Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Aneuploidy, TP53 mutation, and amplification of MYC correlate with increased intratumor heterogeneity and poor prognosis of breast cancer patients.

The clinical course of breast cancer varies from one patient to another. Currently, the choice of therapy relies on clinical parameters and histological and molecular tumor features. Alas, these markers are informative in only a subset of patients. Therefore, additional predictors of disease outcome would be valuable for treatment stratification. Extensive studies showed that the degree of variation of the nuclear DNA content, i.e., aneuploidy, determines prognosis. Our aim was to further elucidate the molecular basis of aneuploidy. We analyzed five diploid and six aneuploid tumors with more than 20 years of follow-up. By performing FISH with a multiplexed panel of 10 probes to enumerate copy numbers in individual cells, and by sequencing 563 cancer-related genes, we analyzed how aneuploidy is linked to intratumor heterogeneity. In our cohort, none of the patients with diploid tumors died of breast cancer during follow-up in contrast to four of six patients with aneuploid tumors (mean survival 86.4 months). The FISH analysis showed markedly increased genomic instability and intratumor heterogeneity in aneuploid tumors. MYC gain was observed in only 20% of the diploid cancers, while all aneuploid cases showed a gain. The mutation burden was similar in diploid and aneuploid tumors, however, TP53 mutations were not observed in diploid tumors, but in all aneuploid tumors in our collective. We conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

Translational predictive biomarker analysis of the phase 1b sorafenib and bevacizumab study expansion cohort.

Predictive biomarkers are needed to triage patients to the best therapy. We prospectively planned examination of sequential blood, biopsy, and functional imaging with which to confirm the mechanism and to identify potential predictive biomarkers in a phase Ib clinical trial expansion of patients with solid tumors receiving sorafenib/bevacizumab. The maximally tolerated doses of sorafenib at 200 mg twice daily with bevacizumab at 5 mg/kg every other week were given to biopsiable patients. Patients were randomized to receive either sorafenib or bevacizumab monotherapy for the first 28-day cycle with the second drug added with cycle 2. Biopsies, dynamic contrast-enhanced MRI, and fluorodeoxyglucose-proton emission tomography were done pre-therapy and at 2 and 6 weeks (2 weeks into combination therapy). Tumor and serum proteomics, Ras/Raf mutational analysis, and functional imaging results were examined individually and across the dataset to identify potential changes predictive of response to therapy and those that confirm the biochemical drug mechanism(s). Therapy with sorafenib/bevacizumab resulted in clinical benefit in 45% of this mixed solid tumor group. ERK activation and microvessel density were decreased with monotherapy treatment with sorafenib or bevacizumab, respectively; whereas a decreased signal over the group of total AKT, phospho(p)-VEGF receptor2, p-endothelial nitric-oxide synthase, b-RAF, and cleaved poly(ADP-ribose) polymerase was associated with earlier progression of disease. Tumor metabolic activity decreased in those patients with clinical benefits lasting longer than 4 months, and activity increased with progression of disease. Cleavage of caspase 3 and poly(ADP-ribose) polymerase was increased, and Ki67 expression decreased in patients with prolonged clinical benefits, consistent with decreased proliferation and increased apoptosis. The conglomerate analysis, incorporating pharmacodynamic and tumor biochemistry, demonstrated sorafenib/bevacizumab-targeted vascular activity in the tumor. Results suggest potential biomarkers for which changes, as a group, during early therapeutic exposure may predict clinical benefit.

Genome-wide DNA methylation profiling in chronic lymphocytic leukaemia.

