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The aim of this study was to determine how TERTp mutations impact glioblastoma prognosis., Materials and Methods: TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients., Results: Overall, there were no differences in outcomes between patients with mutated TERTp -wt or TERTp . However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp -wt ( p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively ( p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes., Conclusions: In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp -wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

RNA-Sequencing (RNA-Seq) can identify gene fusions in tumors, but not all these fusions have functional consequences. Using multiple data bases, we have performed an in silico analysis of fusions detected by RNA-Seq in tumor samples from 139 newly diagnosed glioblastoma patients to identify in-frame fusions with predictable oncogenic potential. Among 61 samples with fusions, there were 103 different fusions, involving 167 different genes, including 20 known oncogenes or tumor suppressor genes (TSGs), 16 associated with cancer but not oncogenes or TSGs, and 32 not associated with cancer but previously shown to be involved in fusions in gliomas. After selecting in-frame fusions able to produce a protein product and running Oncofuse, we identified 30 fusions with predictable oncogenic potential and classified them into four non-overlapping categories: six previously described in cancer; six involving an oncogene or TSG; four predicted by Oncofuse to have oncogenic potential; and 14 other in-frame fusions. Only 24 patients harbored one or more of these 30 fusions, and only two fusions were present in more than one patient: FGFR3::TACC3 and EGFR::SEPTIN14. This in silico study provides a good starting point for the identification of gene fusions with functional consequences in the pathogenesis or treatment of glioblastoma., (© 2022. The Author(s).)

Introduction: Cytological samples obtained by endobronchial ultrasound (EBUS) are capital for diagnosis, staging and molecular profile in non-small cell lung carcinoma (NSCLC)., Objective: To assess the success rate of complete, partial and individual of molecular analysis in samples obtained by EBUS-guided transbronchial needle aspiration (TBNA) and/or by oesophageal ultrasound-guided fine needle aspiration with an echobronchoscope (EUS-B-FNA) in patients with NSCLC., Methods: Prospective study including 90 patients with non-squamous NSCLC, or non-smoking squamous. Cytological samples were classified into two groups. Group 1: PEN membrane slide and/or cell blocks for the determination of mutations of EGFR, KRAS, ERBB2 and BRAF. Group 2: silane coated slides or cell blocks for rearrangements of ALK, ROS1 and MET amplification., Results: The success rate was 78.6% for 4 molecular alterations (EGFR, KRAS, ALK and ROS1), and 44% for 7 determinations. The individual success rate for EGFR was 97%, KRAS 96.3%, ALK 85%, ROS1 82.3%, ERBB2 71.4%, BRAF 67.7% and MET 81.1%. There were no significant differences (p=0.489) in the number of molecular analyses (1-3 vs. 4) in group 1, depending on the types of samples (cell block vs. PEN membrane slide vs. cell block and PEN membrane slide)., Conclusions: In patients with NSCLC, the cytological material obtained by ultrasound-guided needle aspiration is sufficient for individual and partial molecular analysis in the vast majority of cases. Membrane slides such as cell blocks are valid samples for molecular analysis., (Copyright © 2020 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved.)

Glioblastoma (GBM) is the most aggressive brain tumor in adults and is characterized by an immunosuppressive microenvironment. Different factors shaping this tumor microenvironment (TME) regulate tumor initiation, progression, and treatment response. Genetic alterations and metabolism pathways are two main elements that influence tumor immune cells and TME. In this manuscript, we review how both factors can contribute to an immunosuppressive state and overview the strategies being tested.

Biallelic germline mismatch repair (MMR) gene (MLH1, MSH2, MSH6, and PMS2) mutations are an extremely rare event that causes constitutional mismatch repair deficiency (CMMRD) syndrome. CMMRD is underdiagnosed and often debuts with pediatric malignant brain tumors. A high degree of clinical awareness of the CMMRD phenotype is needed to identify new cases. Immunohistochemical (IHC) assessment of MMR protein expression and analysis of microsatellite instability (MSI) are the first tools with which to initiate the study of this syndrome in solid malignancies. MMR IHC shows a hallmark pattern with absence of staining in both neoplastic and non-neoplastic cells for the biallelic mutated gene. However, MSI often fails in brain malignancies. The aim of this report is to draw attention to the peculiar IHC profile that characterizes CMMRD syndrome and to review the difficulties in reaching an accurate diagnosis by describing the case of two siblings with biallelic MSH6 germline mutations and brain tumors. Given the difficulties involved in early diagnosis of CMMRD we propose the use of the IHC of MMR proteins in all malignant brain tumors diagnosed in individuals younger than 25 years-old to facilitate the diagnosis of CMMRD and to select those neoplasms that will benefit from immunotherapy treatment.

Objectives: We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs)., Methods: A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC., Results: Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions., Conclusions: This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival.

