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Introduction: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples., Methods: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated., Results: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation., Conclusions: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice., (© 2024. The Author(s).)

Signet-ring cell (SRC)/poorly cohesive cell carcinoma is an aggressive variant of pancreatic ductal adenocarcinoma (PDAC). This study aimed to clarify its clinicopathologic and molecular profiles based on a multi-institutional cohort of 20 cases. The molecular profiles were investigated using DNA and RNA sequencing. The clinicopathologic parameters and molecular alterations were analyzed based on survival indices and using a validation/comparative cohort of 480 conventional PDAC patients. The primary findings were as follows: (1) clinicopathologic features: SRC carcinomas are highly aggressive neoplasms with poor prognosis, and the lungs are elective metastatic sites; (2) survival analysis: a higher SRC component was indicative of poorer prognosis. In particular, the most clinically significant threshold of SRC was 80%, showing statistically significant differences in both disease-specific and disease-free survival; (3) genomic profiles: SRC carcinomas are similar to conventional PDAC with the most common alterations affecting the classic PDAC drivers KRAS (70% of cases), TP53 (55%), SMAD4 (25%), and CDKN2A (20%). EGFR alterations, RET::CCDC6 fusion gene, and microsatellite instability (3 different cases, 1 alteration per case) represent novel targets for precision oncology. The occurrence of SMAD4 mutations was associated with poorer prognosis; (4) pancreatic SRC carcinomas are genetically different from gastric SRC carcinomas: CDH1, the classic driver gene of gastric SRC carcinoma, is not altered in pancreatic SRC carcinoma; (5) transcriptome analysis: the cases clustered into 2 groups, one classical/exocrine-like, and the other squamous-like; and (6) SRC carcinoma-derived organoids can be successfully generated, and their cultures preserve the histologic and molecular features of parental SRC carcinoma. Although pancreatic SRC carcinoma shares similarities with conventional PDAC regarding the most important genetic drivers, it also exhibits important differences. A personalized approach for patients with this tumor type should consider the clinical relevance of histologic determination of the SRC component and the presence of potentially actionable molecular targets., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypothesis: 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., Materials and Methods: 64 PC GCs were assessed for alterations in 409 genes and 30 cases were subjected to transcriptomic profiling of 20,815 genes., Results: A median of 8.2 mutations per Mb (interquartile range 6.9-10.4) was found and a tumor mutational load >10 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)., Conclusion: 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., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Juvenile polyposis (JP) is a rare familial syndrome characterized by the development of numerous hamartomatous polyps of the gastrointestinal tract and by an increased risk of developing gastrointestinal cancers. It follows a pattern of autosomal dominant inheritance and is associated with germline variants of SMAD4 or BMPR1A genes. Differential diagnosis may be difficult based on histology alone, due to morphological similarities to other familial syndromes. Here we report a case of familial JP diagnosed in a 50-years woman with a familial history positive for gastrointestinal cancers and other tumor types. The patient presented with severe iron deficiency anemia and showed numerous polyps in the stomach and jejunum according to endoscopy and imaging. She underwent an intra-gastric laparoscopic removal of the major gastric polyp, followed by jejunal exploration and resection of a segment with multiple neoformations. Histological examination revealed the presence of hamartomatous polyposis. Gastric and intestinal samples were analyzed with next-generation sequencing. Molecular analysis showed that the patient harbored a germline splicing site variant of SMAD4, c.1139 + 3A > G, which was complemented by different somatic variants of the same gene in the different polyps. Immunohistochemistry for SMAD4 confirmed loss of protein expression in the polyps, with regular expression in normal cells. cDNA sequencing further confirmed the findings. We thus definitively diagnosed the woman as having JP thanks to an integrated approach based on histology, immunohistochemistry and molecular analysis. The identified variants, all previously reported as variants of unknown significance, were classified as pathogenic as they complemented each other leading to SMAD4 loss., (© 2022. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) with microsatellite instability (MSI)/defective mismatch repair (dMMR) is the only subtype of pancreatic cancer with potential response to immunotherapy. Here, we report the histo-molecular characterization of MSI/dMMR PDAC with immunohistochemistry, MSI-based PCR, and next-generation sequencing. Five paradigmatic cases have been identified. The main results include the first report in pancreatic cancer of MSI/dMMR intra-tumor heterogeneity, the presence of microsatellite-stable metastases from MSI/dMMR primary and recurrent B2M gene inactivation, which may confer resistance to immunotherapy. In addition to the classic PDAC drivers, ARID1A was the most common mutated gene in the cohort. Intra-tumor heterogeneity, B2M inactivation, and metastatic sites should be carefully considered in MSI/dMMR PDAC, which should also be investigated in routine diagnostic practice with specific molecular analysis. The chromatin remodeler ARID1A represents another potential driver gene in this context., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

