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Objectives: To i) compare baseline clinical and demographic characteristics of post-acute stroke inpatients who were diagnosed with first-time urinary tract infection (UTI) vs inpatients who were not ii) compare rehabilitation outcomes between both groups and iii) examine associations between time to UTI event and risk factors., Design: Retrospective observational cohort study SETTING: Institution for inpatient neurological rehabilitation., Participants: Inpatients (n=1683) admitted within 3 months post-stroke to a rehabilitation facility between 2005 and 2023., Interventions: Not applicable., Main Outcome Measures: Functional Independence Measure (FIM), Functional Ambulation Categories (FAC) at admission. Cox proportional hazards models analyzed the association between UTI event timing and risk factors., Results: Of the (n=1683) included patients, 196 (11.6%) experienced a UTI. In 32.1% of cases, the UTI occurred during the first week after admission to rehabilitation and 47.9% of UTIs occurred during the first two weeks. Median (IQR) time to UTI was 16 (5-37) days since admission. Most common germs were Escherichia coli (40.5%), Klebsiella pneumoniae (23.7%) and Pseudomonas aeruginosa (6.4%). Patients who acquired a UTI had older age, higher stroke severity, higher proportion of dysphagia, hypertension, neglect, bilateral affectation, atrial fibrillation, hemiplegia, lower levels of functional independence and lower FAC. We identified no differences in sex, type of stroke (ischemic or hemorrhagic), time to admission, aphasia, diabetes, dyslipidemia, chronic obstructive pulmonary disease, dominant side affected and educational level between both groups. Patients with UTI presented significantly poorer rehabilitation outcomes including lower discharge FIM and FAC, larger LOS, lower FIM efficiency and decreased FIM effectiveness. Multivariable Cox proportional hazards identified hypertension HR=1.60(1.13-2.27), admission FIM HR=0.98(0.97-0.99), admission BMI HR=0.96(0.93-0.99) and admitted with catheter HR= 1.80(1.22-2.64) as significant predictors of time to first UTI event (Concordance-index = 0.754)., Conclusions: UTIs identification, characterization, and predictive factors can support post-acute stroke mitigation strategies to minimize UTI-related complications and optimize rehabilitation outcomes., (Copyright © 2024. Published by Elsevier Inc.)

Melanoma is the most aggressive and deadly form of skin cancer, and its incidence has been steadily increasing over the past few decades, particularly in the Caucasian population. Immune checkpoint inhibitors (ICI), anti-PD-1 monotherapy or in combination with anti-CTLA-4, and more recently, anti-PD-1 plus anti-LAG-3 have changed the clinical evolution of this disease. However, a significant percentage of patients do not benefit from these therapies. Therefore, to improve patient selection, it is imperative to look for novel biomarkers. Immune subsets, particularly the quantification of lymphocyte T populations, could contribute to the identification of ICI responders. The main purpose of this review is to thoroughly examine significant published data on the potential role of lymphocyte T subset distribution in peripheral blood (PB) or intratumorally as prognostic and predictive of response biomarkers in advanced melanoma patients treated with ICI regardless of BRAFV600 mutational status.

Targeted NGS allows a fast and efficient multi-gene analysis and the detection of key gene aberrations in melanoma. In this study, we aim to describe the genetic alterations in a series of 87 melanoma cases using the oncomine focus assay (OFA), relate these results with the clinicopathological features of the patients, and compare them with our previous study results in which we used a smaller panel, the oncomine solid tumor (OST) DNA kit. Patients diagnosed with advanced melanoma at our center from 2020 to 2022 were included and DNA and RNA were extracted for sequencing. Common mutated genes were BRAF (29%), NRAS (28%), ALK , KIT , and MAP2K1 (5% each). Co-occurring mutations were detected in 29% of the samples, including BRAF with KIT , CTNNB1 , EGFR , ALK , HRAS , or MAP2K1. Amplifications and rearrangements were detected in 5% of cases. Only BRAF mutation showed a significant statistical association with sun exposure. For patients with a given genetic profile, the melanoma survival and recurrence-free survival rates were equivalent, but not for stage and LDH values. This expanded knowledge of molecular alterations has helped to more comprehensively characterize our patients and has provided relevant information for deciding the best treatment strategy., Competing Interests: C.T. declared no competing non-financial interests but reported advisory and consulting fees from Merck Sharp and Dohme (MSD), Novartis, and AstraZeneca, lecture fees from Roche, Pfizer, Biocartis, and MSD, and research from Novartis and AstraZeneca. Other authors declare no conflicts of interest.