Lynch syndrome (LS), the most frequent form of hereditary colorectal cancer, shows a highly penetrant, autosomal dominant pattern of inheritance. Distinction of LS colorectal carcinoma instances from the much more common sporadic colorectal carcinoma cases is of paramount importance. Revised Bethesda Guidelines were developed to diagnose LS by evaluating a combination of clinical and pathologic data. The aim of the present study was to evaluate the usefulness of the pathology items included in the Revised Bethesda Guidelines. We have prospectively studied a series of 1624 consecutive colorectal carcinomas with an algorithm including immunohistochemical analysis of mismatch repair proteins and molecular study of microsatellite instability and BRAF c.1799 T > A (p.V600E) gene mutations. Patients with tumors showing LS features were referred for germline mutation analysis. By applying our algorithmic approach, we were able to identify LS features in 89 colorectal cancer patients, of whom only 27 met Revised Bethesda Guidelines pathology criteria. Of the 89 patients, 47 were then studied at the Genetic Counseling Unit, and LS was confirmed in 18, of whom 7 had not been identified by the Revised Bethesda Guidelines. Our study shows that the Revised Bethesda Guidelines failed to detect 70% of patients at risk of LS. Our algorithmic approach is a realistic and effective tool for LS identification. We strongly recommend the implementation of universal population screening for LS among all patients with newly diagnosed colorectal carcinoma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: To assess how hybridization probe design may affect MYC status determination in Burkitt lymphoma and diffuse large B-cell lymphoma., Methods and Results: We compared the results obtained with one dual-fusion and two break-apart commercial probes in a retrospective series of 91 aggressive B-cell lymphomas. All three probes were able to detect the IGH-MYC translocation in every case bearing it (13/13). However, seven of 13 (54%) non-IGH-MYC (light-chain immunoglobulin or non-immunoglobulin-MYC) rearrangements were unambiguously detected by just one of the probes tested. On the other hand, when the IGH-MYC dual-fusion probe was used, nine of 15 (60%) cases with a hybridization pattern suggestive of a non-IGH-MYC translocation were attributable to MYC copy gain rather than MYC rearrangement, as demonstrated by both break-apart probes., Conclusions: Taking into account the prognostic and therapeutic implications of the MYC translocation, probe design and limitations should be particularly kept in mind when MYC hybridization patterns are interpreted. In our experience, detection of 8q24 abnormalities could be optimized by a two-probe approach involving the application of both IGH-MYC dual-fusion and MYC break-apart selected kits., (© 2013 John Wiley & Sons Ltd.)

Aims: Although BIOMED-2 polymerase chain reaction (PCR) standardization protocols allow clonality detection in nearly 100% of non-Hodgkin B cell lymphomas, they have not been widely validated for Hodgkin lymphoma (HL). Our aim was to assess BIOMED-2 protocol sensitivity when using non-microdissected, formalin-fixed, paraffin-embedded (FFPE) tissue from HL cases., Methods and Results: We studied 69 consecutive HL cases, of which 61 corresponded to classic HL (cHL) and eight to nodular lymphocyte-predominant HL (NLPHL). CD30-positive cell numbers (<10, 10-25 or >25 per ×200 field), background CD20-positive cell density (low or high) and tumour cell immunophenotype were evaluated. IGH and IGK clonality was assessed on FFPE tissue following BIOMED-2 protocols. Of the 58 assessable cHL cases, 15 (25.9%) exhibited IGH and/or IGK clonality; IGH clonality was shown by nine (15.5%) and IGK clonality by 12 (20.7%). Clonality detection rates in cHL improved as CD30-positive Reed-Sternberg (RS) cell density increased and CD20-positive B cell density decreased, although these correlations did not reach statistical significance. Of the eight NLPHL cases studied, none showed clonal rearrangement., Conclusions: Combined study of IGH and IGK rearrangement according to BIOMED-2 protocols improves clonality detection rate (up to 25% of cases) in HL, even when working on non-microdissected FFPE tissue., (© 2012 Blackwell Publishing Ltd.)

Aims: MYC gene translocation entails a bad prognosis and a poor response to rituximab-cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) in diffuse large B cell lymphomas (DLBCL), and more intensive chemotherapy regimens could be more effective in those cases. Its evaluation requires cytogenetic or fluorescence in-situ hybridization (FISH) studies, which are expensive and not widely available. The aim of this work was to find an immunohistochemical marker able to be used as a screening tool to identify MYC translocations., Methods and Results: Aggressive B cell lymphomas in which MYC status was assessed during their diagnostic work-up between 2007 and 2010 were collected, their immunophenotype was re-evaluated, and were stratified according to the Hans algorithm. Two tissue microarrays were built in order to evaluate MYC protein expression with a commercially available antibody. The study was performed on 56 specimens: nine Burkitt lymphomas (eight translocated), 45 DLBCLs (nine translocated) and two lymphomas with intermediate features (both translocated). Only MYC protein expression detected by immunohistochemistry correlated with MYC translocation. No relationship was seen between MYC gene copies and protein expression., Conclusions: MYC protein expression detected by immunohistochemistry using a commercially available antibody correlates with MYC gene translocation, and could be used as a screening tool to select those cases in which confirmatory genetic testing is mandatory., (2011 Blackwell Publishing Limited.)