We report a case of oligodendroglioma that had consistent histopathological features as well as a distinct change in 1p/19q status in the second recurrence, after temozolomide chemotherapy and radiotherapy. The first tumor recurrence had oligodendroglial morphology, IDH1 R132H and TERT promoter mutations, and 1p/19q codeletion detected by fluorescent in situ hybridization (FISH). Copy number analysis, assessed by next-generation sequencing, confirmed 1p/19q codeletion, and disclosed loss of heterozygosity (LOH) of chromosomes 4 and 9 and chromosome 11 gain. The second recurrence featured not only oligodendroglial morphology but also the appearance of admixed multinucleated giant cells or neoplastic cells having oval nuclei and mitoses and showing microvascular proliferation; it maintained IDH1 R132H and TERT promoter mutations, acquired TP53 mutation, and showed 19q LOH, but disomic 1p, detected by FISH. Copy number analysis depicted LOH of chromosomes 3p, 13, and 19q, 1p partial deletion (1p chr1p34.2-p11), and gain of chromosomes 2p25.3-p24.1, 8q12.2-q24.3, and 11q13.3-q25. B-allele frequency analysis of polymorphic sites disclosed copy-neutral LOH at 1p36.33-p34.2, supporting the initial deletion of 1p, followed by reduplication of 1p36.33-p34.2 alone. These findings suggest that the two tumor recurrences might have originated from an initial neoplastic clone, featuring 1p/19q codeletion and IDH1 and TERT promoter mutations, and have independently acquired other copy number alterations. The reduplication of chromosome 1p might be the result of temozolomide treatment, and gave rise to false negative 1p deletion detected by FISH. The possibility of 1p copy-neutral LOH should be considered in recurrent oligodendrogliomas with altered 1p/19q status detected by FISH., (© 2022 The Authors. Neuropathology published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Neuropathology.)

Mixed neuroendocrine/non-neuroendocrine neoplasms (MiNEN) are rare mixed epithelial neoplasms in which a neuroendocrine component is combined with a non-neuroendocrine component. Here, we provide the clinical, pathologic, and molecular report of a 73-year-old-man presenting with an intestinal MiNEN. The lesion was composed of a well-differentiated G3 neuroendocrine tumor and a colloid adenocarcinoma. The molecular characterization was performed using a multigene next-generation sequencing panel. The neoplasm displayed microsatellite instability due to MLH1 promoter methylation. The extended molecular profile documented the same mutations affecting ARID1A, ASXL1, BLM, and RNF43 genes in both components, indicating a monoclonal origin of the tumor. Regarding component-specific gene mutations, BRCA2 was specifically altered in the neuroendocrine area. It may represent a new actionable target for precision oncology in MiNEN, but the lack of its alteration in the colloid component calls for further considerations on intratumor heterogeneity. The most important finding with potential immediate implications regards the presence of microsatellite instability: it indicates that this molecular alteration should become part of the diagnostic algorithm for these rare neoplasms., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Giant cell glioblastoma (GC-GBM) is a rare variant of IDH-wt GBM histologically characterized by the presence of numerous multinucleated giant cells and molecularly considered a hybrid between IDH-wt and IDH-mutant GBM. The lack of an objective definition, specifying the percentage of giant cells required for this diagnosis, may account for the absence of a definite molecular profile of this variant. This study aimed to clarify the molecular landscape of GC-GBM, exploring the mutations and copy number variations of 458 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability (MSI) in 39 GBMs dichotomized into having 30-49% (15 cases) or ≥ 50% (24 cases) GCs. The type and prevalence of the genetic alterations in this series was not associated with the GCs content (< 50% or ≥ 50%). Most cases (82% and 51.2%) had impairment in TP53/MDM2 and PTEN/PI3K pathways, but a high proportion also featured TERT promoter mutations (61.5%) and RB1 (25.6%) or NF1 (25.6%) alterations. EGFR amplification was detected in 18% cases in association with a shorter overall survival (P = 0.004). Sixteen (41%) cases had a TMB > 10 mut/Mb, including two (5%) that harbored MSI and one with a POLE mutation. The frequency of RB1 and NF1 alterations and TMB counts were significantly higher compared to 567 IDH wild type (P < 0.0001; P = 0.0003; P < 0.0001) and 26 IDH-mutant (P < 0.0001; P = 0.0227; P < 0.0001) GBMs in the TCGA PanCancer Atlas cohort. These findings demonstrate that the molecular landscape of GBMs with at least 30% giant cells is dominated by the impairment of TP53/MDM2 and PTEN/PI3K pathways, and additionally characterized by frequent RB1 alterations and hypermutation and by EGFR amplification in more aggressive cases. The high frequency of hypermutated cases suggests that GC-GBMs might be candidates for immune check-point inhibitors clinical trials., (© 2021. The Author(s).)

