High XIST and Low 53BP1 Expression Predict Poor Outcome after High-Dose Alkylating Chemotherapy in Patients with a BRCA1-like Breast Cancer.

In previous studies, high expression of XIST and low expression of 53BP1 were respectively associated with poor systemic therapy outcome in patients and therapy resistance in BRCA1-deficient mouse tumor models, but have not been evaluated in BRCA1-deficient patients. Previously, we demonstrated that classifying breast cancer copy number profiles as BRCA1-like or non-BRCA1-like identified patients enriched for defects in BRCA1 that benefit from high-dose (HD) alkylating chemotherapy compared with a conventional standard regimen. We investigated whether XIST and 53BP1 expression predicted poor outcome of HD chemotherapy within 28 BRCA1-like patients from a trial randomizing between HD [4 cycles 5-fluorouracil, epirubicin, cyclophosphamide (FEC) followed by 1 cycle HD carboplatin, thiotepa, cyclophosphamide] or conventional chemotherapy (5 cycles FEC), for which both XIST and 53BP1 statuses were available. High RNA expression of XIST (n = 5) and low protein expression of 53BP1 (n = 3) expression did not coincide. Patients with either one had poor outcome after treatment with HD chemotherapy, whereas patients with low expression of XIST and high expression of 53BP1 derived substantial benefit of this regimen on recurrence-free survival, disease-free survival, and overall survival, corroborating preclinical findings. XIST and 53BP1 may be predictive biomarkers in BRCA1-like breast cancer.

Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy.

BACKGROUND: In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation-dependent probe amplification (MLPA) technology. STUDY DESIGN AND METHODS: We analyzed 103 random and 150 selected samples to validate the specificity of the blood-MLPA assay that is able to determine the presence, absence, and copy number of 48 blood group and 112 variant alleles of 18 blood group systems. A total of 4038 serologic typing results, including 52 different antigens, were available for these samples. RESULTS: In 4018 (99.5%) of the 4038 serologic typing results the predicted phenotypes by the blood-MLPA were in concordance with serologic typing. Twenty discordant results were due to false-positive serologic results (n = 2), false-negative serologic results (n = 1), inability of routine serologic typing to detect variant antigens (n = 14), or false-positive prediction from the blood-MLPA due to the presence of a null allele (n = 3). CONCLUSION: The blood-MLPA reliably predicts the presence or absence of blood group antigens, including almost all clinically relevant blood group antigens, except ABO, in patients and donors. Furthermore, it is the first assay that determines copy numbers of blood group alleles in the same test. It even provides more detailed and accurate information than serologic typing, because most variant alleles are immediately recognized. Since only standard laboratory equipment is needed, this assay finally offers the possibility to comprehensively type recipients and makes extensive matching for selected patients groups more feasible.

Identification of Salmonella enterica serovar Typhi genotypes by use of rapid multiplex ligation-dependent probe amplification.

Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic.

Prospective evaluation of three rapid diagnostic tests for diagnosis of human leptospirosis.

BACKGROUND: Diagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively. METHODOLOGY: During 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1(st) submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness. RESULTS: The overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1(st) submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB). CONCLUSIONS: All three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.

RHD and RHCE variant and zygosity genotyping via multiplex ligation-dependent probe amplification.

BACKGROUND: The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity. STUDY DESIGN AND METHODS: We analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases. RESULTS: In 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized. CONCLUSION: The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.

Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors.

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy.

Human heterochromatin proteins form large domains containing KRAB-ZNF genes.

Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1beta) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1-4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3' ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family.

Whole-genome views of chromatin structure.

DNA in eukaryotes is packed into chromatin. The basic component of chromatin is the nucleosome consisting of DNA wrapped around a histone octamer. Inside the cell nucleus, chromatin is folded into higher-order structures through various mechanisms, including repositioning of nucleosomes along the DNA, packing of nucleosomes into more condensed 3-dimensional configurations, looping of chromatin fibres, and tethering of chromosomal regions to nuclear structures. Over the past few years, new microarray-based methods have been developed for the genome-wide mapping of various aspects of chromatin structure. These methods are beginning to provide insights into the different types of chromatin and the architectural principles that govern the 3-dimensional organisation of the genome inside the nucleus.

C-erbB2, p27 and G1/S aberrations in human primary breast cancer.

BACKGROUND: C-erbB2 is overexpressed in approximately one-fourth of human breast cancers. In spite of numerous reports suggesting a connection of c-erbB2 overexpression with cell cycle regulation through p27, D cyclins and c-myc, the relationship between c-erbB2 and proliferation through de-regulation of the pRb pathway in primary breast cancer has not been fully clarified. MATERIALS AND METHODS: For this purpose we compared the expression of c-erbB2 in a total of 105 primary breast tumours with a variety of cell cycle proteins and clinical parameters. RESULTS: C-erbB2 was strongly correlated with down-regulation of p27 and overexpression of c-erbB2 was, as expected, associated with poor survival. However, there was no correlation with proliferation. There was, nevertheless, an association between c-erbB2 and proliferation in certain subtypes of breast cancer, as in oestrogen-receptor-positive tumours, tumours with high cyclin D1, and in tumours with an overall linear pRb pathway, i.e. tumours with a preserved linearity between cyclin D1, pRb phosphorylation and proliferation. CONCLUSION: Our results suggest that c-erbB2 may have alternative functions in different subtypes of breast cancer, and further stress that c-erbB2, in addition to promoting proliferation, also functions through other mechanisms in breast cancer.

The cyclin D1 high and cyclin E high subgroups of breast cancer: separate pathways in tumorogenesis based on pattern of genetic aberrations and inactivation of the pRb node.

In an attempt to identify subtypes of breast cancer and pinpoint patterns of cell cycle regulatory defects associated with clinical behaviour, proliferation and other transformation associated events, a multitude of cell cycle regulatory proteins were analysed in a material of 113 primary breast cancers. Increased proliferation was observed in two different scenarios; (1) with high cyclin D1 and elevated retinoblastoma protein (pRb) phosphorylation, (cyclin D1(high) tumours) or (2) with high cyclin E protein but low cyclin D1 and lack of corresponding pRb phosphorylation (cyclin E(high) tumours) indicative of an interrupted pRb pathway. Characteristic for cyclin E(high) tumours were further defects in p53, p27 and bcl-2, while c-erbB2 overexpression and c-myc amplification was found in both cyclin D1(high) and E(high) tumours. Using transfected cell lines overexpressing cyclin E, cyclin E(high) and D1(high) tumours were mimicked and the cyclin D1(high) cell line normalized the cyclin E kinase activity by an induction and redirection of p21 and p27 to the cyclin E complex whereas cyclin E(high) cell lines obtained increased kinase activity without redirection of inhibitors. Based on differences in genetic aberrations as well as function of the pRb node we therefore propose a model in which cyclin D1(high) and cyclin E(high) tumours represent two alternative mechanisms to inactivate the pRb pathway and thereby achieve unrestrained growth in the tumorogenesis of breast cancer.