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.

Survivin is a member of the inhibitor apoptosis family that is overexpressed in many malignancies. It has five known alternative splice forms, some of which differ in their antiapoptotic properties and expression levels in human cancers. Here we describe a novel donor splice site (DSS), 2B+32 DSS, which is used in conjunction with survivin alternative exon 2B, resulting in the inclusion of 32 additional nucleotides from intron 2 at the 3' end of this exon. Sequence analysis showed that both the classical exon 2B DSS and 2B+32 are provided by an Alu sequence, which is inserted in intron 2 downstream of a functional acceptor splice site, leading to the exonization of part of the repetitive element. Minor transcripts including the 2B+32 alternative exon, or retaining the whole intronic region comprised between exons 2B and 3, were detected in several human cell lines and in some human tissues. Survivin 2B+32 containing variants acquire a premature stop codon (PTC) and may therefore be degraded by the nonsense mediated decay pathway. The implication of these novel isoforms, as well as other PTC+ survivin variants, in the overall regulation of survivin expression is discussed. Sequence analysis of intron 2 which contains the Alu Y element was performed on different primate species in order to trace its insertion and exonization during primate evolution.

JC virus (JCV), the agent of progressive multifocal leucoencephalopathy (PML), exerts an oncogenic effect in several laboratory animal models. Moreover, JCV genomic DNA and early viral protein T-antigen have been detected in various types of human central nervous system (CNS) neoplasms. To further explore this association we have studied paraffin-embedded brain biopsy tissue from 60 neoplasms (55 gliomas and five medulloblastomas) and 15 reactive gliosis cases for the presence of JCV DNA sequences and proteins. Four post mortem cases of HIV-associated PML were used as positive controls. Samples were assessed by polymerase chain reaction (PCR) amplification of early (large T antigen) and late (virion protein 3) sequences and immunohistochemistry (IHC) with both PAb 2024 and anti-SV40 large T antigen monoclonal antibodies. Five cases (three neoplasms and two reactive gliosis instances) showed low viral DNA levels when PCR-tested for VP3 or large T, while no case was immunoreactive for any of the two antibodies used. The four PML cases yielded positive results with both PCR and IHC. Additionally, IHC with both antibodies was applied to a tissue micro-array including 109 CNS tumours and 21 reactive gliosis samples. No immunoreactivity was detected in any of these tissue micro-array samples. The rarity of JCV DNA sequences and early proteins in our brain tumours enriches the controversy over the role of JCV in human neurooncogenesis, whose clarification is in need of further molecular and epidemiologic studies.

In situ hybridization (ISH) for JC virus (JCV) is generally applied for the diagnosis of progressive multifocal leukoencephalopathy (PML). To explore the usefulness of immunohistochemistry (IHC) for JCV early proteins, 14 paraffin-embedded postmortem brain specimens with histologic features compatible with PML were tested for the presence of JCV by means of DNA-DNA ISH with a biotinylated probe corresponding to the entire JCV genome, for JCV early proteins IHC with both PAb 2003 and anti-SV40 large T antigen monoclonal antibodies, and polymerase chain reaction (PCR) amplification of JCV virion protein 3 (VP3) and transcriptional control region (TCR) sequences. ISH was positive in 13 cases and IHC in all 14 cases, the number of IHC-positive cells generally being far in excess of ISH-positive cells. Of the 2 monoclonal antibodies used, PAb 2003 proved to be more sensitive than anti-SV40 large T antigen. Occasional neuronal nuclei were positive for JCV early proteins in 5 cases. As for PCR, VP3 was amplified in all 14 cases and TCR in 9 cases. Consequently, PAb 2003 IHC for JCV early proteins seems to be a powerful tool for viral demonstration in PML and may well become the diagnostic recourse of choice in this setting.

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies.

POU domain proteins are a large family of transcription factors that have been identified in a variety of metazoans, from freshwater sponges, planarians and nematodes to arthropods, echinoderms and vertebrates. Many of these proteins are implicated in the development and establishment of the nervous system. In this paper we describe the identification of the planarian genes GtPOU-1, GtPOU-3 and GtPOU-4, which belong to the subclasses III and IV of POU-domain genes. Their similarity with other members of the POU family is restricted to the POU and homeo domains, plus some peptide sequences scattered in the linker and flanking regions. As with other subclass III POU genes, GtPOU-1 is devoid of introns. Axial transcript distribution by RT-PCR and immunohistochemical assays, performed with a polyclonal antibody raised against the GtPOU-1 fusion protein, indicate that both the GtPOU-1 transcript and protein are continuously expressed along the antero-posterior axis. A monoclonal antibody raised against the same fusion protein indicates that a GtPOU-1-specific epitope, probably obtained by post-translational modification, is present in neural cells from both the central and peripheral nerve systems of the adult planarian's anterior third. Moreover, the GtPOU-1-specific epitope shows a dynamic expression pattern during regeneration, always marking the most anterior region of the planarian nervous system. Both the rapid and general GtPOU-1-specific epitope modification, during posterior regeneration, indicate that regeneration is a global process involving all planarian regions, including those that are far from the wound, by a combination of morphallactic and epimorphic mechanisms., (Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved.)

The repertoire of distinct CD44 protein isoforms is generated by means of alternative pre-mRNA splicing of 10 variable exons located in the central region of the CD44 gene. We have used human breast ductal carcinoma as a model to identify two alternative splicing pathways of the CD44 pre-mRNA variable region that account for the generation of all of the CD44 isoforms described in breast tissue. An alternative splicing pathway that reflects inclusion of variable exons in a gradual 3'-to-5' fashion is evidenced in breast ductal carcinoma and its lymph node metastases. This pathway is compatible with a mechanism that generates the standard form of CD44 (devoid of variable exons) and is distinguishable from an alternative splicing pathway that involves exclusively variant exon 3 and is observable in both normal and carcinoma breast tissue. We show that both pathways are detectable in the same cell type in the breast and provide a speculative model by which these splicing routes could take place.

The genome of the planarian (Platyhelminthes; Turbellaria; Tricladida) Dugesia (Girardia) tigrina includes a paired-like type of homeobox gene. The Dtprd-1 gene encodes a protein of 382 amino acids. The open reading frame of Dtprd-1 is interrupted by two short introns of 65 and 56 bp, and one long intron of 4.8 kb. The intron positions are not located in the homeobox and are not shared with any other known paired-like gene. The Dtprd-1 homeodomain conserves most of the residues characteristic of the paired-like class. Similarity with other members of this class is low, except with the rat, mouse and Caenorhabditis elegans proteins PHD1, Uncx-4.1 and unc-4, with 86-83% of similarity in the homeodomain, plus several peptides in the flanking regions. Such proteins share specific residues in their homeodomains that can be used to define a new family in the paired-like class of genes. The spatial distribution of gene transcript and product in adult tissues, as revealed by RT-PCR, northern blots and polyclonal antibody, demonstrates that Dtprd-1 is highly expressed in cyanophilic gland cells located in the ventral parenchyma close to the nervous system. No expression is observed during the early stages of regeneration (0-3 days). This suggests a possible role for this homeobox gene within these secretory gland cells, but not in the pattern formation mechanisms known to occur at the early stages of regeneration.

Platyhelminthes are widely considered to be the sister group of coelomates (Philippe, H., Chenuil, A. and Adoutte, A. (1994)Development 1994 Supplement, 15-24) and the first organisms to show bilateral symmetry and cephalization. Within this phylum, the freshwater planarians (Turbellaria, Tricladida) have been used as model systems for studying bidirectional regeneration (Slack, J. M. W. (1980) J. Theor. Biol 82, 105-140). We have been attempting to identify potential pattern-control genes involved in the regeneration of planarian heads and tails after amputation. Since Hox cluster genes determine positional identity along the anteroposterior axis in a wide range of animals (Slack, J. M. W., Holland, P. W. H. and Graham, C. F. (1993) Nature 361,490-492), we performed an extensive search for Hox-related genes in the planarian Dugesia(G)tigrina. Sequence analyses of seven planarian Dthox genes (Dthox-A to Dthox-G) reveal high similarities with the homeodomain region of the Hox cluster genes, allowing us to assign planarian Dthox genes to anterior and medial Hox cluster paralogous groups. Whole-mount in situ hybridization studies in regenerating adults showed very early, synchronous and colocalized activation of Dthox-D, Dthox-A, Dthox-C, Dthox-E, Dthox-G and Dthox-F. After one hour of regeneration a clear expression was observed in all Dthox genes studied. In addition, all seemed to be expressed in the same regenerative tissue, although in the last stages of regeneration (9 to 15 days) a differential timing of deactivation was observed. The same Dthox genes were also expressed synchronously and were colocalized during intercalary regeneration, although their expression was delayed. Terminal regeneration showed identical Dthox gene expression in anterior and posterior blastemas, which may prevent these genes from directing the distinction between head and tail. Finally, continuous expression along the whole lateral blastema in sagittal regenerates reflected a ubiquitous Dthox response in all types of regeneration that was not related specifically with the anteroposterior polarity.

Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle-repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.