Hepatoid tumors (HT) are rare neoplasms morphologically resembling hepatocellular carcinoma, which arise in several organs other than the liver. A comprehensive molecular profile of this group of neoplasms is still lacking. Genomic characterization of 19 HTs from different organs (three colon HTs, four esophagogastric HTs, four biliary HTs, six genitourinary HTs, two lung HTs) was performed using a multigene next-generation sequencing panel. NGS unraveled a composite molecular profile of HT. Their genetic alterations were clearly clustered by tumor site: (i) colorectal HT displayed microsatellite instability, high tumor mutational burden, mutations in ARID1A/B genes and NCOA4-RET gene fusion (2/3 cases); (ii) gastric HT had TP53 mutations (2/4); (iii) biliary HT displayed loss of CDKN2A (3/4) and loss of chromosome 18 (2/4); (iv) genital HT showed gain of chromosome 12 (3/6); (v) lung HT had STK11 somatic mutations (2/2). The only commonly mutated gene occurring in HT of different sites was TP53 (8/19 cases: colon 2, esophagogastric 2, biliary 2, genital 1, lungs 1). This study shows that most genetic alterations of HT were clustered by site, indicating that context matters. The novel potential targets for HT precision oncology are also clustered based on the anatomic origin. This study shed light on the biology of these rare cancers and may have important consequences for treatment decisions and clinical trial selection for HT patients., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The mitochondrial metabolism has been associated with pancreatic ductal adenocarcinoma (PDAC) risk. Recent evidence also suggests the involvement of the genetic variability of the mitochondrial function in several traits involved in PDAC etiology. However, a systematic investigation of the genetic variability of mitochondrial genome (mtSNP) and of all the nuclear genes involved in its functioning (n-mtSNPs) has never been reported., Methods: We conducted a two-phase association study of mtSNPs and n-mtSNPs to assess their effect on PDAC risk. We analyzed 35,297 n-mtSNPs and 101 mtSNPs in up to 55,870 individuals (12,884 PDAC cases and 42,986 controls). In addition, we also conducted a gene-based analysis on 1,588 genes involved in mitochondrial metabolism using Multi-marker Analysis of GenoMic Annotation (MAGMA) software., Results: In the discovery phase, we identified 49 n-mtSNPs and no mtSNPs associated with PDAC risk ( P < 0.05). In the second phase, none of the findings were replicated. In the gene-level analysis, we observed that three genes ( TERT , SUGCT , and SURF1 ) involved in the mitochondrial metabolism showed an association below the Bonferroni-corrected threshold of statistical significance ( P = 0.05/1588 = 3.1 × 10 -5 )., Conclusions: Even though the mitochondrial metabolism might be involved in PDAC etiology, our results, obtained in a study with one of the largest sample sizes to date, show that neither n-mtSNPs nor mtSNPs are associated with PDAC risk., Impact: This large case-control study does not support a role of the genetic variability of the mitochondrial function in PDAC risk., (©2021 American Association for Cancer Research.)

