HER2, chromosome 17 polysomy and DNA ploidy status in breast cancer; a translational study.

Breast cancer treatment depends on human epidermal growth factor receptor-2 (HER2) status, which is often determined using dual probe fluorescence in situ hybridisation (FISH). Hereby, also loss and gain of the centromere of chromosome 17 (CEP17) can be observed (HER2 is located on chromosome 17). CEP17 gain can lead to difficulty in interpretation of HER2 status, since this might represent true polysomy. With this study we investigated whether isolated polysomy is present and how this effects HER2 status in six breast cancer cell lines and 97 breast cancer cases, using HER2 FISH and immunohistochemistry, DNA ploidy assessment and multiplex ligation dependent probe amplification. We observed no isolated polysomy of chromosome 17 in any cell line. However, FISH analysis did show CEP17 gain in five of six cell lines, which reflected gains of the whole chromosome in metaphase spreads and aneuploidy with gain of multiple chromosomes in all these cases. In patients' samples, gain of CEP17 indeed correlated with aneuploidy of the tumour (91.1%; p < 0.001). Our results indicate that CEP17 gain is not due to isolated polysomy, but rather due to widespread aneuploidy with gain of multiple chromosomes. As aneuploidy is associated with poor clinical outcome, irrespective of tumour grade, this could improve future therapeutic decision making.

Chromosome 17q21 Genes ORMDL3 and GSDMB in Asthma and Immune Diseases.

Chromosome 17q21 contains a cluster of genes including ORMDL3 and GSDMB, which have been highly linked to asthma in genome-wide association studies. ORMDL3 is localized to the endoplasmic reticulum and regulates downstream pathways including sphingolipids, metalloproteases, remodeling genes, and chemokines. ORMDL3 inhibits serine palmitoyl-CoA transferase, the rate-limiting enzyme for sphingolipid biosynthesis. In addition, ORMDL3 activates the ATF6α branch of the unfolded protein response which regulates SERCA2b and IL-6, pathways of potential importance to asthma. The SNP-linking chromosome 17q21 to asthma is associated with increased ORMDL3 and GSDMB expression. Mice expressing either increased levels of human ORMDL3, or human GSDMB, have an asthma phenotype characterized by increased airway responsiveness and increased airway remodeling (increased smooth muscle and fibrosis) in the absence of airway inflammation. GSDMB regulates expression of 5-LO and TGF-ß1 which are known pathways involved in the pathogenesis of asthma. GSDMB is one of four members of the GSDM family (GSDMA, GSDMB, GSDMC, and GSDMD). GSDMD (located on chromosome 8q24 and not linked to asthma) has emerged as a key mediator of pyroptosis. GSDMD is a key component of the NLPR3 inflammasome and is required for its activation. GSDMD undergoes proteolytic cleavage by caspase-1 to release its N-terminal fragment, which in turn mediates pyroptosis and IL-1ß secretion. Chromosome 17q21 has not only been linked to asthma but also to type 1 diabetes, inflammatory bowel disease, and primary biliary cirrhosis suggesting that future insights into the biology of genes located in this region will increase our understanding of these diseases.

Detection of inherited chromosomally integrated HHV-6 (ciHHV-6) in a marker chromosome.

OBJECTIVES: Inherited chromosomally integrated human herpesvirus-6 (ciHHV-6) is characterised by the complete HHV-6 genome integration into the host germ line genome and is vertically transmitted with a Mendelian inheritance. By now, the only relationship between ciHHV-6 and diseases seems to be with angina pectoris. METHODS: We report a case of an 82-year-old man diagnosed with diffuse large B-cell lymphoma (DLBCL) on October 2014. To substantiate the suspicion of ciHHV-6, we analysed peripheral blood mononuclear cells, bone marrow biopsy and pleural effusion-derived mesothelial cells with PCR, RT-PCR and FISH. RESULTS: Virological routine screening by PCR showed the absence of HHV-8 and EBV infections, while the presence of HHV-6 DNA (ie, U22, U42 and U94 HHV-6 genes), with a viral load of about 1.0 genome per cell, strongly suggests ciHHV-6. The RT-PCR showed the positivity only for the immediate-early U94, at low levels of transcription (100±15 transcripts/1 µg RNA). FISH analysis reported a case of inherited ciHHV-6 in 17p chromosome region and, for the first time, in a marker chromosome. CONCLUSIONS: This is the first case of inherited ciHHV-6 in a marker chromosome, possibly elucidating the role of this abnormality in the biology of DLBCL.

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.

Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions.

Large deletions encompassing the NF1 gene and its flanking regions belong to the group of genomic disorders caused by copy number changes that are mediated by the local genomic architecture. Although nonallelic homologous recombination (NAHR) is known to be a major mutational mechanism underlying such genomic copy number changes, the sequence determinants of NAHR location and frequency are still poorly understood since few high-resolution mapping studies of NAHR hotspots have been performed to date. Here, we have characterized two NAHR hotspots, PRS1 and PRS2, separated by 20 kb and located within the low-copy repeats NF1-REPa and NF1-REPc, which flank the human NF1 gene region. High-resolution mapping of the crossover sites identified in 78 type 1 NF1 deletions mediated by NAHR indicated that PRS2 is a much stronger NAHR hotspot than PRS1 since 80% of these deletions exhibited crossovers within PRS2, whereas 20% had crossovers within PRS1. The identification of the most common strand exchange regions of these 78 deletions served to demarcate the cores of the PRS1 and PRS2 hotspots encompassing 1026 and 1976 bp, respectively. Several sequence features were identified that may influence hotspot intensity and direct the positional preference of NAHR to the hotspot cores. These features include regions of perfect sequence identity encompassing 700 bp at the hotspot core, the presence of PRDM9 binding sites perfectly matching the consensus motif for the most common PRDM9 variant, specific pre-existing patterns of histone modification and open chromatin conformations that are likely to facilitate PRDM9 binding.