Background: DNA methylation aberrations are widespread among the malignant B lymphocytes of patients with chronic lymphocytic leukaemia (CLL), suggesting that DNA methylation might contribute to the pathogenesis of CLL. Aim: We aimed to explore the differentially methylated positions (DMPs) associated with CLL and screen the differentially methylated and expressed genes (DMEGs) by combining public databases. We aimed to observe the direction of each DMEG in CLL based on the DMPs in the promoter and the body region respectively to narrow down DMEGs. We also aimed to explore the methylation heterogeneity of CLL subgroups and the effect of B cells maturation on CLL. Methods: In this population-based case control study, we reported a genome-wide DNA methylation association study using the Infinium HumanMethylation450 BeadChip, profiling the DNA methylation of CD19+ B Cells from 48 CLL cases and 28 healthy controls. By integrating methylation data and expression data from public databases, gene sets were jointly screened, and then the relationship between methylation sites in promoter and body region and expression of each gene was explored. In addition, support vector machine (SVM) classification algorithm was used to identify subgroups of CLL cases based on methylation pattern, and the effect of B-cell differentiation related methylation sites on CLL-related sites was observed. Results: We identified 34,797 DMPs related to CLL across the genome, most of which were hypomethylated; the majority were located in gene body regions. By combining these DMPs with published DNA methylation and RNA sequencing data, we detected 26,244 replicated DMPs associated with 1,130 genes whose expression were significantly different in CLL cases. Among these DMEGs, nine low expressed DMEGs were selected with hypermethylated in promoter and hypomethylated in body region, and 83 high expressed DMEGs were selected with both hypomethylated in promoter and body region. The 48 CLL cases were divided into 3 subgroups based on methylation site by SVM algorithm. Over 92% of CpGs associated with B cell subtypes were found in CLL-related DMPs. Conclusion: The DNA methylation pattern was altered across the genome in CLL patients. The methylation of ZAP70, FMOD, and ADAMTS17 was significantly different between CLL cases and controls. Further studies are warranted to confirm our findings and identify the underlying mechanisms through which these methylation markers are associated with CLL.

A Mentored Pathway into the Sciences for Underrepresented Populations: A Newly Crafted Educational Modality for Women Exploring Career Opportunities in Science, Technology, Engineering, Mathematics, and Medicine.

Purpose: The barriers generally facing women wishing to pursue careers in the disciplines of science, technology, engineering, mathematics, and medicine (STEMM) in the United States have been well described. However, additional layers of cultural beliefs and needs may pose further obstructions to women in certain cultural subgroups who wish to enter STEMM. Recognition of the challenges faced by such subgroups is important and culturally sensitive educational and training approaches may be necessary. Methods: We therefore created a science mentoring and education program incorporating the specific requirements of our test group, young Orthodox Jewish women. Our goals were to facilitate their knowledge, skills, and attitudes to embark on a scientific career in biomedicine. Interventions were designed to target physical, intellectual, emotional, and spiritual areas of growth with each intervention crafted to the sensitivity of the women's cultural and religious backgrounds. Results: Over the course of 6 years, we enrolled 59 Orthodox Jewish women, ages 16-20 years. These women spent their summers as part of the larger Summer Internship Program (SIP) at the National Institutes of Health. They participated in cohort sizes ranging from 6 to 26 in dozens of multilevel experiences in the SIP over 6-10 weeks. Participants reported strengthening interest to pursue careers in health care-related fields. Other graduates committed to pursue careers in the general sciences, and other graduate studies. Conclusion: This unique educational platform shows promise for other intersectional groups approaching educational barriers to careers in STEMM.

Expression of the muscle-associated gene MYF6 in hairy cell leukemia.

Hairy cell leukemia (HCL) is a purine analog-responsive B-cell malignancy containing the BRAF V600E mutation, expressing CD22, CD11c, CD103, tartrate resistant acid phosphatase (TRAP) CD25, CD123, and annexin 1A. BRAF V600E and the latter 4 markers are usually absent in the more aggressive and chemoresistant variant HCLv. To evaluate differences between HCL and HCLv, expression microarrays comparing HCL with HCLv were performed for 24694 genes using 47323 probes. Microarray data from 35 HCL and 27 HCLv purified samples showed the greatest HCL-HCLv difference in the muscle-associated gene MYF6, expressed by its 2 probes 18.5- and 10.8-fold higher in HCL than HCLv (p<0.0001). By real-time quantitative PCR (RQ-PCR), 100% of 152 classic HCL samples were MYF6-positive, vs 5 (6%) of 90 blood donors. MYF6-expression was also detected in 18 (35%) of 51 with HCLv, 11 (92%) of 12 with HCL expressing unmutated IGHV4-34, 35 (73%) of 48 with chronic lymphocytic leukemia (CLL), and 1 (8%) of 12 with mantle cell lymphoma. Hypomethylation status of MYF6 supported expression in HCL more than HCLv. Posttreatment blood samples becoming negative by flow cytometry remained MYF6+ by RQ-PCR in 42 (48%) of 87 HCL patients, and MYF6 RQ-PCR could detect 1 HCL in 105 normal cells. MYF6, universally expressed in HCL and in most CLL samples, may be a useful biomarker for these leukemias. Further studies are underway to determine the role of MYF6 in HCL.

