The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer.

PURPOSE: In HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient's outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors. METHODS: We evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated. RESULTS: FoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively. CONCLUSION: In the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

Nuclei instance segmentation from histopathology images using Bayesian dropout based deep learning.

BACKGROUND: The deterministic deep learning models have achieved state-of-the-art performance in various medical image analysis tasks, including nuclei segmentation from histopathology images. The deterministic models focus on improving the model prediction accuracy without assessing the confidence in the predictions. METHODS: We propose a semantic segmentation model using Bayesian representation to segment nuclei from the histopathology images and to further quantify the epistemic uncertainty. We employ Bayesian approximation with Monte-Carlo (MC) dropout during the inference time to estimate the model's prediction uncertainty. RESULTS: We evaluate the performance of the proposed approach on the PanNuke dataset, which consists of 312 visual fields from 19 organ types. We compare the nuclei segmentation accuracy of our approach with that of a fully convolutional neural network, U-Net, SegNet, and the state-of-the-art Hover-net. We use F1-score and intersection over union (IoU) as the evaluation metrics. The proposed approach achieves a mean F1-score of 0.893 ± 0.008 and an IoU value of 0.868 ± 0.003 on the test set of the PanNuke dataset. These results outperform the Hover-net, which has a mean F1-score of 0.871 ± 0.010 and an IoU value of 0.840 ± 0.032. CONCLUSIONS: The proposed approach, which incorporates Bayesian representation and Monte-Carlo dropout, demonstrates superior performance in segmenting nuclei from histopathology images compared to existing models such as U-Net, SegNet, and Hover-net. By considering the epistemic uncertainty, our model provides a more reliable estimation of the prediction confidence. These findings highlight the potential of Bayesian deep learning for improving medical image analysis tasks and can contribute to the development of more accurate and reliable computer-aided diagnostic systems.

High regional mortality due to malignant melanoma in Eastern Finland may be explained by the increase in aggressive melanoma types.

BACKGROUND: A regional skin cancer prevention program in Eastern Finland revealed a relatively high age-standardized mortality due to malignant melanoma during 2013-2017. An explanation for this is needed. PURPOSE: To analyse the 543 melanoma samples in 524 subjects collected during 2000-2013 at Kuopio University Hospital and reposited in the Biobank of Eastern Finland. A focus was directed to factors related to metastasis. METHODS: The samples were analysed anonymously by examining the histopathological report, referral text and the list of diagnoses. A possible state of immunosuppression was evaluated. RESULTS: The mean age at the diagnosis of malignant melanoma (MM), lentigo maligna (LM) and melanoma in situ was relatively high, i.e., 66.2, 72.1 and 63.3, respectively. Especially the MM type increased markedly during 2000-2013. In further analyses of a representative cohort of 337 samples, the proportion of nodular melanoma and LM/LMM melanoma was relatively high, 35.6 and 22.0%, respectively, but that from superficial spreading melanoma relatively low (33.8%). Metastasis correlated with immunosuppression, male gender, Clark level, Breslow thickness, ulceration, mitosis count, invasion into vessels and/or perineural area, microsatellites, melanoma subtype, body site, recidivism, and the absence of dysplastic nevus cells. CONCLUSION: The marked increase in aggressive melanomas with associated metastasis, and the relatively high age at diagnosis, can partially explain the mortality.

Rare missense mutations in RECQL and POLG associate with inherited predisposition to breast cancer.