Oligodendroglioma is defined by IDH mutation and 1p/19q codeletion. The latter is mutually exclusive to ATRX immunohistochemical loss and has been recently associated with the loss of H3K27me3 immunostaining. We aimed to assess the diagnostic and prognostic value of H3K27me3 immuno-expression in diffuse gliomas with oligodendroglial or mixed oligoastrocytic morphology. H3K27me3 immunostaining was performed in 69 diffuse gliomas with oligodendroglial (n = 62) or oligoastrocytic (n = 7) morphology. The integration with routinely assessed IDH mutations, ATRX immunostaining, and 1p/19q codeletion classified these cases as 60 oligodendroglial and 9 astrocytic. H3K27me3 was lost in 58/60 oligodendrogliomas with retained (n = 47) or non-conclusive (n = 11) ATRX staining, 3/6 IDH-mutant astrocytomas with ATRX loss, and 3/3 IDH-wt astrocytomas. H3K27me3 was retained in 2/60 oligodendrogliomas with retained ATRX, and in 3/6 IDH-mutant astrocytomas, two of which had lost and one retained ATRX. The combination of H3K27me3 and ATRX immunostainings with IDH mutational status correctly classified 55/69 (80%) cases. In IDH-mutant gliomas, ATRX loss indicates astrocytic phenotype, while ATRX retention and H3K27me3 loss identify oligodendroglial phenotype. Only 14 (20%) IDH-mutant cases with retained ATRX and H3K27me3 or inconclusive ATRX immunostaining would have requested 1p/19q codeletion testing to be classified. Furthermore, H3K27me3 retention was associated with significantly shorter relapse-free survival (P < 0.0001), independently from IDH mutation or 1p/19q codeletion (P < 0.005). Our data suggest that adding H3K27me3 immunostaining to the diagnostic workflow of diffuse gliomas with oligodendroglial or mixed morphology is useful for drastically reducing the number of cases requiring 1p/19q codeletion testing and providing relevant prognostic information., (© 2021. The Author(s).)

Purpose: Data regarding the clinical management and follow-up of pancreatic neuroendocrine tumors (PanNETs) associated with Von Hippel-Lindau (VHL) syndrome are limited. This study aimed to assess clinical presentation, genotype-phenotype correlations, treatment and prognosis of PanNETs in a series of VHL syndrome patients., Methods: Retrospective analysis of data of patients observed between 2005 and 2020., Results: Seventeen patients, including 12 probands and 5 relatives (mean age 30.8 ± 18.4; 7 males), were recruited. PanNETs were found in 13/17 patients (77.5%) at a median age of 37 years: 4/13 (30.7%) at the time of VHL diagnosis and 9 (69.3%) during follow up. Six (46.1%) PanNET patients underwent surgery, whereas seven were conservatively treated (mean tumor diameter: 40 ± 10.9 vs. 15 ± 5.3 mm respectively). Four patients (30.7%) had lymph node metastases and a mean tumor diameter significantly larger than the nonmetastatic PanNETs (44.2 ± 9.3 vs. 17.4 ± 7 mm, p = 0.00049, respectively). Five (83.3%) operated patients had stable disease after a median follow up of 3 years whereas one patient showed liver metastases. Six (85.7%) non-resected PanNETs were stable after a median follow-up of 2 years, whereas one patient developed a new small PanNET and a slight increase in diameter of a pre-existing PanNET. No correlation was found between the type of germline mutation and malignant behavior of PanNETs., Conclusions: PanNETs are a common disease of the VHL syndrome and can be the presenting feature. Tumor size rather than genetic mutation is a prognostic factor of malignancy., (© 2021. The Author(s).)