Author Correction: Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

Reducing or eliminating persistent disparities in lung cancer incidence and survival has been challenging because our current understanding of lung cancer biology is derived primarily from populations of European descent. Here we show results from a targeted sequencing panel using NCI-MD Case Control Study patient samples and reveal a significantly higher prevalence of PTPRT and JAK2 mutations in lung adenocarcinomas among African Americans compared with European Americans. This increase in mutation frequency was validated with independent WES data from the NCI-MD Case Control Study and TCGA. We find that patients carrying these mutations have a concomitant increase in IL-6/STAT3 signaling and miR-21 expression. Together, these findings suggest the identification of these potentially actionable mutations could have clinical significance for targeted therapy and the enrollment of minority populations in clinical trials.

Dynamics of Genome Alterations in Crohn's Disease-Associated Colorectal Carcinogenesis.

Purpose: Patients with inflammatory bowel diseases, that is, ulcerative colitis and Crohn's disease (CD), face an increased risk of developing colorectal cancer (CRC). Evidence, mainly from ulcerative colitis, suggests that TP53 mutations represent an initial step in the progression from inflamed colonic epithelium to CRC. However, the pathways involved in the evolution of CRC in patients with CD are poorly characterized.Experimental Design: Here, we analyzed 73 tissue samples from 28 patients with CD-CRC, including precursor lesions, by targeted next-generation sequencing of 563 cancer-related genes and array-based comparative genomic hybridization. The results were compared with 24 sporadic CRCs with similar histomorphology (i.e., mucinous adenocarcinomas), and to The Cancer Genome Atlas data (TCGA).Results: CD-CRCs showed somatic copy-number alterations (SCNAs) similar to sporadic CRCs with one notable exception: the gain of 5p was significantly more prevalent in CD-CRCs. CD-CRCs had a distinct mutation signature: TP53 (76% in CD-CRCs vs. 33% in sporadic mucinous CRCs), KRAS (24% vs. 50%), APC (17% vs. 75%), and SMAD3 (3% vs. 29%). TP53 mutations and SCNAs were early and frequent events in CD progression, while APC, KRAS, and SMAD2/4 mutations occurred later. In four patients with CD-CRC, at least one mutation and/or SCNAs were already present in non-dysplastic colonic mucosa, indicating occult tumor evolution.Conclusions: Molecular profiling of CD-CRCs and precursor lesions revealed an inflammation-associated landscape of genome alterations: 5p gains and TP53 mutations occurred early in tumor development. Detection of these aberrations in precursor lesions may help predicting disease progression and distinguishes CD-associated from sporadic colorectal neoplasia. Clin Cancer Res; 24(20); 4997-5011. ©2018 AACR.

Telomere Length and Survival of Patients with Hepatocellular Carcinoma in the United States.

