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Background: In March 2003, several hospitals in Quebec, Canada, noted a marked increase in the incidence of Clostridium difficile-associated diarrhea.Methods: In 2004 we conducted a prospective study at 12 Quebec hospitals to determine the incidence of nosocomial C. difficile-associated diarrhea and its complications and a case-control study to identify risk factors for the disease. Isolates of C. difficile were typed by pulsed-field gel electrophoresis and analyzed for binary toxin genes and partial deletions in the toxin A and B repressor gene tcdC. Antimicrobial susceptibility was evaluated in a subgroup of isolates.Results: A total of 1703 patients with 1719 episodes of nosocomial C. difficile-associated diarrhea were identified. The incidence was 22.5 per 1000 admissions. The 30-day attributable mortality rate was 6.9 percent. Case patients were more likely than matched controls to have received fluoroquinolones (odds ratio, 3.9; 95 percent confidence interval, 2.3 to 6.6) or cephalosporins (odds ratio, 3.8; 95 percent confidence interval, 2.2 to 6.6). A predominant strain, resistant to fluoroquinolones, was found in 129 of 157 isolates (82.2 percent), and the binary toxin genes and partial deletions in the tcdC gene were present in 132 isolates (84.1 percent).Conclusions: A strain of C. difficile that was resistant to fluoroquinolones and had binary toxin and a partial deletion of the tcdC gene was responsible for this outbreak of C. difficile-associated diarrhea. Exposure to fluoroquinolones or cephalosporins was a risk factor. [ABSTRACT FROM AUTHOR]

Purpose: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology., Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions., Results: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell-type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes., Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290-300. ©2015 AACR., (©2015 American Association for Cancer Research.)

This is an 11-year survey of molecular analysis of APC germline mutations for the province of Quebec done at the Molecular Pathology Unit of the Jewish General Hospital which offers genetic testing for hereditary forms of colorectal cancer for the whole of Quebec province. We report on 47 unique mutations seen in 66 families affected with familial adenomatous polyposis. Of these unique mutations, 60% are short indels, 28% are point mutations, and 6% are whole exon deletions. The absence of founder mutations and the variety of mutations encountered reinforce the value of RNA-based testing and the need for gene dosage techniques such as multiplex ligation-dependent probe amplification.

We compared PCR to conventional culture for the detection of vancomycin-resistant enterococci (VRE) in 30,835 rectal samples over a 3-year period. The positive and negative predictive values of vanB PCR were 1.42% and 99.9%, respectively. A positive vanB result by PCR is poorly predictive and necessitates culture for differentiation of VRE-positive and -negative individuals.

Lynch syndrome is one of the most common autosomal dominantly inherited cancer syndromes. Mutations in MLH1, MSH2, MSH6, and PMS2 account for greater than 98% of reported mutations in Lynch syndrome families. It has been reported that large genomic deletions in MLH1 and MSH2 are a frequent cause of Lynch syndrome in certain populations. Using a multimodal approach, we have identified mutations in MLH1, MSH2, and MSH6 in French Canadian families fulfilling the Amsterdam criteria for Lynch syndrome and who displayed abnormal staining for at least one of the Lynch syndrome proteins. Mutations were identified in 28 of our 29 French Canadian probands (97%). A total of 18 distinct mutations (nine in MLH1, seven in MSH2, two in MSH6) were identified, of which six (33%) were genomic exon deletions. Another four (22%) resulted in exon deletions in cDNA alone. Three (17%) are novel mutations. Five of these 18 mutations were detected in more than one distinct family (four in MLH1, one in MSH2) and haplotype analysis suggests the possibility of founder effects. Fifteen of the 29 (52%) families carried one of these five putative founder mutations. These findings may simplify genetic testing for Lynch syndrome in French Canadians.

Myeloproliferative disorders (MPDs) are often associated with the presence of the JAK2-V617F mutation in hematopoietic cells. It is currently not known if this mutation is carried as well by bone marrow mesenchymal stromal cells (MSCs) in these patients. To test this hypothesis, we recruited seven patients with JAK2-V617F(+) MPD, isolated marrow MSCs and characterized their phenotype and mesenchymal differentiation capacity, and probed for JAK2-V617F genomic DNA mutation. We found that MSCs of most patients could be culture-expanded and had a phenotype and differentiation capacity similar to that of MSCs derived from normal subjects. Using real-time polymerase chain reaction and melting curve analysis with probes specific for the JAK2-V617F DNA mutation, we did not find the mutation in any of the MSC samples studied. These results demonstrate that, in the setting of MPD, MSC do not originate from the mutated hematopoietic progenitor clone.

Background: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas., Methods: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, beta(2)-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058., Results: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes., Conclusions: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.

Sequential treatment with all-trans retinoic acid followed by chemotherapy significantly improves the long-term survival of patients who have acute promyelocytic leukemia (APL). Consequently, a simple and accurate test is needed to establish the diagnosis of APL and to identify those patients having a relapse of the disease. We describe an accurate, 2-hour indirect immunofluorescent assay for identifying APL cells in bone marrow specimens. The assay uses the PML (PG-M3) murine monoclonal antibody that is directed against the amino-terminal portion of the PML gene product. We observed a distinctive, finely speckled pattern of fluorescence in the NB4 cell line (a positive control), as well as in 15 clinical specimens that were confirmed to have APL by cytogenetic, cytochemical, and immunophenotypic studies, including four cases of microgranular variant of APL. By contrast, a coarse globular pattern of fluorescence was observed in 53 other clinical specimens that did not contain APL. When we performed dilution studies using artificial mixtures of APL cells with normal bone marrow cells, we detected as few as 5% APL cells in the mixture. Finally, there was complete concordance between the immunofluorescent assay and a polymerase chain reaction-based assay for the PML-retinoic acid receptor alpha chimeric gene in 12 other clinical specimens. We conclude that the immunofluorescent assay for PML protein is a rapid, sensitive, and accurate method for determining the presence of APL cells in clinical specimens. This assay therefore should be considered as a cost-effective alternative to other diagnostic tests, such as karyotyping or polymerase chain reaction, for the diagnostic evaluation of APL.

Retinoic acid (RA) induces differentiation, followed by apoptosis in acute promyelocytic leukemia (APL) cells, both in vitro and in patients. One problem in understanding these mechanisms is to distinguish molecular events leading to differentiation from those leading to apoptosis. We have identified a leukemic cell line, PLB-985, where RA directly induces apoptosis with no morphologic, genetic, or cell-surface marker evidence of differentiation. These cells differentiate following dimethyl sulfoxide (DMSO), but not RA, treatment. Two-color flow cytometry showed no alteration of the cell cycle after RA treatment, and cell-surface marker analysis of CD11a, CD11b, and CD13 showed no modulation typical of differentiating cells. RNA expression of myeloblastin and transglutaminase, genes regulated by RA-induced differentiation in NB4 cells, was unchanged by RA treatment. Instead, RA induced apoptosis, as shown by typical apoptotic morphological features, genomic DNA laddering, and positive labeling in the TUNEL assay. We found that induction of apoptosis in this model requires a different pattern of retinoid receptor binding and transcriptional activation than is seen in APL cells. As previously described, treatment with retinoid receptor-selective ligands showed that stimulation of RAR alone is sufficient to induce differentiation and apoptosis in NB4 cells, and that stimulation of RXR has no effect on the parameters analyzed. In PLB-985 cells, on the other hand, apoptosis was induced only upon costimulation of both RAR and RXR. Stimulation of either receptor alone had no effect on the cells. Consistent with these findings, bcl-2 RNA and protein levels were downregulated after stimulation of both RAR and RXR, but not with an RAR-specific ligand alone, as in NB4 cells. The expression of several other bcl-2 family members (bcl-X, ich-1, bax, bag, and bak ) and retinoid receptors (RARalpha, RXRalpha, and RXRbeta) was not affected by treatment with RAR- and/or RXR-activating retinoids; RARbeta RNA was undetectable before and after retinoid treatment. Thus, our cell model provides a useful tool in determining the genetic events mediating apoptosis as a response to RA, unobscured by events implicated in differentiation.