1 and JAK3. The most frequently altered gene was TP53 (6/20, 30 % of cases). SMARCB1 mutation was found in 3 (15 %) patients and in all cases the mutational variant was p.T72 K, with known pathogenenic meaning. One (5%) patient presented a pathogenetic mutation of RB1. JAK3 was mutated in 1 (5%) patient and this mutation resulted to have uncertain pathogenetic significance. CONCLUSION: ChRCC is a rare disease still not fully molecularly characterized. Next-generation sequencing analysis could be useful to identify potential mutation with prognostic value or that could be potential therapeutic targets.

SDHA Germline Variants in Adult Patients With SDHA-Mutant Gastrointestinal Stromal Tumor.

BACKGROUND: SDH-deficient gastrointestinal stromal tumors (GIST) account for 20-40% of all KIT/PDGFRA-negative GIST and are due to mutations in one of the four SDH-complex subunits, with SDHA mutations as the most frequent. Here we sought to evaluate the presence and prevalence of SDHA variants in the germline lineage in a population of SDHA-deficient GIST. METHODS: Germline SDHA status was assessed by Sanger sequencing on a series of 14 patients with gastric SDHA-deficient GIST. RESULTS: All patients carried a germline SDHA pathogenic variant, ranging from truncating, missense, or splicing variants. The second hit was the loss of the wild-type allele or an additional somatic mutation. One-third of the patients were over 50 years old. GIST was the only disease presentation in all cases except one, with no personal or familial cancer history. Seven metastatic cases received a multimodal treatment integrating surgery, loco-regional and medical therapy. The mean follow-up time was of 10 years, confirming the indolent clinical course of the disease. CONCLUSION: SDHA germline variants are highly frequent in SDHA-deficient GIST, and the disease may occur also in older adulthood. Genetic testing and surveillance of SDHA-mutation carriers and relatives should be performed.

Gene duplication, rather than epigenetic changes, drives FGF4 overexpression in KIT/PDGFRA/SDH/RAS-P WT GIST.

Gastrointestinal stromal tumours that are wild type for KIT and PDGFRA are referred to as WT GISTs. Of these tumours, SDH-deficient (characterized by the loss of SDHB) and quadruple WT GIST (KIT/PDGFRA/SDH/RAS-P WT) subgroups were reported to display a marked overexpression of FGF4, identifying a putative common therapeutic target for the first time. In SDH-deficient GISTs, methylation of an FGF insulator region was found to be responsible for the induction of FGF4 expression. In quadruple WT, recurrent focal duplication of FGF3/FGF4 was reported; however, how it induced FGF4 expression was not investigated. To assess whether overexpression of FGF4 in quadruple WT could be driven by similar epigenetic mechanisms as in SDH-deficient GISTs, we performed global and locus-specific (on FGF4 and FGF insulator) methylation analyses. However, no epigenetic alterations were detected. Conversely, we demonstrated that in quadruple WT GISTs, FGF4 expression and the structure of the duplication were intimately connected, with the copy of FGF4 closer to the ANO1 super-enhancer being preferentially expressed. In conclusion, we demonstrated that in quadruple WT GISTs, FGF4 overexpression is not due to an epigenetic mechanism but rather to the specific genomic structure of the duplication. Even if FGF4 overexpression is driven by different molecular mechanisms, these findings support an increasing biologic relevance of the FGFR pathway in WT GISTs, both in SDH-deficient and quadruple WT GISTs, suggesting that it may be a common therapeutic target.

An Analysis of Clinical, Surgical, Pathological and Molecular Characteristics of Endometrial Cancer According to Mismatch Repair Status. A Multidisciplinary Approach.

