Tumour Infiltrating Lymphocytes (TILs) and immune composition in breast cancer patients from Kenya: Spatial distributions and associations with risk factors and tumour characteristics.

BACKGROUND: The immune landscape of breast cancer (BC) in patients from Sub Saharan Africa is understudied. Our aims were to describe the distribution of Tumour Infiltrating Lymphocytes (TILs) within the intratumoural stroma (sTILs) and the leading/invasive edge stroma (LE-TILs), and to evaluate TILs across BC subtypes with established risk factors and clinical characteristics in Kenyan women. METHODS: Visual quantification of sTILs and LE-TILs were performed on Haematoxylin and eosin -stained pathologically confirmed BC cases based on the International TIL working group guidelines. Tissue Microarrays were constructed and stained with immunohistochemistry (IHC) for CD3, CD4, CD8, CD68, CD20, and FOXP3. Linear and logistic regression models were used to assess associations between risk factors and tumour features with IHC markers and total TILs, after adjusting for other covariates. RESULTS: A total of 226 invasive BC cases were included. Overall, LE-TIL (mean = 27.9, SD = 24.5) proportions were significantly higher than sTIL (mean = 13.5, SD = 15.8). Both sTILs and LE- TILs were predominantly composed of CD3, CD8, and CD68. We found higher TILs to be associated with high KI67/high grade and aggressive tumour subtypes, although these associations varied by TIL locations. Older age at menarche (≥ 15 vs. < 15 years) was associated with higher CD3 (OR: 2.06, 95%CI:1.26-3.37), but only for the intra-tumour stroma. CONCLUSION: The TIL enrichment in more aggressive BCs is similar to previously published data in other populations. The distinct associations of sTIL/LE-TIL measures with most examined factors highlight the importance of spatial TIL evaluations in future studies.

Nighttime eating and breast cancer among Chinese women in Hong Kong.

BACKGROUND: A novel line of research suggests that eating at nighttime may have several metabolic consequences that are highly relevant to breast cancer. We investigated the association between nighttime eating habits after 10 p.m. and breast cancer in Hong Kong women. METHODS: A hospital-based case-control study was conducted during 2012-2015. A total of 922 patients with incident breast cancer (cases) and 913 hospital controls were recruited and interviewed using a standard questionnaire including information on eating behavior during both daytime and nighttime. We collected the timing, duration, types and frequencies of food intake of eating at nighttime. Odds ratios (ORs) for the risk of breast cancer in relation to nighttime eating-related variables were calculated by unconditional multivariable logistic regression. RESULTS: Eating at night after 10 pm was significantly associated with breast cancer with an adjusted OR of 1.50 (95% confidence interval (CI) 1.06-2.12, P = 0.02), and the associations were stronger in women who had the longest duration of nighttime eating (≥20 years) (adjusted OR = 2.28 (95% CI 1.13-4.61, P = 0.02) and who ate late (midnight to 2 a.m.) (adjusted OR = 2.73, 95% CI 1.01-6.99, P = 0.04). Interestingly, nighttime eating was only associated with breast cancer among women who consumed staple foods (OR = 2.16, 95% CI 1.42-3.29, P < 0.001) but not those who ate vegetables or fruits as nighttime meals. The significant association between nighttime eating and breast cancer was observed among women with body mass index (BMI) <25 (OR = 2.29, 95% CI 1.48-3.52, P < 0.001) but not among women with BMI ≥25. CONCLUSIONS: Results from this study suggest a possible association between nighttime eating behavior and breast cancer. These findings need to be confirmed by independent large studies.

ERß splice variant expression in four large cohorts of human breast cancer patient tumors.

