Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells.

BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. METHODS: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. RESULTS: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. CONCLUSIONS: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.

BACKGROUND: Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. RESULTS: Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. CONCLUSIONS: This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

Tumor microenvironment-associated modifications of alternative splicing.

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Redirecting splicing with bifunctional oligonucleotides.

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5' splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.

PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa.

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays.

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human ß2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

Respiratory tract samples collected from patients in a region of Quebec, Canada, indicate the absence of early circulation of SARS-CoV-2 infection.

Background: The first documented case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Quebec was confirmed on February 27, 2020. Retracing the first cases that occur within a geographical region may provide insight regarding the evolution and spread of SARS-CoV-2 in that region because the spread of undiagnosed cases may facilitate the initial community amplification of the virus. Methods: We performed a retrospective analysis of respiratory tract samples collected for influenza testing in a region of Quebec, Canada, to look for evidence of early circulation of SARS-CoV-2. Frozen nucleic acid extracts initially collected for influenza testing between January 1 and February 20, 2020, were tested for SARS-CoV-2 using a reverse transcription-polymerase chain reaction assay. Results: During the study period, 1,440 of 2,121 (67.9%) nucleic acid extracts from individual patients were available for retrospective testing. None of the samples tested positive for SARS-CoV-2. Conclusions: The results suggest that SARS-CoV-2 was not circulating within the region before February 20, 2020, because many samples, representing more than two-thirds of all samples tested for influenza during early 2020, were tested. Further studies using a similar methodology to determine the date of onset of SARS-CoV-2 in different countries and geographic areas could enhance our understanding of the current pandemic.

Historique: Le premier cas démontré d'infection par le syndrome respiratoire aigu sévère à coronavirus 2 (SARS-CoV-2) au Québec a été confirmé le 27 février 2020. Le retraçage du premier cas survenu dans une région géographique peut donner un aperçu de l'évolution et de la propagation du virus SARS-CoV-2 dans cette région, car la transmission des cas non diagnostiqués peut favoriser l'amplification initiale du virus dans la communauté. Méthodologie: Les chercheurs ont procédé à l'analyse rétrospective des échantillons respiratoires prélevés pour le dépistage de la grippe dans une région du Québec, au Canada, afin de trouver des preuves de circulation précoce du virus SARS-CoV-2D. Les extraits d'acide nucléique congelés entre le 1er janvier et le 20 février 2020 ont été soumis au dépistage du virus SARS-CoV-2 au moyen de l'amplification en chaîne par polymérase après transcriptase inverse. Résultats: Pendant la période de l'étude, 1 440 des 2 121 extraits d'acide nucléique (67,9 %) provenant de patients différents étaient disponibles en vue de tests rétrospectifs. Aucun n'a été positif au virus SARS-CoV-2. Conclusions: D'après les résultats, le virus SARS-CoV-2 n'était pas en circulation dans la région avant le 20 février 2020, car de nombreux échantillons, représentant plus des deux tiers de tous ceux ayant servi au dépistage de la grippe au début de l'année 2020, ont été soumis au dépistage. D'autres études faisant appel à une méthodologie semblable pour déterminer la date d'apparition du virus SARS-CoV-2 dans divers pays et diverses régions géographiques pourraient permettre de mieux comprendre la pandémie en cours.

Modeling RNA tertiary structure motifs by graph-grammars.

A new approach, graph-grammars, to encode RNA tertiary structure patterns is introduced and exemplified with the classical sarcin-ricin motif. The sarcin-ricin motif is found in the stem of the crucial ribosomal loop E (also referred to as the sarcin-ricin loop), which is sensitive to the alpha-sarcin and ricin toxins. Here, we generate a graph-grammar for the sarcin-ricin motif and apply it to derive putative sequences that would fold in this motif. The biological relevance of the derived sequences is confirmed by a comparison with those found in known sarcin-ricin sites in an alignment of over 800 bacterial 23S ribosomal RNAs. The comparison raised alternative alignments in few sarcin-ricin sites, which were assessed using tertiary structure predictions and 3D modeling. The sarcin-ricin motif graph-grammar was built with indivisible nucleotide interaction cycles that were recently observed in structured RNAs. A comparison of the sequences and 3D structures of each cycle that constitute the sarcin-ricin motif gave us additional insights about RNA sequence-structure relationships. In particular, this analysis revealed the sequence space of an RNA motif depends on a structural context that goes beyond the single base pairing and base-stacking interactions.

Dissecting the expression landscape of cytochromes P450 in hepatocellular carcinoma: towards novel molecular biomarkers.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths around the world. Recent advances in genomic technologies have allowed the identification of various molecular signatures in HCC tissues. For instance, differential gene expression levels of various cytochrome P450 genes (CYP450) have been reported in studies performed on limited numbers of HCC tissue samples, or focused on a small subset on CYP450s. In the present study, we monitored the expression landscape of all the members of the CYP450 family (57 genes) in more than 200 HCC tissues using RNA-Seq data from The Cancer Genome Atlas. Using stringent statistical filters and data from paired tissues, we identified significantly dysregulated CYP450 genes in HCC. Moreover, the expression level of selected CYP450s was validated by qPCR on cDNA samples from an independent cohort. Threshold values (sensitivity and specificity) based on dysregulated gene expression were also determined to allow for confident identification of HCC tissues. Finally, a global look at expression levels of the 57 members of the CYP450 family across ten different cancer types revealed specific expression signatures. Overall, this study provides useful information on the transcriptomic landscape of CYP450 genes in HCC and on new potential HCC biomarkers.

hnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the alternative splicing response to oxaliplatin-mediated DNA damage.

Little is known about how RNA binding proteins cooperate to control splicing, and how stress pathways reconfigure these assemblies to alter splice site selection. We have shown previously that SRSF10 plays an important role in the Bcl-x splicing response to DNA damage elicited by oxaliplatin in 293 cells. Here, RNA affinity assays using a portion of the Bcl-x transcript required for this response led to the recovery of the SRSF10-interacting protein 14-3-3ε and the Sam68-interacting protein hnRNP A1. Although SRSF10, 14-3-3ε, hnRNP A1/A2 and Sam68 do not make major contributions to the regulation of Bcl-x splicing under normal growth conditions, upon DNA damage they become important to activate the 5' splice site of pro-apoptotic Bcl-xS. Our results indicate that DNA damage reconfigures the binding and activity of several regulatory RNA binding proteins on the Bcl-x pre-mRNA. Moreover, SRSF10, hnRNP A1/A2 and Sam68 collaborate to drive the DNA damage-induced splicing response of several transcripts that produce components implicated in apoptosis, cell-cycle control and DNA repair. Our study reveals how the circuitry of splicing factors is rewired to produce partnerships that coordinate alternative splicing across processes crucial for cell fate.

[Infertility and testicular seminoma]. / Infertilité et séminome testiculaire.

INTRODUCTION: Infertility in men may be associated with an elevated risk of testicular cancer. The authors report a case of testicular seminoma discovered fortuitously during a workup for infertility. CASE: A 30 year-old male was seen for infertility. Physical examination and testicular ultrasonography were normal. The sperm count found oligoasthenospermia related to the excretory ducts. The patient underwent testicular biopsies for infertility, which showed an intratubular germ cell tumor. Tumor markers (beta HCG, alpha FP, LDH) were normal. Computed tomography was normal for the thorax, abdomen, and pelvis. We performed an inguinal orchiectomy. The pathology examination found seminoma, at a pT1 stage. One course of chemotherapy followed. DISCUSSION: The incidence of testicular cancer is increasing throughout the world. Recent studies show a strong relation between infertility and an increased risk of testicular cancer, and some authors even suggest a causal relation.

[Clinical usefulness of positron emission tomography in prostate cancer]. / Utilité clinique de la tomographie par émission de positons dans le cancer de la prostate.

In prostate cancer, use of FDG, the radiopharmaceutical currently most widely used in oncology, is limited to the most aggressive cancers and, in the absence of another tracer, to attempting to localise occult recurrences detected biochemically (elevated PSA serum levels). Four other PET tracers are currently suggested in various situations of prostate cancer development: for guiding biopsies, for diagnosis and staging of the primary cancer and of local or metastatic recurrences, especially in bone, and for localizing occult biochemical recurrence. This article is illustrated by cases summarising our experience with fluoromethylcholine-(18F) and PET/CT. They cover a wide spectrum of clinical settings: localisation of intraprostatic neoplastic lesions, initial staging, monitoring treatment by ultrasound, detection of occult recurrences and characterisation of images on conventional imaging modalities, which are questionable or difficult to interpret.

[Medium-term results of grade 3 and 4 cystocele repair by porcine xenograft matrix (pelvicol)]. / Résultats à moyen terme du traitement des cystoceles de grade 3 et 4 par plaque de xénogreffe porcine (pelvicol).

OBJECTIVE: To evaluate the medium-term results of grade 3 and 4 (Baden-Walker classification) cystocele repair by transvaginal porcine xenograft matrix (Pelvicol). MATERIALS: Between February 2002 and October 2005, fifty patients with grade 3 or 4 cystocele were treated by Pelvicol matrix. The preoperative grade of prolapse and symptoms (urinary and pelvic heaviness) were recorded and a sexuality questionnaire was completed retrospectively (BISF-W questionnaire). All patients were reviewed in the outpatients department at 1 month and at the date of last follow-up. The success of the surgical procedure was defined by postoperative grades 0 and 1. Preoperative and postoperative symptoms and sexuality were compared. The operative morbidity was recorded. The success of the surgical procedure was compared in patients operated for the first time and in redo patients. RESULTS: The mean age was 69.4 years; 29 patients had a grade 3 cystocele (58%), and 21 had a grade 4 cystocele (42%). Preoperative symptoms consisted of dysuria (32%) and symptoms of overactive bladder (22%), accompanied by symptomatic (36%) or asymptomatic (20%) stress urinary incontinence. Pelvic heaviness was present in 100% of cases. An associated procedure was necessary in 70% of cases (29 suburethral tapes, 6 rectocele repairs, 3 vaginal hysterectomies). Mean follow-up was 27.2 months (95%CI [23.3-31.1]). No intraoperative or postoperative complications were observed. During follow-up, no cases of rejection of material, vaginal erosion or delayed healing were observed. The surgical success rate was 94% (37 grade 0 and 10 grade 1). Dysuria and pelvic heaviness were significantly improved. None of the 10 women who were sexually active preoperatively reported postoperative discomfort. Among the 50 operated patients, 35 (70%) were treated by Pelvicol as first-line procedure and 15 were redo procedures (30%); no significant difference in surgical success rate was observed between these two groups (94.3% versus 93.4%). CONCLUSION: The use of a porcine xenograft matrix (Pelvicol) appears to be a safe and effective technique in the medium term for first-line treatment of grade 3 and 4 cystocele.

Analysis of CTCL cell lines reveals important differences between mycosis fungoides/Sézary syndrome vs. HTLV-1+ leukemic cell lines.

HTLV-1 is estimated to affect ~20 million people worldwide and in ~5% of carriers it produces Adult T-Cell Leukemia/Lymphoma (ATLL), which can often masquerade and present with classic erythematous pruritic patches and plaques that are typically seen in Mycosis Fungoides (MF) and Sézary Syndrome (SS), the most recognized variants of Cutaneous T-Cell Lymphomas (CTCL). For many years the role of HTLV-1 in the pathogenesis of MF/SS has been hotly debated. In this study we analyzed CTCL vs. HTLV-1+ leukemic cells. We performed G-banding/spectral karyotyping, extensive gene expression analysis, TP53 sequencing in the 11 patient-derived HTLV-1+ (MJ and Hut102) vs. HTLV-1- (Myla, Mac2a, PB2B, HH, H9, Hut78, SZ4, Sez4 and SeAx) CTCL cell lines. We further tested drug sensitivities to commonly used CTCL therapies and studied the ability of these cells to produce subcutaneous xenograft tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Our work demonstrates that unlike classic advanced MF/SS cells that acquire many ongoing balanced and unbalanced chromosomal translocations, HTLV-1+ CTCL leukemia cells are diploid and exhibit only a minimal number of non-specific chromosomal alterations. Our results indicate that HTLV-1 virus is likely not involved in the pathogenesis of classic MF/SS since it drives a very different pathway of lymphomagenesis based on our findings in these cells. This study also provides for the first time a comprehensive characterization of the CTCL cells with respect to gene expression profiling, TP53 mutation status, ability to produce tumors in mice and response to commonly used therapies.

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

Gene expression analysis in Cutaneous T-Cell Lymphomas (CTCL) highlights disease heterogeneity and potential diagnostic and prognostic indicators.

Cutaneous T-Cell Lymphomas (CTCL) are rare, but potentially devastating malignancies, whose pathogenesis remains poorly elucidated. Unfortunately, currently it is not possible to predict based on the available criteria in which patients the cancer will progress and which patients will experience an indolent disease course. Furthermore, at early stages this malignancy often masquerades as psoriasis, chronic eczema or other benign inflammatory dermatoses. As a result, it takes on average 6 y to diagnose this lymphoma since its initial presentation. In this study, we performed transcription expression profiling using TruSeq targeted RNA gene expression on 181 fresh and formalin-fixed and paraffin-embedded (FFPE) skin samples from CTCL patients and patients affected by benign inflammatory dermatoses that often mimic CTCL clinically and on histology (e.g., psoriasis, chronic eczema, etc.) We also analyzed multiple longitudinal biopsies that were obtained from the same patients over time. Our results underscore significant molecular heterogeneity with respect to gene expression between different patients and even within the same patients over time. Our study also confirmed TOX, FYB, LEF1, CCR4, ITK, EED, POU2AF, IL26, STAT5, BLK, GTSF1 and PSORS1C2 genes as being differentially expressed between CTCL and benign skin biopsies. In addition, we found that differential expression for a subset of these markers (e.g., TOX, FYB, GTSF1 and CCR4) may be useful in prognosticating this disease. This research, combined with other molecular analyses, prepares the foundation for the development of personalized molecular approach toward diagnosis and management of CTCL.

Endoscopic lithotripsy and the FREDDY laser: initial experience.

BACKGROUND AND PURPOSE: The frequency-doubled double-pulse neodymium:YAG (FREDDY) laser has been developed for endoscopic lithotripsy and combines the characteristics of solid and dye lasers with a thin flexible optical fiber enabling it to be used with flexible ureterorenoscopy. Furthermore, it is less expensive and easier to maintain than other lasers. Our goal was to evaluate its efficacy and role in the ureteroscopic treatment of urinary stones. PATIENTS AND METHODS: We used a FREDDY laser in 26 patients (29 stones). For 4 stone cases, this was the first line of treatment; for the remaining cases, this was the second line of treatment, following SWL in 23 cases and nephrolithotomy in 2 cases. The mean stone size was 9 mm, with a range of 6 to 15 mm. There were 13 renal and 16 ureteral stones. The absence of residual fragments at 3-month postoperative radiography was considered to reflect successful treatment. RESULTS: Twenty-six stones were treated with satisfactory results. Within 3 months, 18 patients were stone free (69%), and 72.4% of the stones (21/29) had been treated completely. Fragments of 8 stones still remained in 8 patients. Of these stones, 5 were >10 mm and persisted at 3 months. Fragmentation was ineffective for 2 cystine stones and poor for 1 calcium oxalate monohydrate stone. Hospitalization, on average, was 1.5 days with a range of 1 to 3 days. A ureteral perforation was observed in the case of an impacted ureteral stone. CONCLUSIONS: Because of the wavelengths used, endoscopic FREDDY laser lithotripsy is an effective and harmless method. This laser can be used as a therapeutic tool because of its moderate cost and ability to be used with flexible ureterorenoscopy. However, it is important to be aware of the FREDDY laser's limited fragmentation capabilities for cystine stones and its inability to treat tissue lesions such as urinary-tract stenosis and tumors.

[Role of endoscopy in the management of upper urinary tract tumors]. / Place de l'endoscopie dans la prise en charge des tumeurs de la voie excrétrice supérieure.

Transitional cell carcinomas of the upper urinary tract are rare tumours that represent about 5% of all transitional cell carcinomas. The reference treatment is currently open nephroureterectomy. Low-grade or superficial urinary tract tumours have a good prognosis, similar to that of noninvasive bladder tumours (80% 5-year specific survival). The surgical management of upper urinary tract tumours is gradually evolving towards complete preservation of the upper urinary tract and renal parenchyma, when compatible with local conditions. Conservative endoscopic treatments (ureteroscopy, percutaneous treatment) provide good oncological results and constitute a possible alternative to nephroureterectomy for the systematic management of good prognosis tumours. The cost of endoscopy equipment and consumable items is currently a limiting factor to the widespread use of these techniques.