Several known breast cancer susceptibility genes with moderate-to-high risk alleles encode proteins involved in DNA damage response (DDR). As these explain less than half of the hereditary breast cancer cases, additional predisposing alleles are likely to be discovered. Many of the previous studies utilizing massive parallel sequencing have focused on the protein-truncating variants, and the role of rare missense mutations has remained poorly addressed. To identify novel susceptibility factors, we have systematically analyzed the data from our parallel sequencing of 796 DDR genes in 189 Northern Finnish hereditary breast cancer patients for rare missense variants, predicted as deleterious. Thirty-five variants were studied here for the disease association using Finnish breast cancer case (n = 492-2,035) and control (n = 277-1,539) cohorts. As a result, two missense variants in genes involved in DNA replication, RECQL p.I156M and POLG p.L392V, the former involving genomic and the latter mitochondrial DNA replication, showed significant association with risk of breast cancer. Rare RECQL p.I156M allele was observed in breast cancer cases only (6/1,946, 0.3%, p = 0.043), whereas POLG p.L392V was two times more frequent in breast cancer cases (53/2,238, 2.4%) compared to controls (18/1,539, 1.2%, OR = 2.1, 95% CI 1.2-3.5, p = 0.010). Based on the current genetic data, both RECQL p.I156M and POLG p.L392V represent novel breast cancer predisposing alleles.

Etiology of hormone receptor positive breast cancer differs by levels of histologic grade and proliferation.

Limited epidemiological evidence suggests that the etiology of hormone receptor positive (HR+) breast cancer may differ by levels of histologic grade and proliferation. We pooled risk factor and pathology data on 5,905 HR+ breast cancer cases and 26,281 controls from 11 epidemiological studies. Proliferation was determined by centralized automated measures of KI67 in tissue microarrays. Odds ratios (OR), 95% confidence intervals (CI) and p-values for case-case and case-control comparisons for risk factors in relation to levels of grade and quartiles (Q1-Q4) of KI67 were estimated using polytomous logistic regression models. Case-case comparisons showed associations between nulliparity and high KI67 [OR (95% CI) for Q4 vs. Q1 = 1.54 (1.22, 1.95)]; obesity and high grade [grade 3 vs. 1 = 1.68 (1.31, 2.16)] and current use of combined hormone therapy (HT) and low grade [grade 3 vs. 1 = 0.27 (0.16, 0.44)] tumors. In case-control comparisons, nulliparity was associated with elevated risk of tumors with high but not low levels of proliferation [1.43 (1.14, 1.81) for KI67 Q4 vs. 0.83 (0.60, 1.14) for KI67 Q1]; obesity among women ≥50 years with high but not low grade tumors [1.55 (1.17, 2.06) for grade 3 vs. 0.88 (0.66, 1.16) for grade 1] and HT with low but not high grade tumors [3.07 (2.22, 4.23) for grade 1 vs. 0.85 (0.55, 1.30) for grade 3]. Menarcheal age and family history were similarly associated with HR+ tumors of different grade or KI67 levels. These findings provide insights into the etiologic heterogeneity of HR+ tumors.

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10-19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10-15 - 5.6 × 10-17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.

Reproductive profiles and risk of breast cancer subtypes: a multi-center case-only study.

BACKGROUND: Previous studies have shown that reproductive factors are differentially associated with breast cancer (BC) risk by subtypes. The aim of this study was to investigate associations between reproductive factors and BC subtypes, and whether these vary by age at diagnosis. METHODS: We used pooled data on tumor markers (estrogen and progesterone receptor, human epidermal growth factor receptor-2 (HER2)) and reproductive risk factors (parity, age at first full-time pregnancy (FFTP) and age at menarche) from 28,095 patients with invasive BC from 34 studies participating in the Breast Cancer Association Consortium (BCAC). In a case-only analysis, we used logistic regression to assess associations between reproductive factors and BC subtype compared to luminal A tumors as a reference. The interaction between age and parity in BC subtype risk was also tested, across all ages and, because age was modeled non-linearly, specifically at ages 35, 55 and 75 years. RESULTS: Parous women were more likely to be diagnosed with triple negative BC (TNBC) than with luminal A BC, irrespective of age (OR for parity = 1.38, 95% CI 1.16-1.65, p = 0.0004; p for interaction with age = 0.076). Parous women were also more likely to be diagnosed with luminal and non-luminal HER2-like BCs and this effect was slightly more pronounced at an early age (p for interaction with age = 0.037 and 0.030, respectively). For instance, women diagnosed at age 35 were 1.48 (CI 1.01-2.16) more likely to have luminal HER2-like BC than luminal A BC, while this association was not significant at age 75 (OR = 0.72, CI 0.45-1.14). While age at menarche was not significantly associated with BC subtype, increasing age at FFTP was non-linearly associated with TNBC relative to luminal A BC. An age at FFTP of 25 versus 20 years lowered the risk for TNBC (OR = 0.78, CI 0.70-0.88, p < 0.0001), but this effect was not apparent at a later FFTP. CONCLUSIONS: Our main findings suggest that parity is associated with TNBC across all ages at BC diagnosis, whereas the association with luminal HER2-like BC was present only for early onset BC.