Aims: The current WHO classification of melanocytic tumours excludes neoplasms showing BRAF or NRAS mutations from the Spitz category. This study aimed to review and reclassify atypical melanocytic tumours with spitzoid morphological features diagnosed between 2009 and 2021 in our hospital after expanding the molecular profile, including BRAF and NRAS mutations in all cases., Methods and Results: A total of 71 neoplasms showing spitzoid features (Spitz-like) and atypia were included. The risk of progression of tumours was first studied by integrating the morphology, immunohistochemistry (p16, Ki67, HMB45 and PRAME) and fluorescence in-situ hybridisation (FISH) results (melanoma multiprobe and 9p21). In a second step, after expanding the molecular study, including BRAF and NRAS mutational status, the neoplasms were finally classified into four subgroups: atypical Spitz tumour (AST, n = 45); BRAF-mutated naevus/low-grade melanocytoma with spitzoid morphology (BAMS, n = 2); Spitz melanoma (SM, n = 14); and BRAF or NRAS mutated melanoma with spitzoid features (MSF, n = 10). Follow-up of patients revealed uneventful results for AST and BAMS. Only one SM presented lymph node metastasis after 134 months. Conversely, patients with MSF showed an unfavourable outcome: three developed lymph node metastases after a mean time of 22 months, with one patient presenting distant metastasis and dying of the disease 64 months from diagnosis. The progression-free survival showed significant differences between the four groups of spitzoid tumours (P < 0.001) and between both melanoma subtypes (P = 0.012)., Conclusions: The classification and prognostication of atypical neoplasms with spitzoid features requires the integration of histomorphology with the molecular investigation of tumours, which should include BRAF and NRAS mutational status., (© 2024 John Wiley & Sons Ltd.)

Liquid biopsy, a minimally invasive approach for detecting tumor biomarkers in blood, has emerged as a leading-edge technique in cancer precision medicine. New evidence has shown that liquid biopsies can incidentally detect pathogenic germline variants (PGVs) associated with cancer predisposition, including in patients with a cancer for which genetic testing is not recommended. The ability to detect these incidental PGV in cancer patients through liquid biopsy raises important questions regarding the management of this information and its clinical implications. This incidental identification of PGVs raises concerns about cancer predisposition and the potential impact on patient management, not only in terms of providing access to treatment based on the tumor molecular profiling, but also the management of revealing genetic predisposition in patients and families. Understanding how to interpret this information is essential to ensure proper decision-making and to optimize cancer treatment and prevention strategies. In this review we provide a comprehensive summary of current evidence of incidental PGVs in cancer predisposition genes identified by liquid biopsy in patients with cancer. We critically review the methodological considerations of liquid biopsy as a tool for germline diagnosis, clinical utility and potential implications for cancer prevention, treatment, and research., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: A combined deep-penetrating tumour redefined as WNT-activated deep-penetrating/plexiform melanocytoma (DPM), may pose challenging clinical and histological diagnoses., Objectives: To review the clinicopathological characteristics of combined DPMs and characterize the molecular profile of atypical and malignant forms., Methods: The study included 51 patients with combined DPMs diagnosed at the Hospital Clinic of Barcelona and the University of Florence between 2012 and 2020. Clinical data, dermoscopy images (when available) and histological characteristics were reviewed. Immunohistochemistry for β-catenin, LEF1, HMB45, Ki67, p16 and PRAME (preferentially expressed antigen in melanoma) was performed. Atypical forms underwent next-generation sequencing (NGS) panel analysis, including driver genes implicated in DPMs, TERT-promoter (p) mutations and the investigation of the 9p21 locus via fluorescence in situ hybridization., Results: Among the 51 patients (32 females and 19 males, age range 4-74 years), 68% with available clinical data (15/22) were initially suspected of having melanoma. Except for one patient, complete excision resulted in no recurrences or metastases. One patient who had an incompletely excised combined DPM developed a lymph node melanoma metastasis 10 years later. In the 51 patients, 10 samples (20%) showed atypical histological features; 7 (14%) exhibited a significant loss of p16 expression; and 2 (4%) showed a high-proliferative index (Ki67 over 5%). NGS analysis in 11 patients revealed a double mutation BRAFV600E and exon 3 CTNNB1; no TERTp mutations were detected., Conclusions: Clinical suspicion of melanoma is common in combined DPMs, but malignant progression is infrequent in tumours lacking high-grade atypia or proliferation. These findings are congruent with the consideration of these lesions as intermediate-grade tumours or melanocytomas., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context.—: The neurotrophic tropomyosin receptor kinase (NTRK) family gene rearrangements have been recently incorporated as predictive biomarkers in a "tumor-agnostic" manner. However, the identification of these patients is extremely challenging because the overall frequency of NTRK fusions is below 1%. Academic groups and professional organizations have released recommendations on the algorithms to detect NTRK fusions. The European Society for Medical Oncology proposal encourages the use of next-generation sequencing (NGS) if available, or alternatively immunohistochemistry (IHC) could be used for screening with NGS confirmation of all positive IHC results. Other academic groups have included histologic and genomic information in the testing algorithm., Objective.—: To apply some of these triaging strategies for a more efficient identification of NTRK fusions within a single institution, so pathologists can gain practical insight on how to start looking for NTRK fusions., Design.—: A multiparametric strategy combining histologic (secretory carcinomas of the breast and salivary gland; papillary thyroid carcinomas; infantile fibrosarcoma) and genomic (driver-negative non-small cell lung carcinomas, microsatellite instability-high colorectal adenocarcinomas, and wild-type gastrointestinal stromal tumors) triaging was put forward., Results.—: Samples from 323 tumors were stained with the VENTANA pan-TRK EPR17341 Assay as a screening method. All positive IHC cases were simultaneously studied by 2 NGS tests, Oncomine Comprehensive Assay v3 and FoundationOne CDx. With this approach, the detection rate of NTRK fusions was 20 times higher (5.57%) by only screening 323 patients than the largest cohort in the literature (0.30%) comprising several hundred thousand patients., Conclusions.—: Based on our findings, we propose a multiparametric strategy (ie, "supervised tumor-agnostic approach") when pathologists start searching for NTRK fusions., (© 2024 College of American Pathologists.)