While many landmark solid tumor immunotherapy studies show clinical benefits for solid tumors with high microsatellite instability (MSI-H) and mismatch repair deficiency (dMMR), the methodologies focus only on confirmatory polymerase chain reaction (PCR) testing for MSI-H. Because some tumors are either dMMR or MSI-H but not the other, clinicians must choose between two testing methods for a broad patient population. We investigated the level of correlation between MMR protein immunohistochemistry (IHC) and microsatellite PCR testing results in 62 cancer patients. Thirty-five of the 62 cases (56.5%) were MSI-H by PCR, whereas 35 (56.5%) were dMMR by IHC. MMR IHC results correlated well with MSI PCR in 32 co-positive cases (91.4%) and 24 co-negative cases (88.9%). Six discrepant cases (9.7%) were identified, among which three were MSI-H and MMR intact, and three were dMMR and microsatellite stable. The results of this study highlight the implications of dMMR/MSI testing strategies on precision oncology. Co-testing with both MMR IHC and MSI PCR may be an effective screening strategy for evaluating immunotherapy eligibility status for solid tumors., (Copyright © 2021. Published by Elsevier Inc.)

Genetic factors play an important role in the susceptibility to pancreatic cancer (PC). However, established loci explain a small proportion of genetic heritability for PC; therefore, more progress is needed to find the missing ones. We aimed at identifying single nucleotide polymorphisms (SNPs) affecting PC risk through effects on micro-RNA (miRNA) function. We searched in silico the genome for SNPs in miRNA seed sequences or 3 prime untranslated regions (3'UTRs) of miRNA target genes. Genome-wide association data of PC cases and controls from the Pancreatic Cancer Cohort (PanScan) Consortium and the Pancreatic Cancer Case-Control (PanC4) Consortium were re-analyzed for discovery, and genotyping data from two additional consortia (PanGenEU and PANDoRA) were used for replication, for a total of 14,062 cases and 11,261 controls. None of the SNPs reached genome-wide significance in the meta-analysis, but for three of them the associations were in the same direction in all the study populations and showed lower value of p in the meta-analyses than in the discovery phase. Specifically, rs7985480 was consistently associated with PC risk (OR = 1.12, 95% CI 1.07-1.17, p = 3.03 × 10 -6 in the meta-analysis). This SNP is in linkage disequilibrium (LD) with rs2274048, which modulates binding of various miRNAs to the 3'UTR of UCHL3 , a gene involved in PC progression. In conclusion, our results expand the knowledge of the genetic PC risk through miRNA-related SNPs and show the usefulness of functional prioritization to identify genetic polymorphisms associated with PC risk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Corradi, Gentiluomo, López de Maturana, Theodoropoulos, Roth, Maiello, Morelli, Archibugi, Izbicki, Sarlós, Kiudelis, Oliverius, Aoki, Vashist, van Eijck, Gazouli, Talar-Wojnarowska, Mambrini, Pezzilli, Bueno-de-Mesquita, Hegyi, Souček, Neoptolemos, Di Franco, Sperti, Kauffmann, Hlaváč, Uzunoğlu, Ermini, Małecka-Panas, Lucchesi, Vanella, Dijk, Mohelníková-Duchoňová, Bambi, Petrone, Jamroziak, Guo, Kolarova, Capretti, Milanetto, Ginocchi, Loveček, Puzzono, van Laarhoven, Carrara, Ivanauskas, Papiris, Basso, Arcidiacono, Izbéki, Chammas, Vodicka, Hackert, Pasquali, Piredda, Costello-Goldring, Cavestro, Szentesi, Tavano, Włodarczyk, Brenner, Kreivenaite, Gao, Bunduc, Vermeulen, Schneider, Latiano, Gioffreda, Testoni, Kupcinskas, Lawlor, Capurso, Malats, Campa and Canzian.)

Colorectal cancer (CRC) with microsatellite instability (MSI) accounts for 15-18 % of all CRCs and represents the category with the best prognosis. This study aimed at determining any possible clinical/pathological features associated with a higher risk of nodal metastasization in MSI-CRC, and at defining any possible prognostic moderators in this setting. All surgically resected CRCs of the last 20 years (mono-institutional series) with a PCR-based diagnosis of MSI, with and without nodal metastasis, have been retrieved for histological review, which was performed following WHO guidelines. Furthermore, the most important prognostic moderators have been investigated with a survival analysis. The study of 33 cases of MSI-CRCs with nodal metastasis highlighted a high fidelity of histology maintenance between primary tumors and matched nodal metastases. At survival analysis, the strongest prognostic variable in MSI-CRCs with nodal metastasis was the extranodal extension (multivariate analysis, HR: 14.4, 95 %CI: 1.46-140.9, p = 0.022). Furthermore, through a comparison between nodal positive (33 cases) and nodal negative (71 cases) MSI-CRCs, right-sided location (p < 0.0001), pT4 stage (p = 0.0004) and signet-ring histology (p = 0.0089) emerged as parameters more commonly associated with nodal metastasization. These findings shed new light on the biology of MSI-CRC and can be of help for the prognostic stratification of MSI-CRC patients., (Copyright © 2021. Published by Elsevier GmbH.)