Gene-environment interactions involving functional variants: Results from the Breast Cancer Association Consortium.

Investigating the most likely causal variants identified by fine-mapping analyses may improve the power to detect gene-environment interactions. We assessed the interplay between 70 single nucleotide polymorphisms identified by genetic fine-scale mapping of susceptibility loci and 11 epidemiological breast cancer risk factors in relation to breast cancer. Analyses were conducted on up to 58,573 subjects (26,968 cases and 31,605 controls) from the Breast Cancer Association Consortium, in one of the largest studies of its kind. Analyses were carried out separately for estrogen receptor (ER) positive (ER+) and ER negative (ER-) disease. The Bayesian False Discovery Probability (BFDP) was computed to assess the noteworthiness of the results. Four potential gene-environment interactions were identified as noteworthy (BFDP < 0.80) when assuming a true prior interaction probability of 0.01. The strongest interaction result in relation to overall breast cancer risk was found between CFLAR-rs7558475 and current smoking (ORint = 0.77, 95% CI: 0.67-0.88, pint = 1.8 × 10-4 ). The interaction with the strongest statistical evidence was found between 5q14-rs7707921 and alcohol consumption (ORint =1.36, 95% CI: 1.16-1.59, pint = 1.9 × 10-5 ) in relation to ER- disease risk. The remaining two gene-environment interactions were also identified in relation to ER- breast cancer risk and were found between 3p21-rs6796502 and age at menarche (ORint = 1.26, 95% CI: 1.12-1.43, pint =1.8 × 10-4 ) and between 8q23-rs13267382 and age at first full-term pregnancy (ORint = 0.89, 95% CI: 0.83-0.95, pint = 5.2 × 10-4 ). While these results do not suggest any strong gene-environment interactions, our results may still be useful to inform experimental studies. These may in turn, shed light on the potential interactions observed.

Breast-cancer risk in families with mutations in PALB2.

BACKGROUND: Germline loss-of-function mutations in PALB2 are known to confer a predisposition to breast cancer. However, the lifetime risk of breast cancer that is conferred by such mutations remains unknown. METHODS: We analyzed the risk of breast cancer among 362 members of 154 families who had deleterious truncating, splice, or deletion mutations in PALB2. The age-specific breast-cancer risk for mutation carriers was estimated with the use of a modified segregation-analysis approach that allowed for the effects of PALB2 genotype and residual familial aggregation. RESULTS: The risk of breast cancer for female PALB2 mutation carriers, as compared with the general population, was eight to nine times as high among those younger than 40 years of age, six to eight times as high among those 40 to 60 years of age, and five times as high among those older than 60 years of age. The estimated cumulative risk of breast cancer among female mutation carriers was 14% (95% confidence interval [CI], 9 to 20) by 50 years of age and 35% (95% CI, 26 to 46) by 70 years of age. Breast-cancer risk was also significantly influenced by birth cohort (P<0.001) and by other familial factors (P=0.04). The absolute breast-cancer risk for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25 to 44) for those with no family history of breast cancer to 58% (95% CI, 50 to 66) for those with two or more first-degree relatives with breast cancer at 50 years of age. CONCLUSIONS: Loss-of-function mutations in PALB2 are an important cause of hereditary breast cancer, with respect both to the frequency of cancer-predisposing mutations and to the risk associated with them. Our data suggest the breast-cancer risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers. (Funded by the European Research Council and others.).

