Chromosomal microarray provides enhanced targetable gene aberration detection when paired with next generation sequencing panel in profiling lung and colorectal tumors.

The development of targeted therapies based on specific genomic alterations has altered the treatment and management of lung and colorectal cancers. Chromosomal microarray (CMA) has allowed identification of copy number variations (CNVs) in lung and colorectal cancers in great detail, and next-generation sequencing (NGS) is used extensively to analyze the genome of cancers for molecular subtyping and use of molecularly guided therapies. The main objective of this study was to evaluate the utility of combining CMA and NGS for a comprehensive genomic assessment of lung and colorectal adenocarcinomas, especially for detecting drug targets. We compared the results from NGS and CMA data from 60 lung and 51 colorectal tumors. From CMA analysis, 33% were amplified, 89% showed gains, 75% showed losses and 41% demonstrated loss of heterozygosity; pathogenic variants were identified in 81% of colon and 67% lung specimens through NGS. KRAS mutations commonly occurred with loss in TP53 and there was significant loss of BRCA1 and NF1 among male patients with lung cancer. For clinically actionable targets, 23% had targetable CNVs when no pathogenic variants were detected by NGS. The data thus indicate that combining the two approaches provides significant benefit in a routine clinical setting not available by NGS alone.

Molecular characterisation of formalin-fixed paraffin-embedded (FFPE) breast tumour specimens using a custom 512-gene breast cancer bead array-based platform.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT-PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel. METHODS: The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data. RESULTS: Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R²=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R²=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes. CONCLUSION: We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.

Associations of progesterone receptor polymorphisms with age at menarche and menstrual cycle length.

BACKGROUND: Age at menarche and menstrual cycle characteristics are indicators of endocrine function and may be risk factors for diseases such as reproductive cancers. The progesterone receptor gene (PGR) has been identified as a candidate gene for age at menarche and menstrual function. METHODS: Women office workers ages 19-41 self-reported age at menarche and participated in a prospective study of menstrual function and fertility. First-morning urine was used as the DNA source. 444 women were genotyped for a functional variant in PGR, rs1042838 (Val660Leu), and 264 women were also genotyped for 29 other SNPs across the extended gene region. RESULTS: Genetic variation across PGR was associated with age at menarche using a global score statistic (p = 0.03 among non-Hispanic whites). Women carrying two copies of the Val660Leu variant experienced menarche 1 year later than women carrying one or no copies of the variant (13.6 ± 0.5 vs. 12.6 ± 0.1; p = 0.03). The Val660Leu variant was also associated with decreased odds of short menstrual cycles (17-24 days) (OR, 95% CI: 0.54 [0.36, 0.80]; p = 0.002). CONCLUSION: Genetic variation in PGR was associated with age at menarche and menstrual cycle length in this population. Further investigation of these associations in a replication dataset is warranted.

Prostate cancer genes associated with TMPRSS2-ERG gene fusion and prognostic of biochemical recurrence in multiple cohorts.

BACKGROUND: Recent studies have indicated that prostate cancer patients with the TMPRSS2-ERG gene fusion have a higher risk of recurrence. To identify markers associated with TMPRSS2-ERG fusion and prognostic of biochemical recurrence, we analysed a cohort of 139 men with prostate cancer for 502 molecular markers. METHODS: RNA from radical prostatectomy tumour specimens was analysed using cDNA-mediated, annealing, selection, extension and ligation (DASL) to determine mRNAs associated with TMPRSS2-ERG T1/E4 fusion and prognostic of biochemical recurrence. Differentially expressed mRNAs in T1/E4-positive tumours were determined using significance analysis of microarrays (false discovery rate (FDR) <5%). Univariate and multivariate Cox regression determined genes, gene signatures and clinical factors prognostic of recurrence (P-value <0.05, log-rank test). Analysis of two prostate microarray studies (GSE1065 and GSE8402) validated the findings. RESULTS: In the 139 patients from this study and from a 455-patient Swedish cohort, 15 genes in common were differentially regulated in T1/E4 fusion-positive tumours (FDR <0.05). The most significant mRNAs in both cohorts coded ERG. Nine genes were found prognostic of recurrence in this study and in a 596-patient Minnesota cohort. A molecular recurrence score was significant in prognosticating recurrence (P-value 0.000167) and remained significant in multivariate analysis of a mixed clinical model considering Gleason score and TMPRSS2-ERG fusion status. CONCLUSIONS: TMPRSS2-ERG T1/E4 fusion-positive tumours had differentially regulated mRNAs observed in multiple studies, the most significant one coded for ERG. Several mRNAs were consistently associated with biochemical recurrence and have potential clinical utility but will require further validation for successful translation.

Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.

Identification and characterization of cvHsp. A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues.

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin.

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p.

A panel of 93 radiation-reduced hybrids have been screened using PCR amplification and oligonucleotide primers for sequence-tagged sites (STSs) specific for 114 single-copy loci mapping to the short arm of chromosome 9. An x-ray dose of 6,000 rads gave an average retention frequency of approximately 23%. We have constructed a framework map containing 31 markers ordered by analyzing coretention patterns, with support for the order greater than 1,000:1. In addition, we have placed the remaining markers which could not be mapped to a single interval with this support to a range of intervals on the framework map. The STS oligonucleotide primers used in the construction of the radiation hybrid (RH) map have been used to isolate and order yeast artificial chromosomes (YACs) assigned to 9p identified from the CEPH megaYAC library. Eighty-nine STS markers have screened positive with at least one YAC. A total of 88 individual YACs (with an average size of 0.9 MB) have been placed on the map in a series of contigs and in some cases mapped cytogenetically by fluorescence in situ hybridization. Additionally, the YAC information has been used in conjunction with the RH framework placements to generate an integrated map containing 65 loci including 51 uniquely positioned markers, with an average resolution of 0.79 Mb.

Construction of a radiation hybrid map of chromosome 9p.

A radiation hybrid panel has been constructed for chromosome 9 using the somatic cell hybrid GM10611 as the donor cell line fused to the hamster cell line A23. The hybrid GM10611 was characterized by fluorescence in situ hybridization and reverse painting onto spreads of normal human metaphase chromosomes; it contains human chromosome 9 as the only cytogenetically detectable human material. GM10611 was irradiated with 6000 rads of X rays prior to fusion, a total of 93 independent clones were selected, and frozen stocks and DNA were prepared from each clone. These clones were screened by PCR amplification with oligonucleotide primers for sequence-tagged sites specific for 50 single-copy loci mapping to the short arm of chromosome 9. The average retention frequency of these hybrids was approximately 23%. The markers were ordered into a framework map by analyzing coretention patterns, minimizing the number of obligatory chromosome breaks, and finally confirming the order by maximum likelihood methods. A framework map ordering 27 markers with odds greater than 1000:1 was constructed. A further 16 markers that could not be uniquely placed on the map with the required support were positioned within a range of adjacent intervals.

Isolation and mapping of three new polymorphic markers to chromosomes 3, 20 and 21.

Screening of single human chromosome plasmid libraries using a digoxygenin labelled (AAAT)15 oligonucleotide probe led to the identification of several positive clones. DNA sequence analysis of these was carried out and showed the presence of a number of simple DNA repeats. Oligonucleotide primers were designed from the sequences flanking these repeats and tested in PCR amplification reactions of human genomic DNA. Three of the markers tested were shown to be polymorphic with heterozygosities ranging from 40% to 69%. The markers were assigned to chromosomes using a panel of monochromosomal somatic cell hybrids combined with linkage analysis using DNA from the CEPH panel of families. The markers designated (AAAT)11, (AAAT)12 and (CA)19 were thus assigned to chromosomes 3, 21 and 20 respectively.

Chromosomal assignment of 79 cDNAs from a range of human tissues.

Single-pass DNA sequencing of cDNAs selected at random from a human mixed tissue cDNA library have generated a series of more than 2000 expressed sequence tags. One hundred twenty-eight unique cDNA fragments with little or no known protein or nucleic acid homologies have been selected for further analysis. Oligonucleotide primer pairs have been designed from the cDNAs and used in PCR amplification in combination with genomic DNA from a panel of monochromosomal somatic cell hybrids. This has allowed us to assign 70 of these transcribed genes to a single chromosome, and a further 9 have been located on two or three chromosomes. Additionally, 3 cDNAs contain short tandem repeats that may allow them to be further localized by linkage analysis.

Development of a panel of monochromosomal somatic cell hybrids for rapid gene mapping.

We have assembled a panel of monochromosomal somatic cell hybrids for use in gene mapping. DNA from each individual hybrid was used as a probe on normal human metaphases to identify the human chromosome and any fragments by reverse painting. To test the efficiency of the panel PCR amplification of DNA from the monochromosomal somatic cell hybrid panel was used in combination with human specific oligonucleotide primers to assign alpha-catenin (CTNNA1) and p21/WAF1 to chromosomes 5 and 6 respectively. These genes were localized further using hybrids containing specific translocations to 5q11-qter and 6p21 respectively. We also developed primers to enable us to assign 17 ESTs sequenced by the HGMP Resource Centre. The hybrid panel was developed with support of the UK HGMP and the DNA is available to all registered users.

Bombesin treatment enhances vasopressin receptors in Swiss 3T3 cells.

Cells possess receptors for multiple different peptides that regulate a wide spectrum of biological processes. Although examples of homologous and heterologous downregulation have been reported, relatively little is known about the interaction between different peptides in modulating cellular activities. Here we demonstrate that pretreatment of Swiss 3T3 fibroblasts with 10 nM bombesin for 48 h enhanced the 45Ca2+ efflux acutely stimulated by vasopressin. The effect was not reciprocal, since preincubation with vasopressin did not affect the bombesin-stimulated Ca2+ efflux. Measurement of displaceable [3H] vasopressin binding demonstrated that bombesin pretreatment increases the hormonal binding by 3.8 +/- 0.2-fold (SE; n = 14) measured at 37 degrees C or at 4 degrees C. Scatchard analysis at 4 degrees C indicated that the increased binding reflects an increase in the number of vasopressin receptors without any significant effect on the apparent affinity of binding. Furthermore, addition of cycloheximide completely prevented the increase in [3H] vasopressin binding induced by bombesin. We conclude that long-term bombesin pretreatment induces heterologous enhancement of vasopressin responsiveness by increasing the number of membrane receptors.

Homologous desensitization of bombesin-induced increases in intracellular Ca2+ in quiescent Swiss 3T3 cells involves a protein kinase C-independent mechanism.

Addition of bombesin to Swiss 3T3 cells causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i), which is followed by desensitization to a subsequent addition of the peptide. The concentrations of bombesin used to study this acute cellular desensitization (0.1-0.5 nM) did not deplete the intracellular pool of Ca2+ released by inositol(1,4,5)trisphosphate, as shown by addition of vasopressin after consecutive additions of bombesin. Two lines of evidence support the conclusion that activation of protein kinase C (PKC) does not mediate the acute homologous desensitization of Ca2+ responses induced by bombesin. First, long-term treatment (48 h) of Swiss 3T3 cells with phorbol 12,13-dibutyrate (PDB) to deplete PKC did not prevent homologous desensitization. The responses to second additions of bombesin at 0.1, 0.25, and 0.5 nM were 42%, 26% and 11% of the initial responses, respectively. Second, the PKC inhibitor GF 109203X did not alter homologous desensitization at concentrations that completely prevented the inhibition of Ca2+ mobilization induced by PDB and blocked PDB-mediated phosphorylation of the prominent PKC substrate 80K/MARCKS. We conclude that acute homologous desensitization of Ca2+ responses induced by bombesin occurs through a PKC-independent mechanism.

Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.