Analysis of CENP-B Boxes as Anchor of Kinetochores in Centromeres of Human Chromosomes.

The kinetochore is a multiprotein structure that attaches at one end to DNA in the centromere and at the other end to microtubules in the mitotic spindle. By connecting centromere and spindle, the kinetochore controls the migration of chromosomes during cell division. The exact position where the kinetochore assembles on each centromere was uncertain because large sections of centromeric DNA had not been sequenced due to highly repetitive alpha-satellite arrays. Embedded in the arrays is a 17 bp consensus sequence, the so-called CENP-B box, which binds the CENP-B protein, the only protein that binds directly to centromeric DNA. Recently, the Telomere-to-Telomere Consortium published the complete centromeric DNA sequences of all chromosomes including their epigenetic modifications in the T2T-CHM13 map. I used data from the T2T-CHM13 map to locate the CENP-B boxes in the centromeres as anchor of kinetochores. Most of the CENP-B boxes in centromeric DNA are methylated with the exception of the so-called centromere dip region (CDR), where CENP-B protein dimers bind to adjacent unmethylated CENP-B boxes and interact with CENP-A and CENP-C proteins to assemble the kinetochore. The centromeres of all chromosomes combined have a size of 407 Mb of which the kinetochores account for 5.0 Mb or 1.2%. There is no correlation between centromere and kinetochore size (P = .77). While the number of CENP-B boxes varies 4-fold between chromosomes, their density (number/Kb) varies less than 2-fold with a mean of 2.61 ± 0.33. The narrow range ensures a uniform pull of the spindle on the centromeres. I illustrate the findings in a model of the human kinetochore anchored at unmethylated CENP-B boxes in the CDR and present circos plots of chromosomes to show the location of kinetochores in their respective centromeres.

Different Tumor Types Share a Common Nuclear Map of Chromosome Territories.

Different tumor types are characterized by unique histopathological patterns including distinctive nuclear architectures. I hypothesized that the difference in nuclear appearance is reflected in different nuclear maps of chromosome territories, the discrete regions occupied by individual chromosomes in the interphase nucleus. To test this hypothesis, I used interchromosomal translocations (ITLs) as an analytical tool to map chromosome territories in 11 different tumor types from the TCGA PanCancer database encompassing 6003 tumors with 5295 ITLs. For each chromosome I determined the number and percentage of all ITLs for any given tumor type. Chromosomes were ranked according to the frequency and percentage of ITLs per chromosome. The ranking showed similar patterns for all tumor types. Chromosomes 1, 8, 11, 17, and 19 were ranked in the top quarter, accounting for 35.2% of 5295 ITLs, whereas chromosomes 13, 15, 18, 21, and X were in the bottom quarter, accounting for only 10.5% ITLs. The correlation between the chromosome ranking in the total group of 6003 tumors and the ranking in individual tumor types was significant, ranging from P < .0001 to .0033. Thus, contrary to my hypothesis, different tumor types share a common nuclear map of chromosome territories. Based on the large number of ITLs in 11 different types of malignancy one can discern a shared pattern of chromosome territories in cancer and propose a probabilistic model of chromosomes 1, 8, 11, 17, 19 in the center of the nucleus and chromosomes 13, 15, 18, 21, X at the periphery.

Mutation Burden Independently Predicts Survival in the Pan-Cancer Atlas.

PURPOSE: Long-standing clinical predictors of cancer survival have included histopathologic type, stage, and grade. We hypothesized that the principal categories of tumor somatic mutations might also portend survival. We investigated this hypothesis using the Pan-Cancer Atlas, encompassing clinical, genomic, and outcome data of 10,652 patients and 32 cancer types. METHODS: We evaluated the prognostic capability of cancer type, stage, grade and the burden of each major mutation category on overall and disease-specific survival. Mutation categories included short substitution and insertion-deletion mutations (SMs), copy number alterations (CNAs), and gene fusions. RESULTS: SM count and CNA fraction proved to be strong independent predictors of survival (joint P = 5.3e-95) that remained highly significant when adjusted for the traditional factors. Importantly, the relationship between mutation burden and survival proved to be nonlinear (P = 9.5e-56); survival improved at both low- and high-burden extremes. In clinically predictive modeling, SM count together with CNA fraction meaningfully distinguished survival even among patients sharing a given cancer type, stage, or grade. CONCLUSION: Burden of somatic mutation is a key index of survival of analogous clinical utility to these traditional factors.