Tumor mutational burden (TMB) is a numeric index that expresses the number of mutations per megabase (muts/Mb) harbored by tumor cells in a neoplasm. TMB can be determined using different approaches based on next-generation sequencing. In the case of high values, it indicates a potential response to immunotherapy. In this systematic review, we assessed the potential predictive role of high-TMB in pancreatic ductal adenocarcinoma (PDAC), as well as the histo-molecular features of high-TMB PDAC. High-TMB appeared as a rare but not-negligible molecular feature in PDAC, being present in about 1.1% of cases. This genetic condition was closely associated with mucinous/colloid and medullary histology ( p < 0.01). PDAC with high-TMB frequently harbored other actionable alterations, with microsatellite instability/defective mismatch repair as the most common. Immunotherapy has shown promising results in high-TMB PDAC, but the sample size of high-TMB PDAC treated so far is quite small. This study highlights interesting peculiarities of PDAC harboring high-TMB and may represent a reliable starting point for the assessment of TMB in the clinical management of patients affected by pancreatic cancer.

This case report describes the history of a 41 year-old woman with a solid pseudopapillary neoplasm (SPN) of the pancreas and a metachronous abdominal desmoid tumor (DT) that occurred two years after the SPN surgical resection. At next-generation sequencing of 174 cancer-related genes, both neoplasms harbored a CTNNB1 somatic mutation which was different in each tumor. Moreover, two BRCA2 pathogenic mutations were found in both tumors, confirmed as germline by the sequencing of normal tissue. The BRCA2 mutations were c.631G>A, resulting in the amino-acid change p.V211I, and c.7008-2A>T, causing a splice acceptor site loss. However, as the two neoplasms showed neither loss of heterozygosity nor somatic mutation in the second BRCA2 allele, they cannot be considered as BRCA -dependent tumors. Nevertheless, this study highlights the important opportunities opened by extensive tumor molecular profiling. In this particular case, it permitted the detection of BRCA2 -germline mutations, essential for addressing the necessary BRCA -related genetic counseling, surveillance, and screening for the patient and her family.

The use of adjuvant therapy is controversial in atypical meningiomas with gross total resection. Predictors of recurrence risk could be useful in selecting patients for additional treatments. The aim of this study was to investigate whether molecular features are associated with recurrence risk of atypical meningiomas. According to WHO classification, the diagnosis of atypical meningioma was based on the presence of one major criteria (mitotic activity, brain invasion) or three or more minor criteria. The molecular profile of 22 cases (eight mitotically active, eight brain-invasive, and six with minor criteria) was assessed exploring the mutational status and copy number variation of 409 genes using next generation sequencing. Of the 22 patients with a median follow up of 53.5 months, 13 had recurrence of disease within 68 months. NF2 mutation was the only recurrent alteration (11/22) and was unrelated to clinical-pathological features. Recurring meningiomas featured a significantly higher proportion of copy number losses than non-recurring ones ( p = 0.027). Chromosome 18q heterozygous loss or CDKN2A/B homozygous deletion was significantly associated with shorter recurrence-free survival ( p = 0.008; hazard ratio: 5.3). Atypical meningiomas could be tested routinely for these genetic alterations to identify cases for adjuvant treatment.

