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Extrahepatic cholangiocarcinoma (ECC) is classified into two subtypes based on anatomic origin: distal extrahepatic (DECC) and perihilar (PHCC) cholangiocarcinoma. This study aimed to shed light on its genomic and transcriptomic profiles.The genomic alterations of 99 ECC (47 PHCC and 52 DECC) were investigated by next-generation sequencing of 96 genes. A subgroup of cases, representative of each subtype, was further investigated using transcriptomic analysis. Bioinformatics tools were applied for clustering and pathway analysis and defining the immune composition of the tumor microenvironment.PHCC had more frequent KRAS mutations (p = 0.0047), whereas TP53 mutations were more common in DECC (p = 0.006). Potentially actionable alterations included high-tumor mutational burden and/or microsatellite instability (7.1%), PI3KCA mutations (8.1%), and MYC (10.1%) and ERBB2 amplification (5.1%). The transcriptomic profiles showed the presence of three distinct clusters, which followed the anatomic origin and differed in immune microenvironment. DECC appeared to contain two distinct tumor subgroups, one enriched for druggable alterations and one lacking actionable opportunities.This study provides new insights into the molecular landscape and the actionable alterations of ECC. Our findings represent a step toward improved ECC molecular taxonomy and therapeutic strategies for precision oncology.

Simple Summary In this manuscript, we offer an integrated molecular analysis of 44 combined large cell neuroendocrine carcinomas (CoLCNECs) in order to deepen the knowledge about these rare histotypes and to clarify their relationship with lung cancers. In the present state of research, molecular studies are still scant, consisting of small and heterogeneous cohorts, and the genomic landscape is poorly characterized. This study shows that CoLCNECs constitute a standalone group of neuroendocrine neoplasm, with three different molecular profiles, two of which overlap with pure LCNEC or adenocarcinoma. CoLCNECs can be considered an independent histologic category with specific genomic and transcriptomic features, different and therefore not comparable to other lung cancers. Indeed, in addition to a histological re-evaluation of lung cancer classification, our study may help to develop a new diagnostic approach for novel and personalized treatments in CoLCNECs. Background: Combined large cell neuroendocrine carcinoma (CoLCNEC) is given by the association of LCNEC with adeno or squamous or any non-neuroendocrine carcinoma. Molecular bases of CoLCNEC pathogenesis are scant and no standardized therapies are defined. Methods: 44 CoLCNECs: 26 with adenocarcinoma (CoADC), 7 with squamous cell carcinoma (CoSQC), 3 with small cell carcinoma (CoSCLC), 4 with atypical carcinoid (CoAC) and 4 napsin-A positive LCNEC (NapA+), were assessed for alterations in 409 genes and transcriptomic profiling of 20,815 genes. Results: Genes altered included TP53 (n = 30), RB1 (n = 14) and KRAS (n = 13). Targetable alterations included six KRAS G12C mutations and ALK-EML4 fusion gene. Comparison of CoLCNEC transcriptomes with 86 lung cancers of pure histology (8 AC, 19 ADC, 19 LCNEC, 11 SCLC and 29 SQC) identified CoLCNEC as a separate entity of neuroendocrine tumours with three different molecular profiles, two of which showed a non-neuroendocrine lineage. Hypomethylation, activation of MAPK signalling and association to immunotherapy signature specifically characterized each of three CoLCNEC molecular clusters. Prognostic stratification was also provided. Conclusions: CoLCNECs are an independent histologic category. Our findings support the extension of routine evaluation of KRAS mutations, fusion genes and immune-related markers to offer new perspectives in the therapeutic management of CoLCNEC.

Poorly cohesive (PC) gastric cancer (GC) exhibits variable clinical behavior, being extremely aggressive in most cases but more indolent at times. We hypothesized that the integrative genomic and gene expression characterization of a PC GC series could help identifying molecular subtypes with potential clinical implications.64 PC GCs were assessed for alterations in 409 genes and 30 cases were subjected to transcriptomic profiling of 20,815 genes.A median of 8.2 mutations per Mb (interquartile range 6.9-10.4) was found and a tumor mutational load10 muts/Mb was significantly associated with patients' worse survival ( P =0.0024). The most frequent mutated genes were CDH1 and TP53 (each 32.8%) followed by PIK3CA (10.9%). In 15 samples (23.4%), at least 1 chromatin remodeling gene was mutated: KMT2D (5 cases); ARID1A and BAP1 (4 cases each); EZH2 , KMT2A , PBRM1 (1 case each). Eight samples (12.5%) had fusion genes involving CLDN18 gene. Gene expression profiling identified 4 different clusters: cluster A associated with epithelial to mesenchymal transition (EMT) signature; cluster B associated to proliferative signature and EMT; cluster C correlated to hedgehog signaling; cluster D showing no enrichment for any of the previous signatures. Notably, cluster A and B showed a worse prognosis compared with clusters C and D ( P =0.0095).integrated genomic and transcriptomic analysis suggest the existence of 4 molecular subtypes of PC GC with prognostic significance where EMT features are associated with a worse outcome.