BACKGROUND: Telomere shortening is an important molecular event in hepatocellular carcinoma (HCC) initiation; however, its role in HCC progression and prognosis is less clear. Our study aimed to examine the association of telomere length with survival of patients with HCC. METHODS: We measured telomere length in tumor and adjacent non-tumor tissues from 126 persons with HCC in the United States (U.S.) who were followed for mortality outcomes. Relative telomere length (RTL) was measured by a monochrome multiplex quantitative polymerase chain reaction assay. Multivariable Cox proportional hazards modeling was used to calculate hazard ratios (HRs) and 95% CIs for the association between telomere length and all-cause mortality. We also examined associations between telomere length and patient characteristics using multiple linear regression. RESULTS: During a mean follow-up of 6.0 years, 79 deaths occurred among 114 individuals for whom survival data were available. The ratio of RTL in tumor relative to non-tumor tissue was greater for individuals with regional or distant stage tumors (0.97) than localized stage tumors (0.77), and for individuals with grade III or IV tumors (0.95) than grade II (0.88) or grade I (0.67) tumors. An RTL ratio ≥1 was not associated with survival (HR 0.92, 95% CI 0.55, 1.55) compared to a ratio <1, after adjusting for age at diagnosis, sex, tumor stage and tumor size. Similarly, RTL in the tumor and non-tumor tissue, respectively, were not associated with survival. CONCLUSIONS: This U.S. based study found that telomeres may be longer in more aggressive HCCs. There was no evidence, however, that telomere length was associated with survival of patients with HCC. Future investigations are warranted to clarify the role of telomere length in HCC prognosis.

Human herpesvirus 8--a novel human pathogen.

In 1994, Chang and Moore reported on the latest of the gammaherpesviruses to infect humans, human herpesvirus 8 (HHV-8). This novel herpesvirus has and continues to present challenges to define its scope of involvement in human disease. In this review, aspects of HHV-8 infection are discussed, such as, the human immune response, viral pathogenesis and transmission, viral disease entities, and the virus's epidemiology with an emphasis on HHV-8 diagnostics.

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.

Long-term stability of total RNA in RNAstable® as evaluated by expression microarray.

Storage of labile RNA in laboratories is accomplished through ultra-low freezing of the nucleic acids. This however requires expensive freezers, convenient storage, reliable electrical power, and increased shipping costs, thereby making it a less viable option. Biomatrica (San Diego, CA) has created RNAstable(®), a stabilization reagent that is used to store RNA in a dehydrated state at room temperature (RT) and protects the RNA from degradation. Our objective was to investigate the sequence integrity and suitability of RNA when stored in RNAstable at extended time periods and at varying temperatures through use of Illumina and Agilent RNA expression microarrays. We observed in Bioanalyzer electropherograms that total RNA extracted from 293 cells stored at RT in RNAstable for 4.5 and 11.5 months is similar in quality to RNA stored at -80°C. Illumina mRNA expression array QC metrics and gene expression patterns from RNAstable-protected RNA, in contrast to RNA stored without RNAstable, correlated well with those of freezer controls. Significantly, when RNA was stored in RNAstable at 45°C for 4.5 months, equivalent to 22 months RT storage, RNA quality, microarray probe signal intensities, probe detection rates, and expression profiles remained similar between RNAstable-protected RNA at RT and the -80°C controls. At 10.5 months, miRNA levels were compared among the storage conditions using miRNA expression arrays. Here too we found strong concordance between miRNA expression patterns when total RNA was stored in RNAstable or at -80°C. Further, Bioanalyzer electrophoresis of RNAstable-protected samples stored at RT for a relative total of 33 months or 50.5 months showed comparable integrity scores to those of -80°C controls. We conclude that use of RNAstable holds promise as an effective stabilization reagent for total RNA and should be useful in situations where shipping and storage options are limited resources.

Impact of telomere length on survival in classic and variant hairy cell leukemia.

Telomeres, which protect the ends of chromosomes, are shortened in several hematologic malignancies, often with adverse prognostic implications, but their effect on prognosis of classic and variant hairy cell leukemia (HCL and HCLv) has not been reported. HCL/HCLv genomic DNA from 46 patients was studied by PCR to determine the ratio of telomere to single copy gene number (T/S). T/S was unrelated to diagnosis of HCL or HCLv (p=0.27), but shorter T/S was associated with unmutated immunoglobulin rearrangements (p=0.033) and age above the median at diagnosis (p=0.017). Low T/S was associated with shorter overall survival from diagnosis (OS), particularly T/S <0.655 (p=0.0064, adjusted p=0.019). Shorter OS was also associated with presence of unmutated (p<0.0001) or IGHV4-34+ (p<0.0001) rearrangements, or increasing age (p=0.0002). Multivariable analysis with Cox modeling showed that short T/S along with either unmutated or IGHV4-34+ rearrangements remained associated with reduced OS (p=0.0071, p=0.0024, respectively) after age adjustment. While T/S is relatively long in HCL and the disease usually indolent with excellent survival, shortened telomeres in HCL/HCLv are associated with decreased survival. Shortened T/S could represent a risk factor needing further investigation/intervention to determine if non-chemotherapy treatment options, in addition to or instead of chemotherapy, might be particularly useful.