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC., Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion-positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay., Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12) , KIF5B(24)-RET(9) and KIF5B(24)-RET(11) . All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively)., Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC., Competing Interests: Dr. Conde has received research funding from Eli Lilly, 10.13039/100004325AstraZeneca, and 10.13039/100011033ThermoFisher Scientific; and honoraria from Pfizer, Roche, AstraZeneca, Janssen, and Eli Lilly. Dr. Hernandez has received research funding from Eli Lilly, AstraZeneca, and ThermoFisher Scientific, and honoraria from Pfizer, Roche, AstraZeneca, ThermoFisher Scientific, and Eli Lilly. Mr. Alonso has received research funding from AstraZeneca, and honoraria from Pfizer, Roche, and AstraZeneca. Dr. Jimenez has received honoraria from Roche. Dr. Garrido has received research grants from 10.13039/100002429Amgen, AstraZeneca, Blueprint, 10.13039/100002491Bristol-Myers Squibb, Boehringer Ingelheim, 10.13039/501100002973Daiichi-Sankyo, 10.13039/100004330GlaxoSmithKline, 10.13039/100005565Janssen, IO Biotech, Eli Lilly, 10.13039/100009947Merck Sharp & Dohme, 10.13039/100004337Roche, Takeda; and honoraria from AbbVie, Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, Boehringer Ingelheim, Daiichi-Sankyo, GlaxoSmithKline, Janssen, Eli Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Sanofi, Takeda, Medscape, and Touch Medical. Dr. Clave has received honoraria from AstraZeneca, Pfizer, Roche, Eli Lilly, and Takeda. Dr. Arriola has received honoraria from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Eli Lilly and Company, Novartis, Takeda, Merck Sharp & Dohme, Bayer, and Bristol Myers Squibb. Dr. Esteban-Rodriguez has received honoraria from AstraZeneca, Pfizer, and Merck Sharp & Dohme. Dr. De Castro has received honoraria from AstraZeneca, Bristol Myers Squibb, Hoffmann- La Roche, Merck Sharp and Dohme, Boehringer-Ingelheim, Janssen, Eli Lilly, Sanofi, Takeda, Pfizer, Glaxo, and Gilead. Dr. Sansano has received honoraria from F. Hoffmann La Roche AG, Merck Sharp & Dohme, Pfizer, Takeda, AstraZeneca, and Boehringer Ingelheim. Dr. Felip has received honoraria from AbbVie, Amgen, AstraZeneca, Bayer, Beigene, Boehringer Ingelheim, Bristol Myers Squibb, Daiichi-Sankyo, Eli Lilly, F. Hoffmann – La Roche, Gilead, Glaxo Smith Kline, Genentech, Janssen, Medical Trends, Medscape, Merck Serono, Merck Sharp & Dohme, Novartis, Peptomyc, Peervoice, Pfizer, Regeneron, Sanofi, Takeda, Turning Point, and Touch Oncology. Dr. Rojo has received research funding from 10.13039/100004337Roche, AstraZeneca, Menarini, 10.13039/100004336Novartis, 10.13039/100004334Merck, Merck Sharp & Dohme, Bristol-Myers Squibb, 10.13039/100004319Pfizer, GlaxoSmithKline, Palex, Amgen, 10.13039/100004322Agilent, and Janssen, and honoraria from Roche, AstraZeneca, Menarini, Novartis, Merck, Merck Sharp & Dohme, Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Palex, Amgen, Agilent, Janssen. Dr. Dómine has received honoraria from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Takeda, Merck Sharp & Dohme, and Bristol Myers Squibb. Dr. Abdulkader has received honoraria from AstraZeneca, Eli Lilly, Pfizer, Roche, Merck Sharp & Dohme, Bristol Myers Squibb, Takeda, and Agilent Technologies S.A. Dr. Garcia-Gonzalez has received honoraria from Amgen, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Roche, Sanofi, Pierre Fabre, Eli Lilly, Pfizer, and Takeda. Dr. Teixido has received honoraria from Novartis, AstraZeneca, Roche, Merck Sharp Dohme, Pfizer, Janssen, Eli Lilly, and, Bristol Myers Squibb. Dr. Reguart has received honoraria from Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, Boehringer, Guardant, Janssen, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Sanofi, and Takeda. Dr. Insa has received honoraria from Roche, Bristol Myers Squibb, Sanofi, Pfizer, Boehringer Ingelheim, AstraZeneca, Takeda, Bayer, Merck Sharp & Dohme, and Eli Lilly. Dr. Mancheño has received honoraria from Roche, AstraZeneca, and Pfizer. Dr. Palanca has received honoraria from Roche Pharma, Pfizer, Amgen, AstraZeneca, Takeda, Eli Lilly, and Janssen. Dr. Juan-Vidal has received honoraria from Boehringer Ingelheim, Bristol Myers Squibb, Merck Sharp & Dohme, Roche/Genetech, AstraZeneca, Pfizer, Eli Lilly, and Takeda. Dr. Baixeras has received honoraria from AstraZeneca and Eli Lilly. Dr. Nadal has received research funding from Roche, Pfizer, Bristol-Myers Squibb and Merck Serono, and honoraria from Roche, Bristol Myers Squibb, Merck Sharp Dohme, Merck Serono, Sanofi, Pfizer, Eli Lilly, Janssen, Amgen, Daiichi-Sankyo, Boehringer Ingelheim, AstraZeneca, Takeda, Sanofi, Pierre Fabre, Qiagen, Janssen, and Bayer. Dr. Calles has received research funding from Merck Sharp & Dome, and honoraria from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Eli Lilly and Company, Novartis, Takeda, Merck Sharp & Dohme, and Bristol Myers Squibb. Dr. Martin has received honoraria from Daiichi-Sankyo and Pfizer. Dr. Salas has received honoraria from Boehringer Ingelheim, Pfizer, and Merck Sharp & Dohme. Dr. Provencio has received honoraria from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Takeda, Merck Sharp & Dohme, and Bristol Myers Squibb. Dr. Massuti has received research funding from Bristol Myers Squibb, and honoraria from Bristol Myers Squibb, Roche, Janssen, Merck Sharp & Dohme, and AstraZeneca. Dr. Majem has received research funding from 10.13039/100002429Amgen Inc., AstraZeneca, Bristol Myers Squibb, and Roche, and honoraria from AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Kyowa Kyrin, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi, and Takeda. Dr. Paz-Ares has received research funding from Merck Sharp & Dohme, AstraZeneca, Pfizer, and Bristol-Myers Squibb, and honoraria from Eli Lilly, Merck Sharp & Dohme, Roche, Pharmamar, Merck, AstraZeneca, Novartis, Servier, Amgen, Pfizer, Sanofi, Bayer, Bristol-Myers Squibb, Mirati, GlaxoSmithKline, Janssen, Takeda, and Mirati. Dr. Lopez-Rios has received research funding from Eli Lilly, AstraZeneca, Roche, Pfizer, and ThermoFisher Scientific, and honoraria from Abbvie, Astellas, AstraZeneca, Bayer, Bristol-Myers Squibb, Daiichi-Sankyo, Janssen, Eli Lilly, Merck Sharp & Dohme, Merck, Pfizer, Roche, Sanofi, Takeda, and Thermo Fisher. The remaining authors declare no conflict of interest., (© 2024 The Authors.)