Pleomorphic xanthoastrocytoma (PXA) is a rare circumscribed glioma, characterized by frequent BRAF p. V600E mutation, and classified as grade 2 or 3. Owing to overlapping clinical-pathological features, the histological distinction from glioblastoma (GBM) with giant cells (GCs) is challenging. Based on the high frequency of TP53 and RB1 alterations in the latter, this study aimed to assess the value of BRAF, p53, and pRB immunostainings in the differential diagnosis.In 37 GBMs with ≥30% GCs and in eight PXAs, we assessed the alterations of 409 cancer-related genes and immunostainings for BRAF, p53, and pRB. GBMs with GCs were TP53-mutated in 30 cases, RB1-altered in 11, and BRAF-mutated in none. PXAs were BRAF-mutated in six cases, TP53-mutated in three, and RB1-altered in none. pRb immunostaining was lost in 25 GBMs (11 RB1-altered and 14 RB1-unaltered), retained in all PXAs and six GBMs, and inconclusive in six GBMs. pRb loss had 100% specificity and 80.6% sensitivity for GBM with GCs. P53 immunostaining was observed in 22 TP53-mutated GBMs and in one TP53-mutated PXA. It showed 87.5% specificity and 60% sensitivity to identify GBM with GCs. BRAF immunostaining corresponded to BRAF mutation status and it had 100% specificity and 75% sensitivity for detecting PXA.This study shows for the first time that loss of pRB immunostaining is sensitive and specific for distinguishing GBM with GCs from PXA in routine practice. Thus, it could complement an immunohistochemical panel that includes BRAF and p53 immunostainings for the differential diagnosis.

Pancreatic intraductal tubulopapillary neoplasm (ITPN) is a recently recognized intraductal neoplasm. This study aimed to clarify the clinicopathologic and molecular features of this entity, based on a multi-institutional cohort of 16 pancreatic ITPNs and associated adenocarcinomas. The genomic profiles were analyzed using histology-driven multi-regional sequencing to provide insight on tumor heterogeneity and evolution. Furthermore, an exploratory transcriptomic characterization was performed on eight invasive adenocarcinomas. The clinicopathologic parameters and molecular alterations were further analyzed based on survival indices. The main findings were as follows: 1) the concomitant adenocarcinomas, present in 75% of cases, were always molecularly associated with the intraductal components. These data definitively establish ITPN as origin of invasive pancreatic adenocarcinoma; 2) alterations restricted to infiltrative components included mutations in chromatin remodeling genes ARID2, ASXL1, and PBRM1, and ERBB2-P3H4 fusion; 3) pancreatic ITPN can arise in the context of genetic syndromes, such as BRCA-germline and Peutz-Jeghers syndrome; 4) mutational profile: mutations in the classical PDAC drivers are present, but less frequently, in pancreatic ITPN; 5) novel genomic alterations were observed, including amplification of the Cyclin and NOTCH family genes and ERBB2, fusions involving RET and ERBB2, and RB1 disruptive variation; 6) chromosomal alterations: the most common was 1q gain (75% of cases); 7) by transcriptome analysis, ITPN-associated adenocarcinomas clustered into three subtypes that correlate with the activation of signaling mechanism pathways and tumor microenvironment, displaying squamous features in their majority; and 8) TP53 mutational status is a marker for adverse prognosis. ITPNs are precursor lesions of pancreatic cancer with a high malignant transformation risk. A personalized approach for patients with ITPN should recognize that such neoplasms could arise in the context of genetic syndromes. BRCA alterations, ERBB2 and RET fusions, and ERBB2 amplification are novel targets in precision oncology. The TP53 mutation status can be used as a prognostic biomarker., This study is supported by Associazione Italiana Ricerca sul Cancro (AIRC IG n. 26343); Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi” (prot. 203885/2017); Fondazione Italiana Malattie Pancreas (FIMP-Ministero Salute J38D19000690001); Italian Ministry of Health (RF CO-2019-12369662: CUP: B39C21000370001).

The current standard of care for advanced non-small-cell lung cancer is based on detecting actionable mutations that can benefit from targeted therapy. Comprehensive genetic tests can have long turn-around times, and because EGFR mutations are the most prevalent actionable mutation, a quick detection would enable a prompt initiation of targeted therapy. Furthermore, the scarcity of diagnostic material means that sometimes only cytologic material is available. The Idylla™ EGFR assay is a real-time PCR–based method able to detect 51 EGFR mutations in 2.5 h. Idylla is validated for use only on FFPE sections, but some researchers described their experiences with cytological material. We reviewed the relevant literature, finding four articles describing 471 cases and many types of cytological input material: smears, cell-block sections, suspensions, and extracted DNA. The sensitivity, specificity, and limit of detection appear comparable to those obtained with histological input material, with one exception: the usage of scraped stained smears as input may reduce the accuracy of the test. In conclusion, usage of cytological material as input to the Idylla EGFR test is possible. A workflow where common mutations are tested first and fast, leaving rarer mutations for subsequent comprehensive profiling, seems the most effective approach.

Advanced non-small cell lung cancer (NSCLC) needs to be managed rapidly; therefore, a rapid assessment of the epidermal growth factor receptor (EGFR) status is mandatory. Computed Tomography (CT)-guided or Ultrasound (US)-guided Fine-Needle Aspiration Cytology (FNAC) allows a rapid diagnosis of both primary and metastatic tumor through rapid on-site evaluation (ROSE) and the proper management of diagnostic material. Idylla (Biocartis, Mechelen, Belgium) is an automated RT-PCR system which evaluates the mutational status of specific genes in less than two hours. In this study, the EGFR mutational status in advanced NSCLC was analyzed on 28 FNAC samples with Idylla. After ROSE, residual FNAC material and/or additional passes were pipetted into the Idylla EGFR cartridge. Patients endorsed a consent form before carrying out the analysis. Results were controlled by pyrosequencing. Adequate EGFR status was obtained in 26/28 cases (22 wild type and 4 mutated). Mutated cases harbored EGFR Exon 19 deletion and L858R point mutation. In 2/28 cases the analysis failed. The combination of FNAC, ROSE and Idylla is a rapid, accurate and effective method that can be conveniently used to assess EGFR status in advanced NSCLC.