Since 2016, our hospital has applied tumor testing with immunohistochemistry (IHC) in endometrial cancer in order to detect mutations of mismatch repair genes (MMR). All cases with MMR deficiency proteins expression are sent for genetic testing, except those with MLH1 protein deficiency, in which case genetic testing is performed if negative for promoter hypermethylation. The primary aim of this study was to investigate the ability of our algorithm to identify Lynch syndrome (LS). The Secondary aims were to investigate the relationship between MMR status and clinicopathological features and prognosis of primary endometrial cancer (EC). From January 2016 to December 2018, 239 patients with EC were retrospectively analyzed and subdivided according to MMR status. Patients were divided in three groups: MMR proficient, LS and Lynch-like cancer (LLC). LS was characterized by a lower age and BMI, more use of contraceptive and less use of hormonal replacement therapy, nulliparity and a trend versus a better prognosis. LLC appeared more related to MMR proficient than LS and exhibited a more aggressive behavior. Our multidisciplinary approach permitted a correct diagnosis of germline mutation in patients with newly diagnosis EC and it confirmed clinicopathologic and prognostic characteristics of LS.

Comparison of Sequential Testing and Next Generation Sequencing in advanced Lung Adenocarcinoma patients - A single centre experience.

OBJECTIVES: Molecular diagnosis determines therapeutic strategies for patients with non-small-cell lung cancer - adenocarcinoma (NSCLC-A) but depends on resources availability. We compared a sequential single-gene testing algorithm to next generation sequencing in NSCLC-A to assess differences in terms of effectiveness, costs, tissue consumption and time. MATERIALS AND METHODS: We analyzed a retrospective cohort of advanced NSCLC-A patients treated at the Sant'Orsola-Malpighi University Hospital. The sequential testing includes a first analysis of EGFR and KRAS status with further molecular testing physician driven. The available NGS panel detects 35 hotspot mutations,19 amplifications and 23 rearrangements. RESULTS: We included 1758 patients; 1221 characterized with the sequential algorithm between January 2014 to February 2019 and 537 with Next Generation Sequencing (NGS) until January 2020. The prevalence of EGFR, ALK and KRAS alterations was similar between the stepwise and NGS group (16.5% vs 14.3%, 6.3% vs 6.3% and 36% vs 33.5%, respectively). Differently, ROS-1 rearrangements prevalence was higher in stepwise respect to NGS group (4.7% vs 0.7%). Similarly, the stepwise group presented higher prevalence than NGS for MET amplification (11.2% vs 2.2%), MET mutations (9.0% vs 2.4%), HER2 amplification (3.3% vs 1.9%) and mutations (9.8% vs 3.0%), and BRAF mutations (4.5% vs 5.6%). Among the NGS group other mutations were found in 141 patients (26.3%) and the presence of concurrent mutations in 131 (24.4%). The stepwise algorithm presented a relevant dropout rate that increased at each step, with 11.4%, 16.4% and 49.3% respectively for ALK, ROS1 and other analysis. Sequential testing's expenditure was 1375 € per patient, vs 770 € for NGS. Moreover, NGS testing can be performed with just a 25 µm slide respect to an estimated 33.3 µm slide for sequential strategy. CONCLUSION: NGS offered a less expensive and more reliable model of diagnosis respect to sequential one for patients affected by NSCLC-A.

Predictive molecular pathology in the time of coronavirus disease (COVID-19) in Europe.

AIMS: Lung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak. METHODS: Fifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time. RESULTS: In most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing. CONCLUSIONS: The workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

Next-generation sequencing revealing TP53 mutation as potential genetic driver in dermal deep-seated melanoma arising in giant congenital nevus in adult patients: A unique case report and review of the literature.

Melanoma in giant congenital nevus (M-GCN) is a rare and potentially lethal neoplasm. In children, M-GCN appears as a dermal/deep-seated melanoma (DDM-GCN) with histopathologic features difficult to distinguish from proliferative nodules (PNs-GCN). DDM-GCN in adults is an anecdotal entity and only 8 cases have been described and genetically characterized. We report the first case of DDM-GCN in a 34-year-old man characterized with a large-panel next-generation sequence (NGS) highlighting a TP53 mutation with a UV-signature (C>T substitution) in DDM but not in PNs-GCN and GCN. Curiously, DDM showed an aberrant p16 overexpression without detection of CDKN2A mutation at NGS. In line with previous studies, it supports a different pathway in children and adults: UV-induced mutations may be involved in the latter not only by CDKN2A but also by TP53 mutations, with a potentially confusing overexpression of p16 protein. While these data need to be confirmed in larger cases series, our results show that NGS could be an additional genetic diagnostic tool in DDM-GCN.