Implementation of a closed-loop reporting system for critical values and clinical communication in compliance with goals of the joint commission.

BACKGROUND: Current practices of reporting critical laboratory values make it challenging to measure and assess the timeliness of receipt by the treating physician as required by The Joint Commission's 2008 National Patient Safety Goals. METHODS: A multidisciplinary team of laboratorians, clinicians, and information technology experts developed an electronic ALERTS system that reports critical values via the laboratory and hospital information systems to alphanumeric pagers of clinicians and ensures failsafe notification, instant documentation, automatic tracking, escalation, and reporting of critical value alerts. A method for automated acknowledgment of message receipt was incorporated into the system design. RESULTS: The ALERTS system has been applied to inpatients and eliminated approximately 9000 phone calls a year made by medical technologists. Although a small number of phone calls were still made as a result of pages not acknowledged by clinicians within 10 min, they were made by telephone operators, who either contacted the same physician who was initially paged by the automated system or identified and contacted alternate physicians or the patient's nurse. Overall, documentation of physician acknowledgment of receipt in the electronic medical record increased to 95% of critical values over 9 months, while the median time decreased to <3 min. CONCLUSIONS: We improved laboratory efficiency and physician communication by developing an electronic system for reporting of critical values that is in compliance with The Joint Commission's goals.

Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation.

The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17beta-estradiol (E2) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E2 in the presence of 2'-deoxyguanosine or 2'-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E2, the 2- and 4-catechol estrogens (2-OHE2, 4-OHE2), and the depurinating adducts 4-OHE(2)-1(alpha,beta)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE(2)-1(alpha,beta)-N3-adenine (4-OHE2-N3-Ade). CYP1B1 oxidized E2 to the catechol 4-OHE2 and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE2-N7-Gua adduct (Km, 4.6+/-0.7 micromol/L; kcat, 45+/-1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (Km, 4.6+/-1.0 micromol/L; kcat, 30+/-1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE2 production. The results present direct proof of CYP1B1-mediated, E2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis.

Interchromosomal Translocations as a Means to Map Chromosome Territories in Breast Cancer.

The genome-wide identification of mutated genes is an important advance in our understanding of tumor biology, but several fundamental questions remain open. How do these genes act together to promote cancer development and, a related question, how are they spatially arranged in the nucleus to allow coordinated expression? We examined the nuclear topography of mutated genes in breast cancer and their relation to chromosome territories (CTs). We performed a literature review and analyzed 1 type of mutation, interchromosomal translocations, in 1546 primary breast cancers to infer the spatial arrangement of chromosomes. The cosegregation of all observed fusion genes was used to create a matrix of genome-wide CT contacts and develop a tentative CT map of breast cancer. Regression analysis was performed to determine the association between CTs and all types of mutations. Chromosomes 17, 11, 8, and 1 had the majority of interchromosomal fusions and are presumably clustered in the nuclear center, whereas chromosomes 22, 21, X, and 18 had the lowest number of contacts, likely reflecting a more peripheral position. Regression analysis revealed that there was no significant association between chromosome length indicated by the number of base pairs per chromosome and the number of total (inter- and intrachromosomal) translocations, point mutations, or copy number aberrations (CNAs). The gene density of chromosomes (genes/Mb) was significantly correlated with total translocations (P = .02), but not with point mutations P = .19 and CNAs P = .62. Finally, the association of the 3 genetic alterations with the CT map deduced from the interchromosomal fusions was significant, ie, total translocations P = 7 × 10-11, point mutations P = .01, CNAs P = .002. In conclusion, we developed a tentative CT map and observed a spatial association with genetic alterations in breast cancer.

Genomic-Epidemiologic Evidence That Estrogens Promote Breast Cancer Development.