Though the role of Estrogen Receptor (ER)α in breast cancer has been studied extensively, there is little consensus about the role of alternative ER isoform ERß in breast cancer biology. ERß has significant sequence homology to ERα but is located on a different chromosome and maintains both overlapping and unique functional attributes. Five variants exist, resulting from alternative splicing of the C-terminal region of ERß. The relevance of ERß variants in breast cancer outcomes and response to therapy is difficult to assess because of conflicting reports in the literature, likely due to variable methods used to assess ERß in patient tumors. Here, we quantitatively assess expression of ERß splice variants on over 2,000 breast cancer patient samples. Antibodies against ERß variants were validated for staining specificity in cell lines by siRNA knockdown of ESR2 and staining reproducibility on formalin-fixed paraffin-embedded tissue by quantitative immunofluorescence (QIF) using AQUA technology. We found antibodies against splice variants ERß1 and ERß5, but not ERß2/cx, which were sensitive, specific, and reproducible. QIF staining of validated antibodies showed both ERß1 and ERß5 QIF scores, which have a normal (bell shaped) distribution on most cohorts assessed, and their expression is significantly associated with each other. Extensive survival analyses show that ERß1 is not a prognostic or predictive biomarker for breast cancer. ERß5 appears to be a context-dependent marker of worse outcome in HER2-positive and triple-negative patients, suggesting an unknown biological function in the absence of ERα.

Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family.

While several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations we identified a novel NRAS variant (c.170A > C, p.D57A) in an Italian melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11,273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2 + binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A > C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

Tumor-Associated Stromal Cellular Density as a Predictor of Recurrence and Mortality in Breast Cancer: Results from Ethnically Diverse Study Populations.

PURPOSE: Tumor-associated stroma is comprised of fibroblasts, tumor-infiltrating lymphocytes (TIL), macrophages, endothelial cells, and other cells that interactively influence tumor progression through inflammation and wound repair. Although gene-expression signatures reflecting wound repair predict breast cancer survival, it is unclear whether combined density of tumor-associated stromal cells, a morphologic proxy for inflammation and wound repair signatures on routine hematoxylin and eosin (H&E)-stained sections, is of prognostic relevance. METHODS: By applying machine learning to digitized H&E-stained sections for 2,084 breast cancer patients from China (n = 596; 24-55 years), Poland (n = 810; 31-75 years), and the United States (n = 678; 55-78 years), we characterized tumor-associated stromal cellular density (SCD) as the percentage of tumor-stroma that is occupied by nucleated cells. Hazard ratios (HR) and 95% confidence intervals (CI) for associations between SCD and clinical outcomes [recurrence (China) and mortality (Poland and the United States)] were estimated using Cox proportional hazard regression, adjusted for clinical variables. RESULTS: SCD was independently predictive of poor clinical outcomes in hormone receptor-positive (luminal) tumors from China [multivariable HR (95% CI)fourth(Q4) vs. first(Q1) quartile = 1.86 (1.06-3.26); P trend = 0.03], Poland [HR (95% CI)Q4 vs. Q1 = 1.80 (1.12-2.89); P trend = 0.01], and the United States [HR (95% CI)Q4 vs. Q1 = 2.42 (1.33-4.42); P trend = 0.002]. In general, SCD provided more prognostic information than most classic clinicopathologic factors, including grade, size, PR, HER2, IHC4, and TILs, predicting clinical outcomes irrespective of menopausal or lymph nodal status. SCD was not predictive of outcomes in hormone receptor-negative tumors. CONCLUSIONS: Our findings support the independent prognostic value of tumor-associated SCD among ethnically diverse luminal breast cancer patients. IMPACT: Assessment of tumor-associated SCD on standard H&E could help refine prognostic assessment and therapeutic decision making in luminal breast cancer.

Identification of modifier genes for cutaneous malignant melanoma in melanoma-prone families with and without CDKN2A mutations.