Background: Preclinical and early clinical data suggest that the irreversible ErbB family blocker afatinib may be effective in urothelial cancers harbouring ERBB mutations., Methods: This open-label, phase II, single-arm trial (LUX-Bladder 1, NCT02780687) assessed the efficacy and safety of second-line afatinib 40 mg/d in patients with metastatic urothelial carcinoma with ERBB1-3 alterations. The primary endpoint was 6-month progression-free survival rate (PFS6) (cohort A); other endpoints included ORR, PFS, OS, DCR and safety (cohorts A and B). Cohort A was planned to have two stages: stage 2 enrolment was based on observed antitumour activity., Results: Thirty-four patients were enroled into cohort A and eight into cohort B. In cohorts A/B, PFS6 was 11.8%/12.5%, ORR was 5.9%/12.5%, DCR was 50.0%/25.0%, median PFS was 9.8/7.8 weeks and median OS was 30.1/29.6 weeks. Three patients (two ERBB2-amplified [cohort A]; one EGFR-amplified [cohort B]) achieved partial responses. Stage 2 for cohort A did not proceed. All patients experienced adverse events (AEs), most commonly (any/grade 3) diarrhoea (76.2%/9.5%). Two patients (4.8%) discontinued due to AEs and one fatal AE was observed (acute coronary syndrome; not considered treatment-related)., Conclusions: An exploratory biomarker analysis suggested that basal-squamous tumours and ERBB2 amplification were associated with superior response to afatinib., Clinical Trial Registration: NCT02780687., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

