Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer.

Familial clustering studies indicate that breast cancer risk has a substantial genetic component. To identify new breast cancer risk variants, we genotyped approximately 300,000 SNPs in 1,600 Icelandic individuals with breast cancer and 11,563 controls using the Illumina Hap300 platform. We then tested selected SNPs in five replication sample sets. Overall, we studied 4,554 affected individuals and 17,577 controls. Two SNPs consistently associated with breast cancer: approximately 25% of individuals of European descent are homozygous for allele A of rs13387042 on chromosome 2q35 and have an estimated 1.44-fold greater risk than noncarriers, and for allele T of rs3803662 on 16q12, about 7% are homozygous and have a 1.64-fold greater risk. Risk from both alleles was confined to estrogen receptor-positive tumors. At present, no genes have been identified in the linkage disequilibrium block containing rs13387042. rs3803662 is near the 5' end of TNRC9 , a high mobility group chromatin-associated protein whose expression is implicated in breast cancer metastasis to bone.

Parental origin of sequence variants associated with complex diseases.

Effects of susceptibility variants may depend on from which parent they are inherited. Although many associations between sequence variants and human traits have been discovered through genome-wide associations, the impact of parental origin has largely been ignored. Here we show that for 38,167 Icelanders genotyped using single nucleotide polymorphism (SNP) chips, the parental origin of most alleles can be determined. For this we used a combination of genealogy and long-range phasing. We then focused on SNPs that associate with diseases and are within 500 kilobases of known imprinted genes. Seven independent SNP associations were examined. Five-one with breast cancer, one with basal-cell carcinoma and three with type 2 diabetes-have parental-origin-specific associations. These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. Furthermore, we observed a novel association between the SNP rs2334499 at 11p15 and type 2 diabetes. Here the allele that confers risk when paternally inherited is protective when maternally transmitted. We identified a differentially methylated CTCF-binding site at 11p15 and demonstrated correlation of rs2334499 with decreased methylation of that site.

A Breast Cancer Candidate Locus at 6q Narrowed to 6q15-q21.

Although a number of high-risk breast cancer genes have been identified, including BRCA1 and BRCA2, the risk profile of many high-risk families cannot be explained using known breast cancer genes. Previously, we have shown strong indications of new breast cancer risk loci at chromosomes 2p, 6q, and 14q in a family of six generations including 10 breast cancer cases. In this study, we identified and traced four new family branches descending from siblings of the parents in the top generation of the studied family. One distantly related branch included four breast cancer cases, two of whom were diagnosed at age < 45 years. DNA samples from the cases were typed at selected polymorphic markers from all three chromosome loci, to test identical origin of the haplotypes. All four cases were shown to segregate a common 6q haplotype with a region identical to the previously identified 6q haplotype. The data strongly support a new breast cancer locus at 6q, and narrow it down to a 17 MB interval at 6q15-q21.

Gene-based burden tests of rare germline variants identify six cancer susceptibility genes.

Discovery of cancer risk variants in the sequence of the germline genome can shed light on carcinogenesis. Here we describe gene burden association analyses, aggregating rare missense and loss of function variants, at 22 cancer sites, including 130,991 cancer cases and 733,486 controls from Iceland, Norway and the United Kingdom. We identified four genes associated with increased cancer risk; the pro-apoptotic BIK for prostate cancer, the autophagy involved ATG12 for colorectal cancer, TG for thyroid cancer and CMTR2 for both lung cancer and cutaneous melanoma. Further, we found genes with rare variants that associate with decreased risk of cancer; AURKB for any cancer, irrespective of site, and PPP1R15A for breast cancer, suggesting that inhibition of PPP1R15A may be a preventive strategy for breast cancer. Our findings pinpoint several new cancer risk genes and emphasize autophagy, apoptosis and cell stress response as a focus point for developing new therapeutics.

The risk allele of SNP rs3803662 and the mRNA level of its closest genes TOX3 and LOC643714 predict adverse outcome for breast cancer patients.