Background: Estrogens are a prime risk factor for breast cancer, yet their causal relation to tumor formation remains uncertain. A recent study of 560 breast cancers identified 82 genes with 916 point mutations as drivers in the genesis of this malignancy. Because estrogens play a major role in breast cancer development and are also known to regulate the expression of numerous genes, we hypothesize that the 82 driver genes are likely to be influenced by estrogens, such as 17ß-estradiol (E2), and the estrogen receptor ESR1 (ERα). Because different types of tumors are characterized by unique sets of cancer driver genes, we also argue that the fraction of driver genes regulated by E2-ESR1 is lower in malignancies not associated with estrogens, e.g., acute myeloid leukemia (AML).Methods: We performed a literature search of each driver gene to determine its E2-ESR1 regulation.Results: Fifty-three of the 82 driver genes (64.6%) identified in breast cancers showed evidence of E2-ESR1 regulation. In contrast, only 19 of 54 mutated driver genes (35.2%) identified in AML were linked to E2-ESR1. Among the 916 driver mutations found in breast cancers, 813 (88.8%) were linked to E2-ESR1 compared with 2,046 of 3,833 in AML (53.4%).Conclusions: Risk assessment revealed that mutations in estrogen-regulated genes are much more likely to be associated with elevated breast cancer risk, while mutations in unregulated genes are more likely to be associated with AML.Impact: These results increase the plausibility that estrogens promote breast cancer development. Cancer Epidemiol Biomarkers Prev; 27(8); 899-907. ©2018 AACR.

Carcinogen-induced histone alteration in normal human mammary epithelial cells.

Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

Mitochondrial DNA G10398A polymorphism and invasive breast cancer in African-American women.

Mitochondria generate oxygen-derived free radicals that damage mitochondrial DNA (mtDNA) as well as nuclear DNA and in turn promote carcinogenesis. The mtDNA G10398A polymorphism alters the structure of Complex I in the mitochondrial electron transport chain, an important site of free radical production. This polymorphism is associated with several neurodegenerative disorders. We hypothesized that the 10398A allele is also associated with breast cancer susceptibility. African mitochondria harbor the 10398A allele less frequently than Caucasian mitochondria, which predominantly carry this allele. Mitochondrial genotypes at this locus were therefore determined in two separate populations of African-American women with invasive breast cancer and in controls. A preliminary study at Vanderbilt University (48 cases, 54 controls) uncovered an association between the 10398A allele and invasive breast cancer in African-American women, [odds ratio (OR), 2.90; 95% confidence interval (95% CI), 0.61-18.3; P = 0.11]. We subsequently validated this finding in a large, population-based, case-control study of breast cancer, the Carolina Breast Cancer Study at the University of North Carolina (654 cases, 605 controls). African-American women in this study with the 10398A allele had a significantly increased risk of invasive breast cancer (OR, 1.60; 95% CI, 1.10-2.31; P = 0.013). The 10398A allele remained an independent risk factor after adjustment for other well-accepted breast cancer risk factors. No association was detectable in white women (879 cases, 760 controls; OR, 1.03; 95% CI, 0.81-1.31; P = 0.81). This study provides novel epidemiologic evidence that the mtDNA 10398A allele influences breast cancer susceptibility in African-American women. mtDNA polymorphisms may be underappreciated factors in breast carcinogenesis.

Estrogens, enzyme variants, and breast cancer: a risk model.