p53 immunohistochemistry (IHC) has been proposed as a surrogate for TP53 mutations in penile squamous cell carcinomas (PSCC). We aimed to evaluate the performance of a pattern-based evaluation of p53 IHC in PSCC. Human papilloma virus (HPV) DNA testing, p16 and p53 IHC, and whole exome sequencing were performed in a series of 40 PSCC. p53 IHC was evaluated following a pattern-based framework and conventional p53 IHC evaluation. Out of 40 PSCC, 12 (30.0%) were HPV-associated, and 28 (70.0%) were HPV-independent. The agreement between the p53 IHC pattern-based evaluation and TP53 mutational status was almost perfect (k = 0.85). The sensitivity and accuracy of the pattern-based framework for identifying TP53 mutations were 95.5% and 92.5%, respectively, which were higher than the values of conventional p53 IHC interpretation (54.5% and 70.0%, respectively), whereas the specificity was the same (88.9%). In conclusions, the pattern-based framework improves the accuracy of detecting TP53 mutations in PSCC compared to the classical p53 IHC evaluation.

Background: Molecular profiling with next-generation sequencing (NGS) has been applied in multiple solid tumors, including melanomas, to identify potential drug targets. However, the association between clinical outcomes and the molecular alterations has not yet been fully clarified., Methods: A total of 108 patients with melanoma were included in this study, 95 of whom had both sequencing data and clinical outcomes were collected. We analyzed the genetic alterations of 108 malignant melanoma patients using the OncoCare panel, which covers 559 genes., Results: A model was also established to predict side effects through a combination analysis of clinical data and somatic variants, yielding an area under the receiver operating characteristic curve (AUROC) score of 0.8. We also identified epidermal growth factor receptor (EGFR) mutation was excellent predictor for progression-free survival (PFS) for patient who received immunotherapy (log-rank P=0.01), while tumor mutation burden (TMB) was found to not be significantly associated with PFS (log-rank P=0.87). Combining clinical features with genetic analysis, we found that patients carrying both DNA POLD1/ALOX12B or POLD1/PTPRT mutations had a significantly lower survival rate., Conclusions: Overall, these results demonstrate the benefits of applying NGS clinical panels and shed light on future directions of personalized therapeutics for the treatment of melanoma., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5346/coif). WS reports funding support from the LinGang Laboratory (Grant No. LG-QS-202205-11), and the National Natural Science Foundation of China (Grant Nos. 82272857 and 81802636). YC reports funding support from the Shanghai Committee of Science and Technology, China (Grant No. 19411951700). JW is from MyGenostics Inc. The other authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

The aims of the study were to investigate and compare the immunophenotype of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in a series of benign, intermediate and malignant Spitzoid lesions showing marked inflammatory lymphoid component, to find out its possible relation with the prognosis of these lesions. Six out of 97 Spitz nevus (SN) (6 %), five out of 26 atypical Spitz tumors (AST) (16 %) and seven out of 37 Spitzoid melanomas (SM) (19 %) showed diffuse, intense inflammatory component and were included in the study. The biological risk of the tumors was assessed in all AST through the melanoma 4 probe-FISH assay and the 9p21 locus exploration. TILs were quantitatively immunophenotyped using CD3, CD4, CD8, CD20, TIA1, FOXP3 and PD1 antibodies. PD-L1 was assessed in tumoral cells and inflammatory cells adjacent to the tumor. No significant differences of TILs immunophenotype were found between SN, AST and SM. However, the classification of tumors according to the biological risk showed that grouped SN plus low-risk AST had a significantly higher number of T-cells CD8+ and TIA-1+, as well as a lower CD4/CD8 relation and B- lymphocyte number than high-risk of progression tumors (grouped high-risk AST plus SM). Immunoregulatory T-cell markers PD1 and FOXP3 only correlated with each other and with PD-L1 expression. In conclusion, The TILs immunoprofile differences between low-risk and high-risk of progression Spitzoid tumors, especially regarding CD8 and the cytotoxic immune response, can add prognostic information about these challenging tumors and impact the clinical management of patients., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