BACKGROUND: The minor allele of SNP rs3803662 has been shown to correlate with increased breast cancer risk and with lower expression of TOX3. The SNP is closely located to TOX3 residing within an uncharacterised gene LOC643714. The aim of the study was to examine the association of the risk allele with expression of TOX3 and LOC643714, and of mRNA levels and genotype with clinical and pathological characteristics. METHODS: The SNP was genotyped in DNA isolated from blood and normal tissue from 160 breast cancer patients and mRNA levels were measured by microarrays and quantitative real-time (qRT)-PCR in breast tumours. Association with clinical and pathological characteristics was analysed by parametric tests. RESULTS: An association of the risk allele of rs3803662 with lower TOX3 expression was confirmed in oestrogen receptor (ER) positive tumours. It was more often observed in lobular tumours (p = 0.04), and carriers of the risk allele who had been diagnosed with luminal A tumours had shorter overall survival (OS) than carriers of the non-risk allele (p = 0.01). Positive correlation between the mRNA levels of TOX3 and LOC643714 was observed (r = 0.44 and p < 0.001). Association analysis with tumour pathology showed that low TOX3 and LOC643714 expression correlated with high Ki67 levels (p = 0.026 and p = 0.002) and the basal subtype (p < 0.001 and p < 0.001), whereas high expression correlated with ER (p = 0.004 and p < 0.001) and progesterone receptor (PgR) (p = 0.005 and p < 0.001) expression. Furthermore, high TOX3 and LOC643714 correlated with positive lymph nodes (p < 0.001 and p = 0.01). Patients with ER positive tumours and high levels of TOX3 mRNA had shorter overall- and distant metastasis free-survival (p = 0.017 and p = 0.021), an effect mostly attributable to patients with luminal B tumours. CONCLUSIONS: The results suggest that the effect of the risk allele of rs3803662 is strongest in luminal A tumours and that the expression levels of TOX3 and/or LOC643714 affect the progression of breast cancer. The effect may vary depending on the subtype and developmental stage of the tumour.

Genomic and Transcriptomic Analyses of Breast Cancer Primaries and Matched Metastases in AURORA, the Breast International Group (BIG) Molecular Screening Initiative.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2- cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC. SIGNIFICANCE: The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659.

High-resolution genomic and expression analyses of copy number alterations in HER2-amplified breast cancer.

INTRODUCTION: HER2 gene amplification and protein overexpression (HER2+) define a clinically challenging subgroup of breast cancer with variable prognosis and response to therapy. Although gene expression profiling has identified an ERBB2 molecular subtype of breast cancer, it is clear that HER2+ tumors reside in all molecular subtypes and represent a genomically and biologically heterogeneous group, needed to be further characterized in large sample sets. METHODS: Genome-wide DNA copy number profiling, using bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH), and global gene expression profiling were performed on 200 and 87 HER2+ tumors, respectively. Genomic Identification of Significant Targets in Cancer (GISTIC) was used to identify significant copy number alterations (CNAs) in HER2+ tumors, which were related to a set of 554 non-HER2 amplified (HER2-) breast tumors. High-resolution oligonucleotide aCGH was used to delineate the 17q12-q21 region in high detail. RESULTS: The HER2-amplicon was narrowed to an 85.92 kbp region including the TCAP, PNMT, PERLD1, HER2, C17orf37 and GRB7 genes, and higher HER2 copy numbers indicated worse prognosis. In 31% of HER2+ tumors the amplicon extended to TOP2A, defining a subgroup of HER2+ breast cancer associated with estrogen receptor-positive status and with a trend of better survival than HER2+ breast cancers with deleted (18%) or neutral TOP2A (51%). HER2+ tumors were clearly distinguished from HER2- tumors by the presence of recurrent high-level amplifications and firestorm patterns on chromosome 17q. While there was no significant difference between HER2+ and HER2- tumors regarding the incidence of other recurrent high-level amplifications, differences in the co-amplification pattern were observed, as shown by the almost mutually exclusive occurrence of 8p12, 11q13 and 20q13 amplification in HER2+ tumors. GISTIC analysis identified 117 significant CNAs across all autosomes. Supervised analyses revealed: (1) significant CNAs separating HER2+ tumors stratified by clinical variables, and (2) CNAs separating HER2+ from HER2- tumors. CONCLUSIONS: We have performed a comprehensive survey of CNAs in HER2+ breast tumors, pinpointing significant genomic alterations including both known and potentially novel therapeutic targets. Our analysis sheds further light on the genomically complex and heterogeneous nature of HER2+ tumors in relation to other subgroups of breast cancer.

Genomic subtypes of breast cancer identified by array-comparative genomic hybridization display distinct molecular and clinical characteristics.

INTRODUCTION: Breast cancer is a profoundly heterogeneous disease with respect to biologic and clinical behavior. Gene-expression profiling has been used to dissect this complexity and to stratify tumors into intrinsic gene-expression subtypes, associated with distinct biology, patient outcome, and genomic alterations. Additionally, breast tumors occurring in individuals with germline BRCA1 or BRCA2 mutations typically fall into distinct subtypes. METHODS: We applied global DNA copy number and gene-expression profiling in 359 breast tumors. All tumors were classified according to intrinsic gene-expression subtypes and included cases from genetically predisposed women. The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was used to identify significant DNA copy-number aberrations and genomic subgroups of breast cancer. RESULTS: We identified 31 genomic regions that were highly amplified in > 1% of the 359 breast tumors. Several amplicons were found to co-occur, the 8p12 and 11q13.3 regions being the most frequent combination besides amplicons on the same chromosomal arm. Unsupervised hierarchical clustering with 133 significant GISTIC regions revealed six genomic subtypes, termed 17q12, basal-complex, luminal-simple, luminal-complex, amplifier, and mixed subtypes. Four of them had striking similarity to intrinsic gene-expression subtypes and showed associations to conventional tumor biomarkers and clinical outcome. However, luminal A-classified tumors were distributed in two main genomic subtypes, luminal-simple and luminal-complex, the former group having a better prognosis, whereas the latter group included also luminal B and the majority of BRCA2-mutated tumors. The basal-complex subtype displayed extensive genomic homogeneity and harbored the majority of BRCA1-mutated tumors. The 17q12 subtype comprised mostly HER2-amplified and HER2-enriched subtype tumors and had the worst prognosis. The amplifier and mixed subtypes contained tumors from all gene-expression subtypes, the former being enriched for 8p12-amplified cases, whereas the mixed subtype included many tumors with predominantly DNA copy-number losses and poor prognosis. CONCLUSIONS: Global DNA copy-number analysis integrated with gene-expression data can be used to dissect the complexity of breast cancer. This revealed six genomic subtypes with different clinical behavior and a striking concordance to the intrinsic subtypes. These genomic subtypes may prove useful for understanding the mechanisms of tumor development and for prognostic and treatment prediction purposes.

The BRCA1 c.4096+3A>G Variant Displays Classical Characteristics of Pathogenic BRCA1 Mutations in Hereditary Breast and Ovarian Cancers, But Still Allows Homozygous Viability.

Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling.

Evidence against PALB2 involvement in Icelandic breast cancer susceptibility.

Several mutations in the PALB2 gene (partner and localizer of BRCA2) have been associated with an increased risk of breast cancer, including a founder mutation, 1592delT, reported in Finnish breast cancer families. Although most often the risk is moderate, it doesn't exclude families with high-risk mutations to exist and such observations have been reported. To see if high-risk PALB2-mutations may be present in the geographically confined population of Iceland, linkage analysis was done on 111 individuals, thereof 61 breast cancer cases, from 9 high-risk non-BRCA1/BRCA2 breast cancer families, targeting the PALB2 region. Also, screening for the 1592delT founder mutation in the 9 high-risk families and in 638 unselected breast cancer cases was performed. The results indicate no linkage in any of the high-risk families and screening for the 1592delT mutation was negative in all samples. PALB2 appears not to be a significant factor in high-risk breast cancer families in Iceland and the 1592delT mutation is not seen to be associated with breast cancer in Iceland.

Counsellee's experience of cancer genetic counselling with pedigrees that automatically incorporate genealogical and cancer database information.

While pedigree drawing software is often utilised in genetic services, the use of genealogical databases in genetic counselling is unusual. This is mainly because of the unavailability of such databases in most countries. Electronically generated pedigrees used for cancer genetic counselling in Iceland create pedigrees that automatically incorporate information from a large, comprehensive genealogy database and nation-wide cancer registry. The aim of this descriptive qualitative study was to explore counsellees' experiences of genetic services, including family history taking, using these electronically generated pedigrees. Four online focus groups with 19 participants were formed, using an asynchronous posting method. Participants were encouraged to discuss their responses to questions posted on the website by the researcher. The main themes arising were motivation, information and trust, impact of testing and emotional responses. Most of the participants expressed trust in the method of using electronically generated pedigrees, although some voiced worries about information safety. Many experienced worry and anxiety while waiting for results of genetic testing, but limited survival guilt was noted. Family communication was either unchanged or improved following genetic counselling. The use of electronically generated pedigrees was well received by participants, and they trusted the information obtained via the databases. Age did not seem to influence responses. These results may be indicative of the particular culture in Iceland, where genealogical information is well known and freely shared. Further studies are needed to determine whether use of similar approaches to genealogical information gathering may be acceptable elsewhere.

The use of genealogy databases for risk assessment in genetic health service: a systematic review.

UNLABELLED: The use of electronic genealogical databases facilitates the construction of accurate and extensive pedigrees for potential use in genetic services. Genealogy databases can be linked to specific disease databases, such as cancer registries, in order to increase the accuracy of pedigrees used, and inform the genetic risk assessment. To review the published literature on the use of genealogy databases to construct pedigrees for risk assessment in genetic health service, a systematic literature search was undertaken using 12 combined search terms to identify all relevant published articles. DATA SOURCES: EbscoHost, PubMed, Web of Science, Ovid and the "grey literature", as well as the reference lists of identified studies. Of 1,035 titles identified, two papers described a study on the use of genealogy databases in cancer risk assessment and two were discussion papers. While authors of the four papers described the potential use of genealogy databases in clinical genetic services, such use has not been adequately investigated and further research is required.

Identification of subtypes in human epidermal growth factor receptor 2--positive breast cancer reveals a gene signature prognostic of outcome.

PURPOSE Human epidermal growth factor receptor 2 (HER2) gene amplification or protein overexpression (HER2 positivity) defines a clinically challenging subgroup of patients with breast cancer (BC) with variable prognosis and response to therapy. We aimed to investigate the heterogeneous biologic appearance and clinical behavior of HER2-positive tumors using molecular profiling. PATIENTS AND METHODS Hierarchical clustering of gene expression data from 58 HER2-amplified tumors of various stage, histologic grade, and estrogen receptor (ER) status was used to construct a HER2-derived prognostic predictor that was further evaluated in several large independent BC data sets. RESULTS Unsupervised analysis identified three subtypes of HER2-positive tumors with mixed stage, histologic grade, and ER status. One subtype had a significantly worse clinical outcome. A prognostic predictor was created based on differentially expressed genes between the subtype with worse outcome and the other subtypes. The predictor was able to define patient groups with better and worse outcome in HER2-positive BC across multiple independent BC data sets and identify a sizable HER2-positive group with long disease-free survival and low mortality. Significant correlation to prognosis was also observed in basal-like, ER-negative, lymph node-positive, and high-grade tumors, irrespective of HER2 status. The predictor included genes associated with immune response, tumor invasion, and metastasis. CONCLUSION The HER2-derived prognostic predictor provides further insight into the heterogeneous biology of HER2-positive tumors and may become useful for improved selection of patients who need additional treatment with new drugs targeting the HER2 pathway.

Chromosome 5 imbalance mapping in breast tumors from BRCA1 and BRCA2 mutation carriers and sporadic breast tumors.

Comparative genomic hybridization (CGH) analysis has shown that chromosome 5q deletions are the most frequent aberration in breast tumors from BRCA1 mutation carriers. To map the location of putative 5q tumor suppressor gene(s), 26 microsatellite markers covering chromosome 5 were used in loss of heterozygosity (LOH) analysis of breast tumors from BRCA1 (n = 42) and BRCA2 mutation carriers (n = 67), as well as in sporadic cases (n = 65). High-density array CGH was also used to map chromosome 5 imbalance in 10 BRCA1 tumors. A high LOH frequency was found in BRCA1 tumors (range 19-82%), as compared to BRCA2 and sporadic tumors (ranges 11-44% and 7-43%, respectively). In all, 11 distinct chromosome 5 regions with LOH were observed, the most frequent being 5q35.3 (82%), 5q14.2 (71%) and 5q33.1 (69%) in BRCA1 tumors; 5q35.3 (44%), 5q31.3 (43%) and 5q13.3 (43%) in BRCA2 tumors and 5q31.3 (43%) in sporadic tumors. Array CGH analysis confirmed the very high frequency of 5q deletions, including candidate tumor suppressor genes such as XRCC4, RAD50, RASA1, APC and PPP2R2B. In addition, 2 distinct homozygous deletions were identified, spanning regions of 0.7-1.5 Mbp on 5q12.1 and 5q12.3-q13.1, respectively. These regions include only a few genes, most notably BRCC3/DEPDC1B (pleckstrin/G protein interacting and RhoGAP domains) and PIK3R1 (PI3 kinase P85 regulatory subunit). Significant association (p < or = 0.05) was found between LOH at certain 5q regions and factors of poor prognosis, including negative estrogen and progesterone receptor status, high grade, large tumor size and high portion of cells in S-phase. In conclusion, our results confirm a very high prevalence of chromosome 5q alterations in BRCA1 tumors, pinpointing new regions and genes that should be further investigated.