Meta-analysis of genome-wide association studies identifies two loci associated with circulating osteoprotegerin levels.

Osteoprotegerin (OPG) is involved in bone homeostasis and tumor cell survival. Circulating OPG levels are also important biomarkers of various clinical traits, such as cancers and atherosclerosis. OPG levels were measured in serum or in plasma. In a meta-analysis of genome-wide association studies in up to 10 336 individuals from European and Asian origin, we discovered that variants >100 kb upstream of the TNFRSF11B gene encoding OPG and another new locus on chromosome 17q11.2 were significantly associated with OPG variation. We also identified a suggestive locus on chromosome 14q21.2 associated with the trait. Moreover, we estimated that over half of the heritability of OPG levels could be explained by all variants examined in our study. Our findings provide further insight into the genetic regulation of circulating OPG levels.

The clinical impact and molecular biology of del(17p) in multiple myeloma treated with conventional or thalidomide-based therapy.

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P < 0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group.

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions.

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.

Tissue microarray for routine clinical breast biomarker analysis. The British Columbia Cancer Agency 2008 experience.

Clinical use of tissue microarrays for immunohistochemical analysis of breast biomarkers, namely estrogen receptor, progesterone receptor, and HER2, was instituted in our laboratory in 2008. The method has proved reliable and cost-effective. We report the results of the initial year of testing with this method.

Comparison of five DNA quantification methods.

Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than expected based on the information by the manufacturers. UV spectrometry, SYBR-Green dye staining, slot blot and RB1 rt-PCR gave 39, 27, 11 and 12%, respectively, higher concentrations than expected based on the manufacturers' information. The DNA preparations were quantified using the Quantifiler Human DNA Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use with the Quantifiler Human DNA Quantification kit. The possible reasons for the problem are discussed and a solution is suggested. The results emphasise the need for standard reference DNA material and standard methods for DNA quantification.

Cloning and characterization of the human BAZF gene, a homologue of the BCL6 oncogene.

The BAZF gene has recently been identified as a novel homologue of the BCL6 oncogene. Here we cloned the human BAZF gene using murine BAZF as a probe. The predicted amino acid sequence was 91% identical to that of murine BAZF. The BTB/POZ and zinc finger domains were almost completely conserved between human and murine BAZF. Fluorescence in situ hybridization analysis revealed that the human BAZF gene is located on chromosome 17p13.1. Although expression of human BAZF mRNA was ubiquitously detected in human tissues, abundant expression was detected in heart and placenta. BAZF mRNA was expressed in some immature B cell lines and erythroleukemia cell lines. The expression in a human erythroleukemia cell line, HEL cells, was upregulated during megakaryocytic differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate. These expression patterns of BAZF mRNA suggest that BAZF may regulate differentiation in stages or lineages that are different from those regulated by BCL6.

Characterization of recurrent homogeneously staining regions in 72 breast carcinomas.

Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.

Assignment of the possible HTLV receptor gene to chromosome 17q21-q23.

We have determined the HTLV (Human T-cell leukemia virus) receptor localization more precisely on the human chromosome 17. Based on the fact that HTLV infection induces syncytium formation of infected cells as a result of interaction between the viral envelope and viral receptor, we performed the sensitive biological assay using recombinant vaccinia expression system. Our results from the induced syncytium pattern of the somatic hybrid cell lines with different deletions indicated that the HTLV receptor gene may reside from q21 to q23 on the long arm of the human chromosome 17.

Enumeration of interphase chromosomes. Comparison of visual in situ hybridization and confocal fluorescence in situ hybridization.

OBJECTIVE: To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites. STUDY DESIGN: Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software. RESULTS: The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections. CONCLUSION: Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.

Molecular genetic alterations of chromosome 17 and p53 nuclear overexpression in human bladder cancer.

We set out to define the alterations of chromosome 17 in human bladder tumors and to correlate p53 nuclear over-expression with 17p deletions in those neoplasms. We studied 60 bladder tumors by restriction fragment-length polymorphism analysis directed at five different loci on chromosome 17. The same tumors were studied with a panel of mouse monoclonal antibodies (PAb1801, PAb240, and PAb1620) to mutant and wild-type p53 proteins using immunohistochemistry. Deletion of 17p correlated with grade (p = 0.039), stage (p = 0.004), and the presence of vascular invasion (p = 0.056). None of the pathologic parameters correlated with 17q deletions. p53 nuclear overexpression correlated with grade (p = 0.027), stage (p = 0.008), vascular invasion (p = 0.021), and the presence of nodal metastases (p = 0.007). In superficial (Ta) lesions, 17p was not deleted, whereas 55% of T1 and T2-T4 tumors showed a loss of heterozygosity. Mutations of p53 as detected by immunohistochemistry were seen in superficial as well as invasive tumors, whereas loss of heterozygosity was seen only in invasive tumors. A strong correlation was found between the presence of mutation and the loss of heterozygosity of the remaining allele (p = 0.0003). Additional follow-up and further studies are required to better define the role of p53 nuclear overexpression and 17p deletions as markers of tumor progression in human bladder cancer.