Objective: Recently, tumours with microsatellite instability (MSI)/defective DNA mismatch repair (dMMR) have gained considerable interest due to the success of immunotherapy in this molecular setting. Here, we aim to clarify clinical-pathological and/or molecular features of this tumour subgroup through a systematic review coupled with a comparative analysis with existing databases, also providing indications for a correct approach to the clinical identification of MSI/dMMR pancreatic ductal adenocarcinoma (PDAC)., Design: PubMed, SCOPUS and Embase were searched for studies reporting data on MSI/dMMR in PDAC up to 30 November 2019. Histological and molecular data of MSI/dMMR PDAC were compared with non-MSI/dMMR PDAC and with PDAC reference cohorts (including SEER database and The Cancer Genome Atlas Research Network - TCGA project)., Results: Overall, 34 studies with 8323 patients with PDAC were included in the systematic review. MSI/dMMR demonstrated a very low prevalence in PDAC (around 1%-2%). Compared with conventional PDAC, MSI/dMMR PDAC resulted strongly associated with medullary and mucinous/colloid histology (p<0.01) and with a KRAS / TP53 wild-type molecular background (p<0.01), with more common JAK genes mutations. Data on survival are still unclear., Conclusion: PDAC showing typical medullary or mucinous/colloid histology should be routinely examined for MSI/dMMR status using specific tests (immunohistochemistry, followed by MSI-PCR in cases with doubtful results). Next-generation sequencing (NGS) should be adopted either where there is limited tissue or as part of NGS tumour profiling in the context of precision oncology, acknowledging that conventional histology of PDAC may rarely harbour MSI/dMMR., Competing Interests: Competing interests: LDW has been a paid consultant for PGDx (Personal Genome Diagnostics, Baltimore, MD, USA). CL has been a paid expert-consultant on microsatellite instability for BioScience Communications (New York, New York, USA)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, whose main molecular trait is the MAPK pathway activation due to KRAS mutation, which is present in 90% of cases.The genetic landscape of KRAS wild type PDAC can be divided into three categories. The first is represented by tumors with an activated MAPK pathway due to BRAF mutation that occur in up to 4% of cases. The second includes tumors with microsatellite instability (MSI) due to defective DNA mismatch repair (dMMR), which occurs in about 2% of cases, also featuring a high tumor mutational burden. The third category is represented by tumors with kinase fusion genes, which marks about 4% of cases. While therapeutic molecular targeting of KRAS is an unresolved challenge, KRAS-wild type PDACs have potential options for tailored treatments, including BRAF antagonists and MAPK inhibitors for the first group, immunotherapy with anti-PD-1/PD-L1 agents for the MSI/dMMR group, and kinase inhibitors for the third group.This calls for a complementation of the histological diagnosis of PDAC with a routine determination of KRAS followed by a comprehensive molecular profiling of KRAS-negative cases.

Background: Glioblastomas (GBMs) are classified into isocitrate dehydrogenase ( IDH ) mutants and IDH wild-types ( IDH -wt). This study aimed at identifying the mutational assets of IDH -wt GBMs in patients aged 18-54 years for which limited data are available., Methods: Sixteen IDH -wt GBMs from adults < 55 years old were explored for mutations, copy number variations, tumour mutational load (TML), and mutational spectrum by a 409 genes TML panel., Results: Eight (50%) IDH -wt GBMs were hypermutated (TML > 9 mutations/Mb) and two (12.5%) were ultra-mutated (TML > 100 mutations/Mb). One ultra-mutated GBM had microsatellite instability (MSI), a somatic MSH6 mutation, and a germline POLE mutation. The other ultra-mutated GBMs had MSI and two somatic mutations in MSH2. Both ultra-mutated GBMs featured at least 25% giant cells. The overall survival of eight patients with hypermutated GBMs was significantly longer than that of patients with non-hypermutated GBMs ( p = 0.04)., Conclusions: We identified a hyper-mutated subgroup among IDH-wt GBMs in adults < 55 years that had improved prognosis. Two cases were ultra-mutated and characterized by the presence of at least 25% giant cells, MMR mutations, and MSI. Since high TML has been associated with response to immune checkpoint inhibition in paediatric gliomas, the identification of a subtype of ultra-mutated IDH-wt GBM may have implications for immunotherapy., Competing Interests: The authors declare no conflict of interest.

Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 μM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

Acute promyelocitic leukemia (APL) is characterized by the pathognomonic presence in leukemic blasts of the hybrid protein PML/RARA, that acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid differentiation. Here, we show that primary blasts from APL patients express lower levels of the oncosuppressor protein PTEN, as compared to blast cells from other AML subtypes or normal bone marrow, and demonstrate that PML-RARA directly inhibits PTEN expression. We show that All-Trans Retinoic Acid (ATRA) triggers in APL cells an active chromatin status at the core regulatory region of the PTEN promoter, that allows the binding of the myeloid-regulating transcription factor PU.1, and, in turn, the transcriptional induction of PTEN. ATRA, via PML/RARA degradation, also promotes PTEN nuclear re-localization and decreases expression of the PTEN target Aurora A kinase. In conclusion, our findings support the notion that PTEN is one of the primary targets of PML/RARA in APL.

Chronic lymphocytic leukemia (CLL) is characterized by extremely variable clinical course indicating substantial differences in the biology of the disease. Molecular characterization provides new insights useful for treatment decision making. We report on a patient diagnosed with CLL, whose disease was characterized by episodes of rapid progression and disease stabilization, and in which a SRSF2 gene mutation was identified in the absence of other commonly known mutations of CLL. To the best of our knowledge this is the first case of SRSF2 gene mutation ever reported in CLL.

Somatic mutations of the spliceosome machinery have been recently identified by whole genome analysis in hematologic diseases, such as myelodysplastic syndrome, chronic lymphocytic leukemia, myeloproliferative neoplasms, acute myeloid leukemia, and advanced forms of mastocytosis, and also in nonhematologic conditions. SRSF2 is a member of the serine/arginine-rich family pre-mRNA splicing factors that plays a role in mRNA export from the nucleus and translation. We describe a high-resolution melting (HRM) curve analysis to screen for SRSF2 hotspot mutations in a fast, sensitive, and reliable way. Fifty bone marrow samples from patients with myelodysplastic syndrome were analyzed by the HRM assay and by direct sequencing. HRM screening identified four melting patterns corresponding to a negative (wild-type) group and three different mutated groups. Each mutated group was identified according to the positive control used: P95H, P95L, and P95R, respectively. An HRM mutated pattern was identified in seven patients. Positive and negative results from HRM were compared with direct sequencing results with a sensitivity and specificity of 100% (95% CI, 0.56-1, and 95% CI, 0.89-1, respectively). Analytical sensitivity analysis revealed a detection threshold of up to 1:9 (mutated/wild type) dilution. This rapid screening method may provide useful information for clinical decision making and be helpful to optimize laboratory resources and reduce turnaround time., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Recent studies have shown that language processing is grounded in actions. Multiple independent research findings indicate that children with specific language impairment (SLI) show subtle difficulties beyond the language domain. Uncertainties remain on possible association between body-mediated, non-linguistic expression of verbs and early manifestation of SLI during verb acquisition. The present study was conducted to determine whether verb production through non-linguistic modalities is impaired in children with SLI. Children with SLI (mean age 41 months) and typically developing children (mean age 40 months) were asked to recognize target verbs while viewing video clips showing the action associated with the verb (verb-recognition task) and to enact the action corresponding to the verb (verb-enacting task). Children with SLI performed more poorly than control children in both tasks. The present study demonstrates that early language impairment emerges at the bodily level. These findings are consistent with the embodied theories of cognition and underscore the role of action-based representations during language development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We investigated whether the bodily-mediated production of verbs emerges earlier than verb recognition and oral production during early language acquisition. Children (aged 18-22, 23-27, 28-32, and 33-37 months) viewed animated pictures representing actions related to transitive and intransitive verbs and were asked to (i) orally indicate the verb presented, (ii) recognize the target verb among other verbs, and (iii) perform the actions corresponding to the target verb enunciated by the experimenter. Children 18-22 months showed a capacity to enact the verbs, while their recognition and oral production abilities were not comparably developed. Until 27 months of age, children produced more transitive than intransitive verbs across tasks. The gap between verb recognition and verb oral production was found in all ages tested. This is the first study to directly demonstrate that the ability to produce verbs, especially transitive verbs, via overt body actions emerges ontogenetically earlier than recognition and oral production., (Copyright © 2013 Wiley Periodicals, Inc.)