FANCM mutation c.5791C>T is a risk factor for triple-negative breast cancer in the Finnish population.

PURPOSE: The FANCM c.5101C>T nonsense mutation was previously found to associate with breast cancer in the Finnish population, especially among triple-negative cases. Here, we studied the prevalence of three other FANCM variants: c.5791C>T, which has been reported to predispose to familial breast cancer, and the c.4025_4026delCT and c.5293dupA variants recently identified in Finnish cancer patients. METHODS: We genotyped the FANCM c.5791C>T mutation in 4806 invasive breast cancer patients, including BRCA1/2 mutation negative familial cases and unselected cases, and in 2734 healthy population controls from four different geographical areas of Finland. The association of the mutation with breast cancer risk among patient subgroups was statistically evaluated. We further analyzed the combined risk associated with c.5101C>T and c.5791C>T mutations. We also genotyped 526 unselected ovarian cancer patients for the c.5791C>T mutation and 862 familial breast cancer patients for the c.4025_4026delCT and c.5293dupA variants. RESULTS: The frequency of the FANCM c.5791C>T mutation was higher among breast cancer cases than in controls (OR 1.94, 95% CI 0.87-4.32, P = 0.11), with a statistically significant association with triple-negative breast cancer (OR 5.14, 95% CI 1.65-16.0, P = 0.005). The combined analysis for c.5101C>T and c.5791C>T carriers confirmed a strong association with breast cancer (OR 1.86, 95% CI 1.32-2.49, P = 0.0002), especially among the triple-negative patients (OR 3.08, 95% CI 1.77-5.35, P = 0.00007). For the other variants, only one additional c.4025_4026delCT carrier and no c.5293dupA carriers were observed. CONCLUSIONS: These results support the role of FANCM as a breast cancer susceptibility gene, particularly for triple-negative breast cancer.

Patient survival and tumor characteristics associated with CHEK2:p.I157T - findings from the Breast Cancer Association Consortium.

BACKGROUND: P.I157T is a CHEK2 missense mutation associated with a modest increase in breast cancer risk. Previously, another CHEK2 mutation, the protein truncating c.1100delC has been associated with poor prognosis of breast cancer patients. Here, we have investigated patient survival and characteristics of breast tumors of germ line p.I157T carriers. METHODS: We included in the analyses 26,801 European female breast cancer patients from 15 studies participating in the Breast Cancer Association Consortium. We analyzed the association between p.I157T and the clinico-pathological breast cancer characteristics by comparing the p.I157T carrier tumors to non-carrier and c.1100delC carrier tumors. Similarly, we investigated the p.I157T associated risk of early death, breast cancer-associated death, distant metastasis, locoregional relapse and second breast cancer using Cox proportional hazards models. Additionally, we explored the p.I157T-associated genomic gene expression profile using data from breast tumors of 183 Finnish female breast cancer patients (ten p.I157T carriers) (GEO: GSE24450). Differential gene expression analysis was performed using a moderated t test. Functional enrichment was investigated using the DAVID functional annotation tool and gene set enrichment analysis (GSEA). The tumors were classified into molecular subtypes according to the St Gallen 2013 criteria and the PAM50 gene expression signature. RESULTS: P.I157T was not associated with increased risk of early death, breast cancer-associated death or distant metastasis relapse, and there was a significant difference in prognosis associated with the two CHEK2 mutations, p.I157T and c.1100delC. Furthermore, p.I157T was associated with lobular histological type and clinico-pathological markers of good prognosis, such as ER and PR expression, low TP53 expression and low grade. Gene expression analysis suggested luminal A to be the most common subtype for p.I157T carriers and CDH1 (cadherin 1) target genes to be significantly enriched among genes, whose expression differed between p.I157T and non-carrier tumors. CONCLUSIONS: Our analyses suggest that there are fundamental differences in breast tumors of CHEK2:p.I157T and c.1100delC carriers. The poor prognosis associated with c.1100delC cannot be generalized to other CHEK2 mutations.

Prognostic value of automated KI67 scoring in breast cancer: a centralised evaluation of 8088 patients from 10 study groups.

BACKGROUND: The value of KI67 in breast cancer prognostication has been questioned due to concerns on the analytical validity of visual KI67 assessment and methodological limitations of published studies. Here, we investigate the prognostic value of automated KI67 scoring in a large, multicentre study, and compare this with pathologists' visual scores available in a subset of patients. METHODS: We utilised 143 tissue microarrays containing 15,313 tumour tissue cores from 8088 breast cancer patients in 10 collaborating studies. A total of 1401 deaths occurred during a median follow-up of 7.5 years. Centralised KI67 assessment was performed using an automated scoring protocol. The relationship of KI67 levels with 10-year breast cancer specific survival (BCSS) was investigated using Kaplan-Meier survival curves and Cox proportional hazard regression models adjusted for known prognostic factors. RESULTS: Patients in the highest quartile of KI67 (>12 % positive KI67 cells) had a worse 10-year BCSS than patients in the lower three quartiles. This association was statistically significant for ER-positive patients (hazard ratio (HR) (95 % CI) at baseline = 1.96 (1.31-2.93); P = 0.001) but not for ER-negative patients (1.23 (0.86-1.77); P = 0.248) (P-heterogeneity = 0.064). In spite of differences in characteristics of the study populations, the estimates of HR were consistent across all studies (P-heterogeneity = 0.941 for ER-positive and P-heterogeneity = 0.866 for ER-negative). Among ER-positive cancers, KI67 was associated with worse prognosis in both node-negative (2.47 (1.16-5.27)) and node-positive (1.74 (1.05-2.86)) tumours (P-heterogeneity = 0.671). Further classification according to ER, PR and HER2 showed statistically significant associations with prognosis among hormone receptor-positive patients regardless of HER2 status (P-heterogeneity = 0.270) and among triple-negative patients (1.70 (1.02-2.84)). Model fit parameters were similar for visual and automated measures of KI67 in a subset of 2440 patients with information from both sources. CONCLUSIONS: Findings from this large-scale multicentre analysis with centrally generated automated KI67 scores show strong evidence in support of a prognostic value for automated KI67 scoring in breast cancer. Given the advantages of automated scoring in terms of its potential for standardisation, reproducibility and throughput, automated methods appear to be promising alternatives to visual scoring for KI67 assessment.

FANCM c.5101C>T mutation associates with breast cancer survival and treatment outcome.

Breast cancer (BC) is a heterogeneous disease, and different tumor characteristics and genetic variation may affect the clinical outcome. The FANCM c.5101C > T nonsense mutation in the Finnish population associates with increased risk of breast cancer, especially for triple-negative breast cancer patients. To investigate the association of the mutation with disease prognosis, we studied tumor phenotype, treatment outcome, and patient survival in 3,933 invasive breast cancer patients, including 101 FANCM c.5101C > T mutation carriers and 3,832 non-carriers. We also examined association of the mutation with nuclear immunohistochemical staining of DNA repair markers in 1,240 breast tumors. The FANCM c.5101C > T mutation associated with poor 10-year breast cancer-specific survival (hazard ratio (HR)=1.66, 95% confidence interval (CI) 1.09-2.52, p = 0.018), with a more pronounced survival effect among familial cases (HR = 2.93, 95% CI 1.5-5.76, p = 1.80 × 10-3 ). Poor disease outcome of the carriers was also found among the estrogen receptor (ER) positive subgroup of patients (HR = 1.8, 95% CI 1.09-2.98, p = 0.021). Reduced survival was seen especially among patients who had not received radiotherapy (HR = 3.43, 95% CI 1.6-7.34, p = 1.50 × 10-3 ) but not among radiotherapy treated patients (HR = 1.35, 95% CI 0.82-2.23, p = 0.237). Significant interaction was found between the mutation and radiotherapy (p = 0.040). Immunohistochemical analyses show that c.5101C > T carriers have reduced PAR-activity. Our results suggest that FANCM c.5101C > T nonsense mutation carriers have a reduced breast cancer survival but postoperative radiotherapy may diminish this survival disadvantage.

Genetic variation in mitotic regulatory pathway genes is associated with breast tumor grade.

Mitotic index is an important component of histologic grade and has an etiologic role in breast tumorigenesis. Several small candidate gene studies have reported associations between variation in mitotic genes and breast cancer risk. We measured associations between 2156 single nucleotide polymorphisms (SNPs) from 194 mitotic genes and breast cancer risk, overall and by histologic grade, in the Breast Cancer Association Consortium (BCAC) iCOGS study (n = 39 067 cases; n = 42 106 controls). SNPs in TACC2 [rs17550038: odds ratio (OR) = 1.24, 95% confidence interval (CI) 1.16-1.33, P = 4.2 × 10(-10)) and EIF3H (rs799890: OR = 1.07, 95% CI 1.04-1.11, P = 8.7 × 10(-6)) were significantly associated with risk of low-grade breast cancer. The TACC2 signal was retained (rs17550038: OR = 1.15, 95% CI 1.07-1.23, P = 7.9 × 10(-5)) after adjustment for breast cancer risk SNPs in the nearby FGFR2 gene, suggesting that TACC2 is a novel, independent genome-wide significant genetic risk locus for low-grade breast cancer. While no SNPs were individually associated with high-grade disease, a pathway-level gene set analysis showed that variation across the 194 mitotic genes was associated with high-grade breast cancer risk (P = 2.1 × 10(-3)). These observations will provide insight into the contribution of mitotic defects to histological grade and the etiology of breast cancer.

Heterogeneity of luminal breast cancer characterised by immunohistochemical expression of basal markers.

BACKGROUND: Luminal A breast cancer defined as hormone receptor positive and human epidermal growth factor receptor 2 (HER2) negative is known to be heterogeneous. Previous study showed that luminal A tumours with the expression of basal markers ((cytokeratin (CK) 5 or CK5/6) or epidermal growth factor receptor (EGFR)) were associated with poorer prognosis compared with those that stained negative for basal markers. Prompted by this study, we assessed whether tumour characteristics and risk factors differed by basal marker status within luminal A tumours. METHODS: We pooled 5040 luminal A cases defined by immunohistochemistry (4490 basal-negative ((CK5 (or CK5/6))- and EGFR-) and 550 basal-positive ((CK5 (or CK5/6+)) or EGFR+)) from eight studies participating in the Breast Cancer Association Consortium. Case-case comparison was performed using unconditional logistic regression. RESULTS: Tumour characteristics and risk factors did not vary significantly by the expression of basal markers, although results suggested that basal-positive luminal tumours tended to be smaller and node negative, and were more common in women with a positive family history and lower body mass index. CONCLUSIONS: Most established breast cancer risk factors were similar in basal-positive and basal-negative luminal A tumours. The non-significant but suggestive differences in tumour features and family history warrant further investigations.

Genetic variation in the immunosuppression pathway genes and breast cancer susceptibility: a pooled analysis of 42,510 cases and 40,577 controls from the Breast Cancer Association Consortium.

Immunosuppression plays a pivotal role in assisting tumors to evade immune destruction and promoting tumor development. We hypothesized that genetic variation in the immunosuppression pathway genes may be implicated in breast cancer tumorigenesis. We included 42,510 female breast cancer cases and 40,577 controls of European ancestry from 37 studies in the Breast Cancer Association Consortium (2015) with available genotype data for 3595 single nucleotide polymorphisms (SNPs) in 133 candidate genes. Associations between genotyped SNPs and overall breast cancer risk, and secondarily according to estrogen receptor (ER) status, were assessed using multiple logistic regression models. Gene-level associations were assessed based on principal component analysis. Gene expression analyses were conducted using RNA sequencing level 3 data from The Cancer Genome Atlas for 989 breast tumor samples and 113 matched normal tissue samples. SNP rs1905339 (A>G) in the STAT3 region was associated with an increased breast cancer risk (per allele odds ratio 1.05, 95 % confidence interval 1.03-1.08; p value = 1.4 × 10(-6)). The association did not differ significantly by ER status. On the gene level, in addition to TGFBR2 and CCND1, IL5 and GM-CSF showed the strongest associations with overall breast cancer risk (p value = 1.0 × 10(-3) and 7.0 × 10(-3), respectively). Furthermore, STAT3 and IL5 but not GM-CSF were differentially expressed between breast tumor tissue and normal tissue (p value = 2.5 × 10(-3), 4.5 × 10(-4) and 0.63, respectively). Our data provide evidence that the immunosuppression pathway genes STAT3, IL5, and GM-CSF may be novel susceptibility loci for breast cancer in women of European ancestry.

Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes.

Distinct subtypes of triple negative (TN) breast cancer have been identified by tumor expression profiling. However, little is known about the relationship between histopathologic features of TN tumors, which reflect aspects of both tumor behavior and tumor microenvironment, and molecular TN subtypes. The histopathologic features of TN tumors were assessed by central review and 593 TN tumors were subjected to whole genome expression profiling using the Illumina Whole Genome DASL array. TN molecular subtypes were defined based on gene expression data associated with histopathologic features of TN tumors. Gene expression analysis yielded signatures for four TN subtypes (basal-like, androgen receptor positive, immune, and stromal) consistent with previous studies. Expression analysis also identified genes significantly associated with the 12 histological features of TN tumors. Development of signatures using these markers of histopathological features resulted in six distinct TN subtype signatures, including an additional basal-like and stromal signature. The additional basal-like subtype was distinguished by elevated expression of cell motility and glucose metabolism genes and reduced expression of immune signaling genes, whereas the additional stromal subtype was distinguished by elevated expression of immunomodulatory pathway genes. Histopathologic features that reflect heterogeneity in tumor architecture, cell structure, and tumor microenvironment are related to TN subtype. Accounting for histopathologic features in the development of gene expression signatures, six major subtypes of TN breast cancer were identified.

Evidence of gene-environment interactions between common breast cancer susceptibility loci and established environmental risk factors.

Various common genetic susceptibility loci have been identified for breast cancer; however, it is unclear how they combine with lifestyle/environmental risk factors to influence risk. We undertook an international collaborative study to assess gene-environment interaction for risk of breast cancer. Data from 24 studies of the Breast Cancer Association Consortium were pooled. Using up to 34,793 invasive breast cancers and 41,099 controls, we examined whether the relative risks associated with 23 single nucleotide polymorphisms were modified by 10 established environmental risk factors (age at menarche, parity, breastfeeding, body mass index, height, oral contraceptive use, menopausal hormone therapy use, alcohol consumption, cigarette smoking, physical activity) in women of European ancestry. We used logistic regression models stratified by study and adjusted for age and performed likelihood ratio tests to assess gene-environment interactions. All statistical tests were two-sided. We replicated previously reported potential interactions between LSP1-rs3817198 and parity (Pinteraction = 2.4 × 10(-6)) and between CASP8-rs17468277 and alcohol consumption (Pinteraction = 3.1 × 10(-4)). Overall, the per-allele odds ratio (95% confidence interval) for LSP1-rs3817198 was 1.08 (1.01-1.16) in nulliparous women and ranged from 1.03 (0.96-1.10) in parous women with one birth to 1.26 (1.16-1.37) in women with at least four births. For CASP8-rs17468277, the per-allele OR was 0.91 (0.85-0.98) in those with an alcohol intake of <20 g/day and 1.45 (1.14-1.85) in those who drank ≥ 20 g/day. Additionally, interaction was found between 1p11.2-rs11249433 and ever being parous (Pinteraction = 5.3 × 10(-5)), with a per-allele OR of 1.14 (1.11-1.17) in parous women and 0.98 (0.92-1.05) in nulliparous women. These data provide first strong evidence that the risk of breast cancer associated with some common genetic variants may vary with environmental risk factors.

Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with prognosis of estrogen receptor-negative breast cancer after chemotherapy.

INTRODUCTION: Tumor lymphocyte infiltration is associated with clinical response to chemotherapy in estrogen receptor (ER) negative breast cancer. To identify variants in immunosuppressive pathway genes associated with prognosis after adjuvant chemotherapy for ER-negative patients, we studied stage I-III invasive breast cancer patients of European ancestry, including 9,334 ER-positive (3,151 treated with chemotherapy) and 2,334 ER-negative patients (1,499 treated with chemotherapy). METHODS: We pooled data from sixteen studies from the Breast Cancer Association Consortium (BCAC), and employed two independent studies for replications. Overall 3,610 single nucleotide polymorphisms (SNPs) in 133 genes were genotyped as part of the Collaborative Oncological Gene-environment Study, in which phenotype and clinical data were collected and harmonized. Multivariable Cox proportional hazard regression was used to assess genetic associations with overall survival (OS) and breast cancer-specific survival (BCSS). Heterogeneity according to chemotherapy or ER status was evaluated with the log-likelihood ratio test. RESULTS: Three independent SNPs in TGFBR2 and IL12B were associated with OS (P <10⁻³) solely in ER-negative patients after chemotherapy (267 events). Poorer OS associated with TGFBR2 rs1367610 (G > C) (per allele hazard ratio (HR) 1.54 (95% confidence interval (CI) 1.22 to 1.95), P = 3.08 × 10⁻4) was not found in ER-negative patients without chemotherapy or ER-positive patients with chemotherapy (P for interaction <10-3). Two SNPs in IL12B (r² = 0.20) showed different associations with ER-negative disease after chemotherapy: rs2546892 (G > A) with poorer OS (HR 1.50 (95% CI 1.21 to 1.86), P = 1.81 × 10⁻4), and rs2853694 (A > C) with improved OS (HR 0.73 (95% CI 0.61 to 0.87), P = 3.67 × 10⁻4). Similar associations were observed with BCSS. Association with TGFBR2 rs1367610 but not IL12B variants replicated using BCAC Asian samples and the independent Prospective Study of Outcomes in Sporadic versus Hereditary Breast Cancer Study and yielded a combined HR of 1.57 ((95% CI 1.28 to 1.94), P = 2.05 × 10⁻5) without study heterogeneity. CONCLUSIONS: TGFBR2 variants may have prognostic and predictive value in ER-negative breast cancer patients treated with adjuvant chemotherapy. Our findings provide further insights into the development of immunotherapeutic targets for ER-negative breast cancer.

Annexin A1 expression in a pooled breast cancer series: association with tumor subtypes and prognosis.

BACKGROUND: Annexin A1 (ANXA1) is a protein related with the carcinogenesis process and metastasis formation in many tumors. However, little is known about the prognostic value of ANXA1 in breast cancer. The purpose of this study is to evaluate the association between ANXA1 expression, BRCA1/2 germline carriership, specific tumor subtypes and survival in breast cancer patients. METHODS: Clinical-pathological information and follow-up data were collected from nine breast cancer studies from the Breast Cancer Association Consortium (BCAC) (n = 5,752) and from one study of familial breast cancer patients with BRCA1/2 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. RESULTS: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P <0.0001. ANXA1 was also highly expressed in BCAC tumors that were poorly differentiated, triple negative, EGFR-CK5/6 positive or had developed in patients at a young age. In the first 5 years of follow-up, patients with ANXA1 positive tumors had a worse breast cancer-specific survival (BCSS) than ANXA1 negative (HRadj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). CONCLUSIONS: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases.