Oskar Th Johannsson

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: ∼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.

Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer.

We carried out a genome-wide association study of breast cancer predisposition with replication and refinement studies involving 6,145 cases and 33,016 controls and identified two SNPs (rs4415084 and rs10941679) on 5p12 that confer risk, preferentially for estrogen receptor (ER)-positive tumors (OR = 1.27, P = 2.5 × 10−12 for rs10941679). The nearest gene, MRPS30, was previously implicated in apoptosis, ER-positive tumors and favorable prognosis. A recently reported signal in FGFR2 was also found to associate specifically with ER-positive breast cancer.

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

IntroductionBreast 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.MethodsWe 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.ResultsWe 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.ConclusionsGlobal 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.

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.

Ancestry-shift refinement mapping of the C6orf97-ESR1 breast cancer susceptibility locus.

We used an approach that we term ancestry-shift refinement mapping to investigate an association, originally discovered in a GWAS of a Chinese population, between rs2046210[T] and breast cancer susceptibility. The locus is on 6q25.1 in proximity to the C6orf97 and estrogen receptor α (ESR1) genes. We identified a panel of SNPs that are correlated with rs2046210 in Chinese, but not necessarily so in other ancestral populations, and genotyped them in breast cancer case∶control samples of Asian, European, and African origin, a total of 10,176 cases and 13,286 controls. We found that rs2046210[T] does not confer substantial risk of breast cancer in Europeans and Africans (OR = 1.04, P = 0.099, and OR = 0.98, P = 0.77, respectively). Rather, in those ancestries, an association signal arises from a group of less common SNPs typified by rs9397435. The rs9397435[G] allele was found to confer risk of breast cancer in European (OR = 1.15, P = 1.2×10−3), African (OR = 1.35, P = 0.014), and Asian (OR = 1.23, P = 2.9×10−4) population samples. Combined over all ancestries, the OR was 1.19 (P = 3.9×10−7), was without significant heterogeneity between ancestries (Phet = 0.36) and the SNP fully accounted for the association signal in each ancestry. Haplotypes bearing rs9397435[G] are well tagged by rs2046210[T] only in Asians. The rs9397435[G] allele showed associations with both estrogen receptor positive and estrogen receptor negative breast cancer. Using early-draft data from the 1,000 Genomes project, we found that the risk allele of a novel SNP (rs77275268), which is closely correlated with rs9397435, disrupts a partially methylated CpG sequence within a known CTCF binding site. These studies demonstrate that shifting the analysis among ancestral populations can provide valuable resolution in association mapping.

The BARD1 Cys557Ser Variant and Breast Cancer Risk in Iceland

Background Most, if not all, of the cellular functions of the BRCA1 protein are mediated through heterodimeric complexes composed of BRCA1 and a related protein, BARD1. Some breast-cancer-associated BRCA1 missense mutations disrupt the function of the BRCA1/BARD1 complex. It is therefore pertinent to determine whether variants of BARD1 confer susceptibility to breast cancer. Recently, a missense BARD1 variant, Cys557Ser, was reported to be at increased frequencies in breast cancer families. We investigated the role of the BARD1 Cys557Ser variant in a population-based cohort of 1,090 Icelandic patients with invasive breast cancer and 703 controls. We then used a computerized genealogy of the Icelandic population to study the relationships between the Cys557Ser variant and familial clustering of breast cancer. Methods and Findings The Cys557Ser allele was present at a frequency of 0.028 in patients with invasive breast cancer and 0.016 in controls (odds ratio [OR] = 1.82, 95% confidence interval [CI] 1.11–3.01, p = 0.014). The alleleic frequency was 0.037 in a high-predisposition group of cases defined by having a family history of breast cancer, early onset of breast cancer, or multiple primary breast cancers (OR = 2.41, 95% CI 1.22–4.75, p = 0.015). Carriers of the common Icelandic BRCA2 999del5 mutation were found to have their risk of breast cancer further increased if they also carried the BARD1 variant: the frequency of the BARD1 variant allele was 0.047 (OR = 3.11, 95% CI 1.16–8.40, p = 0.046) in 999del5 carriers with breast cancer. This suggests that the lifetime probability of a BARD1 Cys557Ser/BRCA2 999del5 double carrier developing breast cancer could approach certainty. Cys557Ser carriers, with or without the BRCA2 mutation, had an increased risk of subsequent primary breast tumors after the first breast cancer diagnosis compared to non-carriers. Lobular and medullary breast carcinomas were overrepresented amongst Cys557Ser carriers. We found that an excess of ancestors of contemporary carriers lived in a single county in the southeast of Iceland and that all carriers shared a SNP haplotype, which is suggestive of a founder event. Cys557Ser was found on the same SNP haplotype background in the HapMap Project CEPH sample of Utah residents. Conclusions Our findings suggest that BARD1 Cys557Ser is an ancient variant that confers risk of single and multiple primary breast cancers, and this risk extends to carriers of the BRCA2 999del5 mutation.

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 ≤ 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.

The Retinoblastoma Gene Undergoes Rearrangements in BRCA1-Deficient Basal-like Breast Cancer

Breast tumors from BRCA1 germ line mutation carriers typically exhibit features of the basal-like molecular subtype. However, the specific genes recurrently mutated as a consequence of BRCA1 dysfunction have not been fully elucidated. In this study, we used gene expression profiling to molecularly subtype 577 breast tumors, including 73 breast tumors from BRCA1/2 mutation carriers. Focusing on the RB1 locus, we analyzed 33 BRCA1-mutated, 36 BRCA2-mutated, and 48 non-BRCA1/2-mutated breast tumors using a custom-designed high-density oligomicroarray covering the RB1 gene. We found a strong association between the basal-like subtype and BRCA1-mutated breast tumors and the luminal B subtype and BRCA2-mutated breast tumors. RB1 was identified as a major target for genomic disruption in tumors arising in BRCA1 mutation carriers and in sporadic tumors with BRCA1 promoter methylation but rarely in other breast cancers. Homozygous deletions, intragenic breaks, or microdeletions were found in 33% of BRCA1-mutant tumors, 36% of BRCA1 promoter-methylated basal-like tumors, 13% of non-BRCA1-deficient basal-like tumors, and 3% of BRCA2-mutated tumors. In conclusion, RB1 was frequently inactivated by gross gene disruption in BRCA1 hereditary breast cancer and BRCA1-methylated sporadic basal-like breast cancer but rarely in BRCA2 hereditary breast cancer and non-BRCA1-deficient sporadic breast cancers. Together, our findings show the existence of genetic heterogeneity within the basal-like breast cancer subtype that is based upon BRCA1 status.

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

BackgroundThe 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.MethodsThe 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.ResultsAn 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.ConclusionsThe 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.