CDKN2A is a major susceptibility gene for cutaneous malignant melanoma (CMM), but the variable penetrance and clinical manifestations among mutation carriers suggest the existence of modifier factors. The goal of this study was to identify modifier genes for CMM in CMM-prone families with or without CDKN2A mutations. We genotyped 537 individuals (107 CMM) from 28 families (19 CDKN2A+, 9 CDKN2A-) for 1,536 SNPs in 152 genes involved in DNA repair, apoptosis and immune response pathways. We used conditional logistic regression to account for family ascertainment and differences in disease prevalence among families. Pathway- and gene-based permutation analyses were used to assess the risk of CMM associated with genes in the 5 pathways (DNA repair, apoptosis, TNF/NFkappaB, TH1:TH2 and other immune regulation). Our analyses identified some candidate genes such as FAS, BCL7A, CASP14, TRAF6, WRN, IL9, IL10RB, TNFSF8, TNFRSF9 and JAK3 that were associated with CMM risk (p<0.01, gene-based test). After correction for multiple comparisons, IL9 remained significant (Bonferroni p<0.05). The effects of some genes were stronger in CDKN2A-positive families (BCL7A and IL9), while some were stronger in CDKN2A-negative families (BCL2L1). Our findings support the hypothesis that common genetic polymorphisms in DNA repair, apoptosis and immune response pathways may modify the risk of CMM in CMM-prone families with or without CDKN2A mutations.

Construction and validation of tissue microarrays of ductal carcinoma in situ and terminal duct lobular units associated with invasive breast carcinoma.

Construction of tissue microarrays (TMAs) to efficiently characterize large sets of noninvasive epithelial lesions in the breast by immunohistochemistry is an appealing investigative approach, but presents technical challenges. We report methodologic studies performed to optimize methods for building TMAs from noninvasive breast tissues collected in a large case-control study of breast cancer. Using a manual arraying technique with 2.0-mm diameter needles, we constructed TMAs from specimens obtained from 32 women with breast cancer containing the following targets: (1) 28 terminal duct lobular units (TDLUs); (2) 28 ductal carcinomas in situ, and (3) 23 invasive carcinomas. Using careful target selection, we achieved representation of approximately 80% of noninvasive targets with sustained preservation through section 30 of the TMAs. Immunohistochemical staining of TDLU targets demonstrated positive staining for estrogen receptor (ER) in 30.8% of tubules and for progesterone receptor (PR) in 50.0%. To establish an efficient method to evaluate staining results in TDLUs, we created a categorical scoring system to approximate the percentage of tubules containing positive stained cells (<10%, 10% to 50%, >or=50%), and compared the results with those obtained by tubule counting. Comparison between the two methods demonstrated exact agreement for 70.8% of ER and 79.2% of PR stains without two-category discrepancies. ER/PR expression levels in multiple (up to 4) noninvasive targets of the same tissue type (TDLU or DCIS) from a single block showed good correlation. These data suggest that it is feasible to produce TMAs of noninvasive breast structures, albeit with careful selection of targets, and that immunostains of such cores may permit efficient immunohistochemical characterization of peritumoral tissues. Additional exploration of this approach is needed.

Evaluation of risk factors for nasopharyngeal carcinoma in high-risk nasopharyngeal carcinoma families in Taiwan.

A study of nasopharyngeal carcinoma (NPC) families with two or more affected members was conducted in Taiwan (265 families with 2,444 individuals, 502 affected and 1,942 unaffected) to determine the association between NPC and potential etiologic factors in NPC high-risk families. Similar to results from a previous case-control study in Taiwan, Guangdong salted fish consumption during childhood, exposure to wood, and betel nut consumption were all associated with elevated NPC risk using conditional logistic regression, although these associations were not as strong as in the case-control study possibly due to shared environment among family members. Risk associated with cumulative wood exposure and salted fish consumption before age 10 was stronger in families with early NPC age-onset [odds ratio (OR(wood)), 5.10; 95% confidence interval (95% CI), 1.50-17.34; OR(fish), 3.94; 95% CI, 1.47-10.55] or three or more affected members (OR(wood), 4.41; 95% CI, 1.58-12.30; OR(fish), 4.27; 95% CI, 1.10-16.47). In contrast, a tendency for elevated risk was noted for betel nut use in late age-onset families (OR, 2.44; 95% CI, 1.16-5.13) and the CYP2E1 c2 allele in families with less than three affected members (OR, 2.06; 95% CI, 1.04-3.35). Risk estimates associated with these exposures were similar when the analyses were restricted to EBV-seropositive subjects. To better adjust for degree of relationship among family members and residual genetic correlations, we also calculated ORs using a variance components model. The results from the two methods were similar indicating that the risk estimates from conditional logistic regression were unbiased.

Identification of susceptibility loci for complex diseases in a case-control association study using the Genetic Analysis Workshop 14 dataset.

Although current methods in genetic epidemiology have been extremely successful in identifying genetic loci responsible for Mendelian traits, most common diseases do not follow simple Mendelian modes of inheritance. It is important to consider how our current methodologies function in the realm of complex diseases. The aim of this study was to determine the ability of conventional association methods to fine map a locus of interest. Six study populations were selected from 10 replicates (New York) from the Genetic Analysis Workshop 14 simulated dataset and analyzed for association between the disease trait and locus D2. Genotypes from 45 single-nucleotide polymorphisms in the telomeric region of chromosome 3 were analyzed by Pearson's chi-square tests for independence to test for association with the disease trait of interest. A significant association was detected within the region; however, it was found 3 cM from the documented location of the D2 disease locus. This result was most likely due to the method used for data simulation. In general, this study showed that conventional case-control association methods could detect disease loci responsible for the development of complex traits.

Linkage analysis of the GAW14 simulated dataset with microsatellite and single-nucleotide polymorphism markers in large pedigrees.

Recent studies have suggested that a high-density single nucleotide polymorphism (SNP) marker set could provide equivalent or even superior information compared with currently used microsatellite (STR) marker sets for gene mapping by linkage. The focus of this study was to compare results obtained from linkage analyses involving extended pedigrees with STR and single-nucleotide polymorphism (SNP) marker sets. We also wanted to compare the performance of current linkage programs in the presence of high marker density and extended pedigree structures. One replicate of the Genetic Analysis Workshop 14 (GAW14) simulated extended pedigrees (n = 50) from New York City was analyzed to identify the major gene D2. Four marker sets with varying information content and density on chromosome 3 (STR [7.5 cM]; SNP [3 cM, 1 cM, 0.3 cM]) were analyzed to detect two traits, the original affection status, and a redefined trait more closely correlated with D2. Multipoint parametric and nonparametric linkage analyses (NPL) were performed using programs GENEHUNTER, MERLIN, SIMWALK2, and S.A.G.E. SIBPAL. Our results suggested that the densest SNP map (0.3 cM) had the greatest power to detect linkage for the original trait (genetic heterogeneity), with the highest LOD score/NPL score and mapping precision. However, no significant improvement in linkage signals was observed with the densest SNP map compared with STR or SNP-1 cM maps for the redefined affection status (genetic homogeneity), possibly due to the extremely high information contents for all maps. Finally, our results suggested that each linkage program had limitations in handling the large, complex pedigrees as well as a high-density SNP marker set.

Familial risks and estrogen receptor-positive breast cancer in Hong Kong Chinese women.

PURPOSE: The role of family history to the risk of breast cancer was analyzed by incorporating menopausal status in Hong Kong Chinese women, with a particular respect to the estrogen receptor-positive (ER+) type. METHODS: Seven hundred and forty seven breast cancer incident cases and 781 hospital controls who had completed information on family cancer history in first-degree relatives (nature father, mother, and siblings) were recruited. Odds ratio for breast cancer were calculated by unconditional multiple logistic regression, stratified by menopausal status (a surrogate of endogenous female sex hormone level and age) and type of relative affected with the disease. Further subgroup analysis by tumor type according to ER status was investigated. RESULTS: Altogether 52 (6.96%) breast cancer cases and 23 (2.95%) controls was found that the patients' one or more first-degree relatives had a history of breast cancer, showing an adjusted odds ratio (OR) of 2.41 (95%CI: 1.45-4.02). An excess risk of breast cancer was restricted to the ER+ tumor (OR = 2.43, 95% CI: 1.38-4.28), with a relatively higher risk associated with an affected mother (OR = 3.97, 95%CI: 1.46-10.79) than an affected sister (OR = 2.06, 95%CI: 1.07-3.97), while the relative risk was more prominent in the subgroup of pre-menopausal women. Compared with the breast cancer overall, the familial risks to the ER+ tumor increased progressively with the number of affected first-degree relatives. CONCLUSIONS: This study provides new insights on a relationship between family breast cancer history, menopausal status, and the ER+ breast cancer. A separate risk prediction model for ER+ tumor in Asian population is desired.

Genomic regions linked to alcohol consumption in the Framingham Heart Study.

BACKGROUND: Pedigree, demographic, square-root transformed maximum alcohol (SRMAXAPD) and maximum cigarette (MAXCPD) consumption, and genome-wide scan data from the Framingham Heart Study (FHS) were used to investigate genetic factors that may affect alcohol and cigarette consumption in this population-based sample. RESULTS: A significant sister:sister correlation greater than spouse correlation was observed for MAXCPD only. Single-point sib-pair regression analysis provided nominal evidence for linkage of loci to both SRMAXAPD and MAXCPD consumption traits, with more significant evidence of linkage to SRMAXAPD than to MAXCPD. One genomic region, chr9q21.11, exhibits significant multi-point sib-pair regression to SRMAXAPD. CONCLUSION: SRMAXAPD exhibits greater evidence for genetic linkage than does MAXCPD in the FHS sample. Four regions of the genome exhibiting nominal evidence for linkage to SRMAXAPD in the FHS sample correspond to regions of the genome previously identified as linked to alcoholism or related traits in the family data set ascertained on individuals affected with alcohol dependence known as COGA.

Constitutional promoter methylation and risk of familial melanoma.

Constitutional epigenetic changes detected in blood or non-disease involving tissues have been associated with disease susceptibility. We measured promoter methylation of CDKN2A (p16 and p14ARF) and 13 melanoma-related genes using bisulfite pyrosequencing of blood DNA from 114 cases and 122 controls in 64 melanoma-prone families (26 segregating CDKN2A germline mutations). We also obtained gene expression data for these genes using microarrays from the same blood samples. We observed that CDKN2A epimutation is rare in melanoma families, and therefore is unlikely to cause major susceptibility in families without CDKN2A mutations. Although methylation levels for most gene promoters were very low (<5%), we observed a significantly reduced promoter methylation (odds ratio = 0.63, 95% confidence interval = 0.50, 0.80, P<0.001) and increased expression (fold change = 1.27, P = 0.048) for TNFRSF10C in melanoma cases. Future research in large prospective studies using both normal and melanoma tissues is required to assess the significance of TNFRSF10C methylation and expression changes in melanoma susceptibility.

Variants associated with susceptibility to pancreatic cancer and melanoma do not reciprocally affect risk.

BACKGROUND: Melanoma cases may exist in pancreatic cancer kindreds, whereas there is increased risk of pancreatic cancer in familial melanoma. The two cancers may share genetic susceptibility variants in common. METHODS: Three dbGaP (datasets in Genotypes and Phenotypes)-deposited GWAS (genome-wide association study) datasets (MD Anderson melanoma, PanScan 1, and PanScan 2 for pancreatic cancer) were used. Thirty-seven melanoma susceptibility variants in 22 genomic regions from published GWAS, plus melanoma-related genes and pathways were examined for pancreatic cancer risk in the PanScan datasets. Conversely, nine known pancreatic cancer susceptibility variants were examined for melanoma risk in the MD Anderson dataset. RESULTS: In the PanScan data, initial associations were found with melanoma susceptibility variants in NCOA6 [rs4911442; OR, 1.32; 95% confidence interval (CI), 1.03-1.70; P = 0.03], YWHAZP5 (rs17119461; OR, 2.62; 95% CI, 1.08-6.35; P = 0.03), and YWHAZP5 (rs17119490; OR, 2.62; 95% CI, 1.08-6.34; P = 0.03), TYRP1 (P = 0.04), and IFNA13 (P = 0.04). In the melanoma dataset, two pancreatic cancer susceptibility variants were associated: NR5A2 (rs12029406; OR, 1.39; 95% CI, 1.01-1.92; P = 0.04) and CLPTM1L-TERT (rs401681; OR, 1.16; 95% CI, 1.01-1.34; P = 0.04). None of these associations remained significant after correcting for multiple comparisons. CONCLUSION: Reported variants of melanoma genes and pathways do not play a role in pancreatic cancer predisposition. Reciprocally, pancreatic cancer susceptibility variants are not associated with melanoma risk. IMPACT: Known melanoma-related genes and pathways, as well as GWAS-derived susceptibility variants of melanoma and pancreatic cancer, do not explain the shared genetic etiology of these two cancers. Cancer Epidemiol Biomarkers Prev; 23(6); 1121-4. ©2014 AACR.

Associations of 9p21 variants with cutaneous malignant melanoma, nevi, and pigmentation phenotypes in melanoma-prone families with and without CDKN2A mutations.

Chromosome 9p21 has been implicated in the pathogenesis of cutaneous malignant melanoma (CMM). In addition to CDKN2A, the major known high-risk susceptibility gene for CMM, recent studies suggest that other 9p21 genes may be involved in melanoma/nevi development. To identify 9p21 variants that influence susceptibility to CMM and number of nevi in CMM-prone families with and without CDKN2A mutations, we analyzed 562 individuals (183 CMM) from 53 families (23 CDKN2A+, 30 CDKN2A-) for 233 tagging SNPs in 21 genes at 9p21. Single SNP- and gene-based regression analyses were used to assess the risk of CMM, nevi count, skin complexion, and tanning ability associated with these SNPs and genes. We found that SNP rs7023329 in the MTAP gene was associated with number of nevi (P (trend) = 0.003) confirming a recent finding by a genome-wide association study. In addition, three SNPs in the ACO1 gene, rs7855483 (P (trend) = 0.002), rs17288067 (P (trend) = 0.0009), and rs10813813 (P (trend) = 0.005), showed the strongest associations with CMM risk. None of the examined 9p21 SNPs was associated with skin complexion, whereas two SNPs, rs10964862 in IFNW1 (P (trend) = 0.003), and rs13290968 in TUSC1 (P (trend) = 0.0006), were associated with tanning ability. Gene-based analyses suggested that the ACO1 gene was significantly associated with CMM (P = 0.0004); genes IFNW1 (P = 0.002) and ACO1 (P = 0.0002) were significantly associated with tanning ability. Our findings are consistent with recent proposals that additional 9p21 genes may contribute to CMM susceptibility in CMM-prone families. These genetic variants may, at least partially, exert their effects through nevi and tanning ability.

Association of human leukocyte antigens with nasopharyngeal carcinoma in high-risk multiplex families in Taiwan.

An association between specific human leukocyte antigens (HLA) alleles and nasopharyngeal carcinoma (NPC) has been reported for sporadic NPC, but studies of familial NPC are lacking. We evaluated this association with familial NPC in a study of 301 NPC cases and 1010 family and community controls from Taiwan. Class I HLA alleles were characterized using a sequence-based typing protocol. Allele frequencies between case and control groups were compared by chi(2) or exact tests. For alleles associated with NPC, odds ratios (OR) and 95% confidence intervals (CI) were calculated. Similar allelic frequency distribution and HLA associations were found as those previously reported for sporadic NPC: protective effect for HLA-A*1101 and increased risk for HLA-A*0207, HLA-A*3303, HLA-B*3802, and HLA-B*5801. Overall, the magnitude of observed associations was weakest when cases were compared with sibling controls and strongest when compared with unrelated community controls. Evaluating the joint effect of HLA-A*0207 and HLA-B*4601, individuals who were carriers of HLA-A*0207 with or without the presence of HLA-B*4601 had a 1.9-fold (95% CI = 1.0-3.4) and 2.1-fold (95% CI = 0.83-5.3) risk of NPC, respectively. Conversely, carriers of HLA-B*4601 in the absence of HLA-A*0207 had a 50% reduction in NPC risk (95% CI = 0.27-0.93). Comparable findings from our family study and those from previous sporadic studies were found with the notable exception of a lack of positive association between HLA-B*4601 and familial NPC in the absence of HLA-A*0207. This finding requires replication in larger studies.

Estimating age-specific breast cancer risks: a descriptive tool to identify age interactions.

OBJECTIVE: Clarifying age-specific female breast cancer risks and interactions may provide important etiologic clues. METHOD: Using a population-based case-control study in Poland (2000-2003) of 2,386 incident breast cancer cases and 2,502 control subjects aged 25-74 years, we estimated age-specific breast cancer incidence rates according to risk factors. RESULTS: Breast cancer risks were elevated among women with positive family history (FH), younger age at menarche, older age at first full-term birth, nulliparity, exogenous hormonal usage, and reduced physical activity (PA). Notwithstanding overall risks, we observed statistically significant quantitative (non-crossover) and qualitative (crossover) age interactions for all risk factors except for FH and PA. For example, nulliparity compared to parity reduced breast cancer risk among women ages 25-39 years then rates crossed or reversed, after which nulliparity increased relative risks among women ages 40-74 years. CONCLUSION: Though quantitative age interactions could be expected, qualitative interactions were somewhat counterintuitive. If confirmed in other populations, qualitative interactions for a continuous covariate such as age will be difficult to reconcile in a sequential (multistep or monolithic) 'stochastic' breast cancer model. Alternatively, the reversal of relative risks among younger and older women suggests subgroup heterogeneity with different etiologic mechanisms for early-onset and late-onset breast cancer types.

Genetic variation of Cytochrome P450 1B1 (CYP1B1) and risk of breast cancer among Polish women.

Four single nucleotide polymorphisms (SNPs) in CYP1B1 (Ex2 + 143 C > G, Ex2 + 356 G > T, Ex3 + 251 G > C, Ex3 + 315 A > G) cause amino acid changes (R48G, A119S, L432V and N453S, respectively) and are associated with increased formation of catechol estrogens; however, epidemiologic evidence only weakly supports an association between these variants and breast cancer risk. Because genetic variability conferring increased susceptibility could exist beyond these putative functional variants, we comprehensively examined the common genetic variability within CYP1B1. A total of eight haplotype-tagging (ht)SNPs (including Ex3 + 315 A > G), in addition to two putatively functional SNPs (Ex2 + 143 C > G and Ex3 + 251 G > C), were selected and genotyped in a large case-control study of Polish women (1995 cases and 2296 controls). Haplotypes were estimated using the expectation-maximization algorithm, and overall differences in the haplotype distribution between cases and controls were assessed using a global score test. We also evaluated levels of tumor CYP1B1 protein expression in a subset of 841 cases by immunohistochemistry, and their association with genetic variants. In the Polish population, we observed two linkage disequilibrium (LD)-defined blocks. Neither haplotypes (global P-value of 0.99 and 0.67 for each block of LD, respectively), nor individual SNPs (including three putatively functional SNPs) were associated with breast cancer risk. CYP1B1 was expressed in most tumor tissues (98%), and the level of expression was not related to the studied genetic variants. We found little evidence for modification of the estimated effect of haplotypes or individual SNPs by age, family history of breast cancer, or tumor hormone receptor status. The present study provides strong evidence against the existence of a substantial overall association between common genetic variation in CYP1B1 and breast cancer risk.