An altered sense of self-awareness and agency has been proposed to underlie symptoms of schizophrenia. In this study, we used the enfacement illusion paradigm - in which perception of another person's face leads to changes in perception of one's own peri-personal space - to examine the brain correlates of the sense of agency and its potential disruption in schizophrenia. Thirty-three schizophrenic patients and 27 healthy controls underwent fMRI scanning during performance of a task designed to elicit the enfacement illusion. Activations were examined using whole-brain analysis and also in an a priori identified region of interest (ROI) in the temporoparietal junction (TPJ), a region that has been described as involved in self/other differentiation and sense of agency. Both groups showed a pattern of cortical activation involving the pre and postcentral cortex, Rolandic operculum, insula, parietal, temporal and occipital cortex bilaterally as well as TPJ (but only right-side in patients). Examination of the TPJ ROI revealed significantly reduced activation on the left in the patients that was associated with poorer insight. The findings suggest brain functional abnormality in schizophrenia related to the formation or maintenance of processes related to self and/or agency. Decreased function in the TPJ may have a role in the impaired insight seen in patients with the disorder., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Melanoma develops as a result of several genetic alterations, with UV radiation often acting as a mutagenic risk factor. Deep knowledge of the molecular signaling pathways of different types of melanoma allows better characterization and provides tools for the development of therapies based on the intervention of signals promoted by these cascades. The latest World Health Organization classification acknowledged the specific genetic drivers leading to melanoma and classifies melanocytic lesions into nine distinct categories according to the associate cumulative sun damage (CSD), which correlates with the molecular alterations of tumors. The largest groups are melanomas associated with low-CSD or superficial spreading melanomas, characterized by frequent presentation of the BRAF V600 mutation. High-CSD melanomas include lentigo maligna type and desmoplastic melanomas, which often have a high mutation burden and can harbor NRAS , BRAF non-V600E, or NF1 mutations. Non-CSD-associated melanomas encompass acral and mucosal melanomas that usually do not show BRAF , NRAS , or NF1 mutations (triple wild-type), but in a subset may have KIT or SF3B1 mutations. To improve survival, these driver alterations can be treated with targeted therapy achieving significant antitumor activity. In recent years, relevant improvement in the prognosis and survival of patients with melanoma has been achieved, since the introduction of BRAF / MEK tyrosine kinase inhibitors and immune checkpoint inhibitors. In this review, we describe the current knowledge of molecular pathways and discuss current and potential therapeutic targets in melanoma, focusing on their clinical relevance of development.

Anti-programmed cell death (PD1)/ligand-1 (PD-L1) checkpoint inhibitors have improved the survival of non-small cell lung cancer (NSCLC) patients. Additionally, PD-L1 has emerged as a predictive biomarker of response. Our goal was to examine the histological features of all PD-L1 cases of NSCLC analyzed in our center between 2017 and 2020, as well as to correlate the expression values of the same patient in different tested samples. PD-L1 immunohistochemistry (IHC) was carried out on 1279 external and internal samples: 482 negative (tumor proportion score, TPS < 1%; 37.7%), 444 low-expression (TPS 1-49%; 34.7%) and 353 high-expression (TPS ≥ 50%; 27.6%). Similar results were observed with samples from our institution (N = 816). Significant differences were observed with respect to tumor histological type ( p = 0.004); squamous carcinoma was positive in a higher proportion of cases than other histological types. There were also differences between PD-L1 expression and the type of sample analyzed (surgical, biopsy, cytology; p < 0.001), with a higher frequency of negative cytology. In addition, there were cases with more than one PD-L1 determination, showing heterogeneity. Our results show strong correlation with the literature data and reveal heterogeneity between tumors and samples from the same patient, which could affect eligibility for treatment with immunotherapy.

Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Targeted therapies are a new paradigm in lung cancer management. Next-generation sequencing (NGS) techniques have allowed for simultaneous testing of several genes in a rapid and efficient manner; however, there are other molecular diagnostic tools such as the nCounter ® Vantage 3D single nucleotide variants (SNVs) solid tumour panel which also offer important benefits regarding sample input and time-to-response, making them very attractive for daily clinical use. This study aimed to test the performance of the Vantage panel in the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients and to validate and compare its outputs with the Oncomine Solid Tumor (OST) panel DNA kit, the standard technique in our institution. Two parallel multiplexed approaches were performed based on DNA NGS and direct digital detection of DNA with nCounter ® technology to evaluate SNVs. A total of 42 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 95% of samples were successfully characterized by both technologies. The Vantage panel accounted for a sensitivity of 95% and a specificity of 82%. In terms of predictive values, the probability of truly presenting the SNV variant when it is detected by the nCounter panel was 82%, whereas the probability of not presenting the SNV variant when it is not detected by the platform was 95%. Finally, Cohen's Kappa coefficient was 0.76, indicating a substantial correlation grade between OST and Vantage panels. Our results make nCounter an analytically sensitive, practical and cost-effective tool.

Background: Desmoplastic melanoma (DM) is a rare subtype of spindle cell malignant melanoma characterized by frequent local recurrences and hematogenous spread, but without molecular classification. The aim of the study was to investigate in a DM series the incidence of relevant gene alterations in cancer, the programmed death-ligand 1 (PD-L1) expression status and the association with clinicopathological features and melanoma progression., Methods: A total of 38 patients were included. Clinical follow-up and the histopathological features of all cases were retrospectively collected. PD-L1 expression by immunohistochemistry (IHC) and BRAF genomic alterations by real-time PCR were determined in 34 samples. Additionally, a molecular analysis by next-generation sequencing was performed in 25 DMs., Results: Tumors occurred predominantly in men (76%) and in the head and neck region (50%). Most tumors were pure DMs (66%), containing less than 10% of conventional melanoma. Overall, 48% of our cohort harbored TP53 mutations, most of them showing a molecular signature associated with ultraviolet (UV)-oncogenesis, and 29%, BRAF mutations. A positive correlation between TP53 with depth of invasion (P=0.005) and presence of elastosis (P=0.002) was found. High-expression of PD-L1 in tumor cells was observed in 38% of cases and correlated with depth of tumoral infiltration (P=0.003), TP53 (P=0.016), PD-1 (P<0.001) and tumor-infiltrating lymphocytes (TILS) (P<0.001). PD-L1 expression in immune cells correlated with PD-1 (P=0.006), tumoral PD-L1 expression (P=0.029) and TP53 mutation (P=0.002). Survival correlated with depth of invasion (P=0.003), stage of tumors (P=0.015), positive sentinel lymph node (P=0.004), lymph node metastasis (P=0.024) and distant metastasis (P<0.001)., Conclusions: Our results suggest that progressed DMs with deep tumoral infiltration frequently harbor TP53 mutations, PD-L1 expression and present a high inflammatory response, probably related to adaptive immune resistance in this tumor-type., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-1846). Dr. AA reports personal fees and other from BMS, NOVARTIS, PIERRE FABRE, MSD, ROCHE, MERCK, AMGEM, and SANOFI, outside the submitted work. Dr. SP reports grants, personal fees and non-financial support from Almirall, grants and personal fees from Amgen, grants, personal fees and non-financial support from Isdin, non-financial support from Lilly, personal fees from BMS, grants from Novartis, grants, personal fees and non-financial support from Sanofi, grants from Sunpharma, grants, personal fees and non-financial support from La Roche Posay, grants and personal fees from Leo Pharma, grants from Melagenics, grants from Castle, non-financial support from Abbie, personal fees and non-financial support from Pfizer, grants and personal fees from Ojerpharma, personal fees and non-financial support from Roche, outside the submitted work. Dr. CT reports grants and personal fees from Pfizer, grants and personal fees from Novartis, personal fees from Takeda, personal fees from MSD, personal fees from BMS, personal fees from Roche, personal fees from Diaceutics, outside the submitted work. The other authors have no conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)

Personalized medicine is nowadays a paradigm in lung cancer management, offering important benefits to patients. This study aimed to test the feasibility and utility of embedding two multiplexed genomic platforms as the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients. Two parallel multiplexed approaches were performed based on DNA sequencing and direct digital detection of RNA with nCounter ® technology to evaluate gene mutations and fusions. The results were used to guide genotype-directed therapies and patient outcomes were collected. A total of 224 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 85% of samples were successfully characterized at DNA and RNA levels and oncogenic drivers were found in 68% of patients, with KRAS , EGFR , MET Δex14, BRAF , and ALK being the most frequent (31%, 19%, 5%, 4%, and 4%, respectively). Among all patients with complete genotyping results and follow-up data ( n = 156), the median overall survival (OS) was 1.90 years (confidence interval (CI) 95% 1.69-2.10) for individuals harbouring an actionable driver treated with a matched therapy, compared with 0.59 years (CI 95% 0.39-0.79) in those not eligible for any targeted therapy and 0.61 years (CI 95% 0.12-1.10) in patients with no drivers identified ( p < 0.001). Integrating DNA and RNA multiplexing technologies into the routine molecular testing of advanced NSCLC patients is feasible and useful and highlights the necessity of widespread integrating comprehensive molecular diagnosis into lung cancer care.

Small-cell lung cancer has defied our scientific community for decades. Chemotherapy has been the mainstay treatment for small-cell lung cancer (SCLC) and unlike its counterpart, non-small cell lung cancer, no significant therapeutic breakthroughs have been made since the 1970s. Among the reasons for this slow-paced therapeutic development, one that stands out is the distinctive and almost universal loss of function of the tumour suppressor genes TP53 and RB1 in this disease, for which pharmacological activation has yet to be achieved, despite having been highly sought after. Although no molecularly targeted approach has been approved for clinical practice thus far, several strategies are currently exploring the potential to drug the tumour's "Achilles heel" that stems from essential pathways regulating DNA-damage response. Most recently, we have witnessed newfound reasons to hope, as the combination of immunotherapy and systemic chemotherapy has improved survival outcomes, representing the first landmark achievement in decades and a new standard of care for patients with extensive disease SCLC. However, continuous efforts are still needed towards a better understanding of the molecular pathways that singularise this tumour to eventually identify the predictive biomarkers that might result in the development of a more rational therapeutic approach, including the use of immunotherapy combinations. In this review we aim to uncover critical aspects of the immune microenvironment and biology of SCLC and provide an overview of the current and future landscape of promising therapeutic opportunities. The challenge still stands, but regardless, we are living in exciting times to finally check SCLC off the "bucket list" of our scientific community.

Aim: Single biomarker diagnostic test of BRAF V600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma., Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing., Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAF V600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%)-53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA , ERBB4 , CTNNB1 , STK11 , FGFR1 , SMAD4 , KRAS , FGFR3 , PTEN and AKT . Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03)., Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2020.)

Aims: The clinical implications of the programmed cell death 1 (PD1)/programmed cell death-ligand 1 (PD-L1) axis in patients with post-transplant lymphoproliferative disorders are largely unknown, and its association with Epstein-Barr virus (EBV) status and PD-L1 copy number alterations (CNAs) has not been thoroughly studied., Methods and Results: PD1/PD-L1 expression was studied in 50 adult post-transplant lymphoproliferative disorders, and the correlations with PD-L1 CNAs, EBV, clinicopathological features and outcome were evaluated. Thirty-seven (74%) cases were classified as diffuse large B-cell lymphoma (DLBCL), nine (18%) cases were classified as polymorphic, and four (8%) cases were classified as classic Hodgkin lymphoma. Thirty-four cases were EBV-positive, with 29 of 34 (85%) having latency II or III, and 15 of 34 (44%) having viral replication. PD-L1 expression in tumour cells and tumour-associated macrophages was observed in 30 (60%) and 37 (74%) cases, respectively. PD1 positivity was seen in 16 (32%) cases. PD-L1 expression was associated with EBV with latency II or III (P = 0.001) and organ rejection (P = 0.04), and, in DLBCL, with non-germinal centre type DLBCL (P < 0.001). Cases with PD-L1-positive tumour cells showed a higher number of PD-L1 CNAs than PD-L1-negative cases (P = 0.001). Patients with EBV/latency III/replication and simultaneous PD-L1 expression showed the worst overall survival (P < 0.001)., Conclusions: The PD1/PD-L1 axis is deregulated in post-transplant lymphoproliferative disorders, with frequent PD-L1 expression and PD1 negativity. PD-L1 expression is associated with EBV latency II or III and PD-L1 CNAs, and probably reflects a proinflammatory tumour microenvironment. The combined analysis of EBV status and PD-L1 expression may help to identify deeply immunosuppressed patients who can benefit from immune reconstitution approaches., (© 2019 John Wiley & Sons Ltd.)

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data., Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific)., Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively)., Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Background: Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-γ). We have explored whether the expression of IFNG, the gene encoding IFN-γ, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined., Methods: Total RNA from 17 NSCLC and 21 melanoma patients was analyzed by quantitative reverse transcription PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, IFNG, PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8 + T-cells were also evaluated. Progression-free survival and overall survival were estimated., Results: A total of 17 NSCLC patients received nivolumab and 21 melanoma patients received pembrolizumab. Progression-free survival with nivolumab was significantly longer in NSCLC patients with high versus low IFNG expression (5.1 months versus 2 months, p = 0.0124). Progression-free survival with pembrolizumab was significantly longer in melanoma patients with high versus low IFNG expression (5.0 months versus 1.9 months, p = 0.0099). Significantly longer overall survival was observed for melanoma patients with high versus low IFNG expression (not reached versus 10.2 months p = 0.0183). There was a trend for longer overall survival for NSCLC patients with high versus low IFNG expression., Conclusions: IFN-γ is an important marker for prediction of response to immune checkpoint blockade. Further research is warranted in order to validate whether IFNG is more accurate than PD-L1., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest.