Citations

ABSTRACT A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.

ABSTRACT The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA + and 135 were MecA − . For the coagulase-negative staphylococci, 150 were MecA + and 111 were MecA − . The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA + but oxacillin sensitive and 1 isolate was MecA − but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA + isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.

Background: Seventy percent of patients with light chain (AL) amyloidosis present with renal involvement. The phases of the disease include asymptomatic seeding, increasing proteinuria, and progression to end stage renal disease. In an attempt to identify genes relevant to renal reprogramming due to persistent disease,renal biopsies from treated AL patients were obtained for systematic histologic evaluation and transcriptional profiling. Correlates were sought between these two approaches. Methods: Renal biopsies from 10 patients with persistent proteinuria (>500mg/day) following hematologic responses to anti-plasma cell therapy were scored independently by 2 expert renal pathologists employing a novel histologic tool that assessed scarring, fibrosis and the distribution of amyloid, and were successfully evaluated by transcriptional profiling in the Michigan Kidney Translational Medicine Core lab. Clinical data and histologic scores (CSIC = composite scarring injury score, AS = amyloid score) were correlated to the expression profiles of specific tubular and glomerular gene sets. Results: Baseline characteristics and histologic scores are in Table 1. Using a false discovery rate corrected P-value of < 0.10, numerous genes of interest were identified (Table 2). Transcriptional profiling revealed 2 distinct patient clusters (G1 and G2) within the tubular and glomerular gene expression sets. The histopathologic features were different between G1 and G2; the amyloid score was significantly higher in G2 in both the tubular (7.0 vs. 4.25; p=0.03) and the glomerular (6.92 vs. 4.38; p= 0.04) compartments. The differential expression for the genes of interest between G1 and G2 were also determined (Table 2). Conclusion: This effort has generated multiple genes of interest. ICQD and PODXL, in particular, correlated with the distribution of amyloid in the capillaries and mesangium. We are currently comparing these expression profiles to those of other nephropathies to identify genes whose expression levels may be specific to AL nephropathy, hopefully leading to further novel histologic and protein-based investigations. Disclosures Comenzo: Takeda: Research Funding; Prothena Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Caelum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Unum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Myself: Patents & Royalties: Patent 9593332, Pending 20170008966.

Abstract It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we established a subclone of the U937 cell line designated F5, in which a single copy of the rare-cutting meganuclease I-SceI recognition site has been integrated into chromosome 7. We transfected an I-SceI expression vector into these cells and analyzed misrepair events. We recovered no GCR, but instead found that most DSBs were repaired by small (<50bp) indels (91.8%). We also noticed some DSBs were repaired by large (<50bp) deletions (6.4%) and rare DSBs were repaired by large (100-500 bp) insertions (1.8%). Surprisingly, the nucleotide sequence of these insertions matched distant regions of the genome. To determine if this templated sequence insertion (TSI) was peculiar to I-SceI induced DNA DSB, we repeated the experiment using TALENs to produce DNA DSB. Again, we found insertions that were derived from distant genomic regions. To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed structural variations identified by whole genome sequence (WGS) of two myeloma cell lines (KP6, MC1286PE1), and identified 15 independent TSIs. These TSIs were verified by Sanger sequencing, and lengths of inserted fragment were 38 to 666 bp (median 189 bp). Six insertions were noted to be identical between the 2 cell lines, suggesting that these TSIs were germline polymorphic events. These TSIs were compared to a catalog of known structural variations identified by WGS of 52 healthy individuals; 13 of 15 TSI junctions were identified, suggesting that these represented polymorphic insertions in the human genome. Two TSIs were potential somatic mutations in the KP6 cell line, including one that had 85bp insertion at the junction of an acquired t(6;12) chromosomal translocation. Six TSIs showed the hallmarks of L1 retrotransposon-mediated insertions, with a 5’-TTTT/A-3’ integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was neither a LINE nor a SINE, but instead was a transcribed, genic region mapping to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these insertions were caused by the L1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSI showed no preference for a 5’-TTTT/A-3’ integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junctions. This second class is similar to TSI detected at I-SceI induced DNA DSB, and we predict that these TSI were generated by reverse transcription of pre-mRNA creating a “patch” for a spontaneous DNA DSB that occurred in a germ cell. To determine if TSI at I-SceI induced DNA DSB could be repaired by RNA-templated sequences, we cotransfected murine RNA and an I-SceI expression vector into F5 cells. Approximately 10% of the TSI recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. To further support the contention that TSIs were derived from RNA, we again used I-SceI to induce a DNA DSB, and treated the cells with reverse transcriptase inhibitors. The TSI frequency was reduced by 70%. Finally, a subset of insertions were due to telomere repeat sequences, lending further support for the hypothesis that RNA could serve as a template for the repair of DNA DSB. TSI is novel form of potentially mutagenic DNA DSB repair, which should be considered as an alternative to a balanced chromosomal translocation in the interpretation of reciprocal interchromosomal structural variations identified by short-read deep sequencing. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Spinal chordomas, a subtype of primary spinal column malignancies (PSCM), are rare tumors with poor prognosis, and we have limited understanding of the molecular drivers of neoplasia. Methods Study design was a retrospective review of prospectively collected data with cross-sectional survival. Archived paraffin embedded pathologic specimens were collected for 133 patients from 6 centers within Europe and North America between 1987 and 2012. Tumor DNA was extracted and the human telomerase reverse transcriptase (hTERT) promoter was sequenced. The hTERT mutational status was correlated with overall survival (OS) and time to first local recurrence. Results Ninety-two chordomas, 26 chondrosarcomas, 7 osteosarcomas, 3 Ewing’s sarcomas, and 5 other malignant spinal tumors were analyzed. Median OS following surgery was 5.8 years (95% CI: 4.6 to 6.9) and median time to first local recurrence was 3.9 years (95% CI: 2.5 to 6.7). Eight chordomas, 2 chondrosarcomas, 1 Ewing’s sarcoma, and 1 other malignant spinal tumor harbored either a C228T or C250T mutation in the hTERT promoter. In the overall cohort, all patients with hTERT mutation were alive at 10 years postoperative with a median OS of 5.1 years (95% CI: 4.5 to 6.6) (P = 0.03). hTERT promoter mutation was observed in 8.7% of spinal chordomas, and 100% of chordoma patients harboring the mutation were alive at 10 years postoperative compared with 67% patients without the mutation (P = 0.05). Conclusions We report for the first time that hTERT promoter mutations C228T and C250T are present in approximately 8.7% of spinal chordomas. The presence of hTERT mutations conferred a survival benefit and could potentially be a valuable positive prognostic molecular marker in spinal chordomas.

Abstract Current standards for husbandry and maintenance of zebrafish and other aquatic species in the laboratory are diverse, and are subject to laboratory performance, engineering, and practice standards (the Guide ), institutional interpretation, national animal welfare laws, and cultural differences. Consequently, it is difficult, and probably not advantageous, to establish a single standard in view of the hardy nature of zebrafish and the diversity of research requirements it is used to address. Based on their natural habitat, zebrafish can thrive in a variety of environmental conditions, which is a specific advantage for working with this laboratory organism. However, it also makes reporting and reproducibility difficult, because variations in the husbandry and environmental conditions, including the environmental conditions before and during experiments, are often underreported in the scientific literature. This lack of consistency presents a potential problem for research reproducibility. To begin addressing this emerging scientific gap, the National Institutes of Health's (NIH) Office of Research Infrastructure Programs (ORIP), Division of Construction and Instruments (DCI), hosted a workshop in late 2017, entitled “Zebrafish and Other Aquatic Models: Reporting of Environmental Husbandry Conditions for Rigorous Experiments and Reproducible Results,” that was attended by ∼60 participants. The objectives of the workshop were to bring together a diverse group of stakeholders—researchers, facility managers, veterinarians, journal editors, commercial vendors, and others to (1) review current husbandry and environmental management practices for the care of zebrafish and other aquatic organisms in the laboratory and to (2) propose a process for the development of a minimal set of environmental parameters that should be reported in publications to ensure rigor and robustness of experiments and reproducible outcomes. The participants also discussed how these recommendations, as an initial step, might be collected, disseminated, implemented, and improved upon after future iteration.

Abstract Background The treatment of oligometastatic (≤5 metastases) spinal disease has trended toward ablative therapies, yet to the authors’ knowledge little is known regarding the prognosis of patients presenting with oligometastatic spinal disease and the value of this approach. The objective of the current study was to compare the survival and clinical outcomes of patients with cancer with oligometastatic spinal disease with those of patients with polymetastatic (>5 metastases) disease. Methods The current study was an international, multicenter, prospective study. Patients who were admitted to a participating spine center with a diagnosis of spinal metastases and who underwent surgical intervention and/or radiotherapy between August 2013 and May 2017 were included. Data collected included demographics, overall survival, local control, and treatment information including surgical, radiotherapy, and systemic therapy details. Health‐related quality of life (HRQOL) measures included the EuroQOL 5 dimensions 3‐level questionnaire (EQ‐5D‐3L), the 36‐Item Short Form Health Survey (SF‐36v2), and the Spine Oncology Study Group Outcomes Questionnaire (SOSGOQ). Results Of the 393 patients included in the current study, 215 presented with oligometastatic disease and 178 presented with polymetastatic disease. A significant survival advantage of 90.1% versus 77.3% at 3 months and 77.0% versus 65.1% at 6 months from the time of treatment was found for patients presenting with oligometastatic disease compared with those with polymetastatic disease. It is important to note that both groups experienced significant improvements in multiple HRQOL measures at 6 months after treatment, with no differences in these outcome measures noted between the 2 groups. Conclusions The treatment of oligometastatic disease appears to offer a significant survival advantage compared with polymetastatic disease, regardless of treatment choice. HRQOL measures were found to improve in both groups, demonstrating a palliative benefit for all treated patients.

BACKGROUNDThe Spine Oncology Study Group Outcomes Questionnaire (SOSGOQ) was developed as the first spine oncology‐specific health‐related quality of life (HRQOL) measure. This study evaluated the psychometric properties and clinical validity of the SOSGOQ in a diverse cohort of patients with spinal metastases.METHODSAn international, multicenter, prospective observational cohort study including patients with spinal metastases who underwent surgery and/or radiotherapy was conducted by the AOSpine Knowledge Forum Tumor. Demographic, tumor, and treatment data were collected. HRQOL was evaluated using the SOSGOQ and Medical Outcomes Study Questionnaire Short Form 36 Health Survey (SF‐36) at baseline and fixed follow‐up times. Construct validity was assessed using multitrait scaling analyses, confirmatory factor analyses, and correlation with the SF‐36 and NRS pain score. Test‐retest reliability was assessed in a subgroup of patients between 12 weeks after treatment and the retest 4 to 9 days later.RESULTSA total of 238 patients were enrolled at 9 centers across North America; 153 of these patients had HRQOL data available at 12 weeks after treatment. Multitrait scaling analyses and confirmatory factor analyses resulted in a refined version of the SOSGOQ with 4 domains and 4 single items. The revised SOSGOQ (SOSGOQ2.0) demonstrated strong correlations with SF‐36 and the ability to discriminate between clinically distinct patient groups. Reliability of the SOSGOQ2.0 was demonstrated to be good, with an intraclass correlation coefficient ranging from 0.58 to 0.92 for the different domains.CONCLUSIONSThe SOSGOQ2.0 is a reliable and valid measure with which to evaluate HRQOL in patients with spinal metastases. It is recommended to use the SOSGOQ2.0 together with a generic HRQOL outcome measure to comprehensively assess HRQOL and increase sensitivity and specificity. Cancer 2018;124:1828‐38. © 2018 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.