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers.

INTRODUCTION: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. METHODS: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. RESULTS: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10). All associations were confirmed in two independently collected cohorts. CONCLUSION: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

Real-time PCR provides improved detection and titer determination of bacteriophage.

The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.

Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen.

Presently, the assay that attains maximal sensitivity and dynamic range of HIV-1 viral copy number (50 copies per milliliter) is nucleic acid amplification of HIV RNA in plasma. Enzyme-linked immunosorbent assay (ELISA) methods for quantification of HIV-1 p24 antigen have been relatively insensitive. In this report, we show data that indicate real-time immuno-polymerase chain reaction (IPCR), a combination of the ELISA and PCR techniques, is more sensitive for HIV-1 p24 antigen detection than other currently reported methods. When derived from an IPCR standard curve, a dose response was observed from patient samples with known viral loads diluted within a 3-log range (1.68-6,514 viral RNA copies per milliliter). IPCR detected 42% (22/52) of patient samples that had fewer than 50 viral RNA copies per milliliter by reverse transcriptase-PCR. IPCR shows the potential to become the most analytically sensitive test available for determination of HIV-1 viral load by the detection of HIV-1 p24 antigen.

Hepatitis C virus transfusion-transmitted infection in Brazilian cardiac surgery patients.

In the past two decades, major improvements in blood safety have been achieved, particularly for HIV and hepatitis C virus (HCV). A prospective study was carried out between 1996 and 1999 in Brazil to determine the incidence of post-transfusion infection in surgery patients caused by HCV. One hundred sixty-four patients who received a blood transfusion during cardiac surgery were followed for six months and blood samples collected before and after surgery were assessed to investigate HCV infection. Alanine aminotransferase levels were serially determined, as well as clinical data related to hepatitis. Prior to surgery, HCV infection was detected by anti-HCV ELISA III in 6 patients. Any post-surgical samples which were positive by a third generation ELISA test were confirmed by immunoblot and reverse-transcription polymerase chain reaction (RT-PCR), as were the pre-transfusion samples to exclude pre-transfusion HCV infection not detected by ELISA screening. Results indicated that one patient who was previously considered negative for HCV antibody in the pre-surgical sample was later found to be positive for HCV by RT-PCR in that sample. Seroconversion for HCV antibody after surgery was observed in two patients, one of them with clinical hepatitis; their genotypes were 1a and 1b. The overall prevalence of HCV infection was 4.26% (7/164) and the incidence rate of HCV infection after surgery was 1.27% (2/157). This study shows a high rate of HCV infection acquired post-transfusion in a cohort of surgery patients in Brazil and suggests that better screening methods such as viral RNA assessment may be effective in lowering this rate.

Genetic and epigenetic analysis of monozygotic twins discordant for testicular cancer.

Despite the notion that monozygotic (identical) twins share 100% identical genetic information, genetic differences among monozygotic twin pairs do occur and can be explained by mechanisms occurring during post-zygotic events. Despite such twins being fundamentally "identical", these post-zygotic genetic changes may give rise to phenotypic differences and genetic diseases. Consequently, studies of monozygotic twin pairs discordant for specific genetic diseases represent an important tool for the identification of disease genes. We used array comparative genomic hybridization (aCGH) and methylation arrays to search for genetic and epigenetic differences in blood drawn from four monozygotic twin pairs discordant for testicular germ cell tumors. No consistent differences were identified. A larger twin study would be required to achieve confident discovery of very subtle differences between monozygotic twins discordant for testicular germ cell tumors.