Oxidative metabolites of estrogens have been implicated in the development of breast cancer, yet relatively little is known about the metabolism of estrogens in the normal breast. We developed a mathematical model of mammary estrogen metabolism based on the conversion of 17beta-estradiol (E(2)) by the enzymes cytochrome P450 (CYP) 1A1 and CYP1B1, catechol-O-methyltransferase (COMT), and glutathione S-transferase P1 into eight metabolites [i.e., two catechol estrogens, 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)); three methoxyestrogens, 2-methoxyestradiol, 2-hydroxy-3-methoxyestradiol, and 4-methoxyestradiol; and three glutathione (SG)-estrogen conjugates, 2-OHE(2)-1-SG, 2-OHE(2)-4-SG, and 4-OHE(2)-2-SG]. When used with experimentally determined rate constants with purified enzymes, the model provides for a kinetic analysis of the entire metabolic pathway. The predicted concentration of each metabolite during a 30-minute reaction agreed well with the experimentally derived results. The model also enables simulation for the transient quinones, E(2)-2,3-quinone (E(2)-2,3-Q) and E(2)-3,4-quinone (E(2)-3,4-Q), which are not amenable to direct quantitation. Using experimentally derived rate constants for genetic variants of CYP1A1, CYP1B1, and COMT, we used the model to simulate the kinetic effect of enzyme polymorphisms on the pathway and identified those haplotypes generating the largest amounts of catechols and quinones. Application of the model to a breast cancer case-control population identified a subset of women with an increased risk of breast cancer based on their enzyme haplotypes and consequent E(2)-3,4-Q production. This in silico model integrates both kinetic and genomic data to yield a comprehensive view of estrogen metabolomics in the breast. The model offers the opportunity to combine metabolic, genetic, and lifetime exposure data in assessing estrogens as a breast cancer risk factor.

Methoxyestrogens exert feedback inhibition on cytochrome P450 1A1 and 1B1.

Cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) catalyze the oxidative metabolism of 17 beta-estradiol (E2) to catechol estrogens (2-OHE2 and 4-OHE2) and estrogen quinones, which may lead to DNA damage. Catechol-O-methyltransferase catalyzes the methylation of catechol estrogens to methoxyestrogens (2-MeOE2, 2-OH-3-MeOE2, and 4-MeOE2), which simultaneously lowers the potential for DNA damage and increases the concentration of 2-MeOE2, an antiproliferative metabolite. In this study, we showed that CYP1A1 and CYP1B1 recognized as substrates both the parent hormone E2 and the methoxyestrogens. Using purified recombinant enzymes, we demonstrated that CYP1A1 and CYP1B1 O-demethylated the methoxyestrogens to catechol estrogens according to Michaelis-Menten kinetics. Both CYP1A1 and CYP1B1 demethylated 2-MeOE2 and 2-OH-3-MeOE2 to 2-OHE2, whereas CYP1B1 additionally demethylated 4-MeOE2 to 4-OHE2. Because the P450-mediated oxidation of E2 and the O-demethylation of methoxyestrogens both yielded identical catechol estrogens as products, we used deuterated E2 (E2-d4), unlabeled methoxyestrogens, and gas chromatography/mass spectrometry to examine both reactions simultaneously. Kinetic analysis revealed that methoxyestrogens acted as noncompetitive inhibitors of E2 oxidation with K(i) ranging from 27 to 153 micro M. For both enzymes, the order of inhibition by methoxyestrogens was 2-OH-3-MeOE2 > or = 2-MeOE2 > 4-MeOE2. Thus, methoxyestrogens exert feedback inhibition on CYP1A1- and CYP1B1-mediated oxidative estrogen metabolism, thereby reducing the potential for estrogen-induced DNA damage.

Sequential action of phase I and II enzymes cytochrome p450 1B1 and glutathione S-transferase P1 in mammary estrogen metabolism.

The Phase I enzyme cytochrome p450 1B1 (CYP1B1) has been postulated to play a key role in estrogen-induced mammary carcinogenesis by catalyzing the oxidative metabolism of 17beta-estradiol (E(2)) to catechol estrogens (2-OHE(2) and 4-OHE(2)) and highly reactive estrogen quinones (E(2)-2,3-Q and E(2)-3,4-Q). The potential of the quinones to induce mutagenic DNA lesions is expected to be decreased by their conjugation with glutathione (GSH) either nonenzymatically or catalyzed by glutathione S-transferase P1 (GSTP1), a Phase II enzyme. Because the interaction of the Phase I and Phase II enzymes is not well defined in this setting, we prepared recombinant purified CYP1B1 and GSTP1 to examine their individual and combined roles in the oxidative pathway and used gas and liquid chromatography/mass spectrometry to measure the parent hormone E(2), the catechol estrogens, and the GSH conjugates. 2-OHE(2) and 4-OHE(2) did not form conjugates with GSH alone or in the presence of GSTP1. However, incubation of GSH and CYP1B1 with 2-OHE(2) resulted in nearly linear conjugation through C-4 and C-1 (i.e., 2-OHE(2)-4-SG and 2-OHE(2)-1-SG), whereas the reaction of 4-OHE(2) yielded only 4-OHE(2)-2-SG. When CYP1B1 and GSTP1 were added together, the rate of conjugation was accelerated with a hyperbolic pattern of product formation in the order 4-OHE(2)-2-SG > 2-OHE(2)-4-SG >> 2-OHE(2)-1-SG. Incubation of E(2) with CYP1B1 and GSTP1 resulted in the formation of 4-OHE(2), 2-OHE(2), 4-OHE(2)-2-SG, 2-OHE(2)-4-SG, and 2-OHE(2)-1-SG. The production of GSH-estrogen conjugates was dependent on the concentrations of E(2) and GSTP1 but overall yielded only one-tenth of the catechol estrogen production. The concentration gap between catechol estrogens and GSH-estrogen conjugates may result from nonenzymatic reaction of the labile quinones with other nucleophiles besides GSH or may reflect the lower efficiency of GSTP1 compared with CYP1B1. In summary, both reactions are coordinated qualitatively in terms of product formation and substrate utilization, but the quantitative gap would leave room for the accumulation of estrogen quinones and their potential for DNA damage as part of estrogen-induced mammary carcinogenesis.

Association of homozygous wild-type glutathione S-transferase M1 genotype with increased breast cancer risk.

More than 500 studies have examined the association of the glutathione S-transferase M1 (GSTM1) genotype with various malignancies yielding inconsistent results. The genotyping was based on a PCR assay that identified the GSTM1 null (-/-) genotype but did not distinguish homozygous wild-type (+/+) and heterozygous (+/-) individuals. We developed an assay that allowed the definition of +/+, +/-, and -/- genotypes by separate identification of wild-type and null alleles, which were found with frequencies of 0.225 and 0.775, respectively, in Caucasian women. We applied the new assay to a breast cancer case-control study and identified the +/+ genotype in 14 (6.9%) of 202 control subjects compared with 37 (18.2%) of 203 patients. Compared with women with the -/- genotype, the relative risk of breast cancer for the +/+ genotype was 2.83 (95% confidence interval, 1.45-5.59; P = 0.002), suggesting a protective effect of the GSTM1 deletion.

Estrogen receptor genotypes and haplotypes associated with breast cancer risk.

Nearly one in eight US women will develop breast cancer in their lifetime. Most breast cancer is not associated with a hereditary syndrome, occurs in postmenopausal women, and is estrogen and progesterone receptor-positive. Estrogen exposure is an epidemiologic risk factor for breast cancer and estrogen is a potent mammary mitogen. We studied single nucleotide polymorphisms (SNPs) in estrogen receptors in 615 healthy subjects and 1011 individuals with histologically confirmed breast cancer, all from New York City. We analyzed 13 SNPs in the progesterone receptor gene (PGR), 17 SNPs in estrogen receptor 1 gene (ESR1), and 8 SNPs in the estrogen receptor 2 gene (ESR2). We observed three common haplotypes in ESR1 that were associated with a decreased risk for breast cancer [odds ratio (OR), approximately O.4; 95% confidence interval (CI), 0.2-0.8; P < 0.01]. Another haplotype was associated with an increased risk of breast cancer (OR, 2.1; 95% CI, 1.2-3.8; P < 0.05). A unique risk haplotype was present in approximately 7% of older Ashkenazi Jewish study subjects (OR, 1.7; 95% CI, 1.2-2.4; P < 0.003). We narrowed the ESR1 risk haplotypes to the promoter region and first exon. We define several other haplotypes in Ashkenazi Jews in both ESR1 and ESR2 that may elevate susceptibility to breast cancer. In contrast, we found no association between any PGR variant or haplotype and breast cancer. Genetic epidemiology study replication and functional assays of the haplotypes should permit a better understanding of the role of steroid receptor genetic variants and breast cancer risk.

Immunohistochemical expression of estrogen receptor in enlarged lobular units with columnar alteration in benign breast biopsies: a nested case-control study.

The prognostic and therapeutic implications of estrogen receptor (ER) status in breast cancer are well known. Whether ER status plays a role in benign breast lesions and the progression to malignancy has not been proven. Enlarged lobular units with columnar alteration (ELUCA), also known as unfolded lobular units, have been associated with mild elevations in subsequent breast cancer risk. We examined the association of ERalpha expression in ELUCA with invasive breast cancer risk. A nested case-control study was performed of women with ELUCA who had undergone benign breast surgery. Eighty-two women who developed invasive breast cancer on follow-up were matched by age and year of biopsy with 166 women who did not develop invasive breast cancer. Entry biopsies were stained for ERalpha (clone 6F11) without knowledge of subsequent cancer outcome. ELUCA lesions were scored as positive if greater than 10% of epithelial nuclei stained with ERalpha and at least one ELUCA contained >50% of cells staining for ERalpha. Relative risks of breast cancer were estimated by odds ratios derived from conditional logistic regression analyses. Thirty-nine percent of cases and 56% of controls had positive ERalpha staining in ELUCA. The relative risk of invasive breast cancer in women with ERalpha-negative ELUCA was 1.85 times that of women with ERalpha-positive lesions (95% confidence interval, 1.0-3.4, P=0.04). The presence of ERalpha staining in women with ELUCA is associated with a lower risk of developing subsequent invasive carcinoma. These findings have implications for risk assessment in benign breast biopsies and are of particular interest given the controversy currently surrounding hormone replacement therapy.

Glutathione S-transferase genotypes and cancer risk.

Over 500 studies have examined the association of genetic variants of glutathione S-transferases with various malignancies yielding inconsistent results. The genotyping was based on PCR assays that identified the GSTM1 and GSTT1 null (-/-) genotypes but did not distinguish homozygous wild-type +/+ and heterozygous +/- individuals. Complete GSTM1 and GSTT1 genotyping can be accomplished by recently developed assays [Cancer Res. 64 (2004) 1233-1236; Pharmacogenetics 10 (2000) 557-565] that allow the definition of +/+, +/-, and -/- genotypes by separate identification of the respective GSTM1 and GSTT1 wild-type and null alleles. Application of the new GSTM1 assay to a breast cancer case-control study revealed that the relative risk of breast cancer for the +/+ genotype compared to the -/- genotype was 2.83 (95% confidence interval 1.45-5.59; P=0.002), suggesting a protective effect of the GSTM1 deletion [Cancer Res. 64 (2004) 1233-1236]. Regardless of the explanation for the association between the +/+ genotype and increased breast cancer risk, these results warrant application of true GSTM1 and GSTT1 genotyping to additional or previously analyzed groups with breast cancer or other malignancies.

Increased prevalence of the HFE C282Y hemochromatosis allele in women with breast cancer.

Individuals with the major hemochromatosis (HFE) allele C282Y and iron overload develop hepatocellular and some extrahepatic malignancies at increased rates. No association has been previously reported between the C282Y allele and breast cancer. We hypothesized that due to the pro-oxidant properties of iron, altered iron metabolism in C282Y carriers may promote breast carcinogenesis. Because 1 in 10 Caucasians of Northern European ancestry carries this allele, any impact it may have on breast cancer burden is potentially great. We determined C282Y genotypes in 168 patients who underwent high-dose chemotherapy and blood cell transplantation for cancer: 41 with breast cancer and 127 with predominantly hematological cancers (transplant cohort). Demographic, clinical, and tumor characteristics were reviewed in breast cancer patients. The frequency of C282Y genotypes in breast cancers was compared with the frequency in nonbreast cancers, an outpatient sample from Tennessee (n = 169), and a published United States national sample. The frequency of at least one C282Y allele in breast cancers was higher (36.6%, 5 homozygotes/10 heterozygotes) than frequencies in Tennessee (12.7%, P < 0.001), the general population (12.4%, P < 0.001), and similarly selected nonbreast cancers (17.0%, P = 0.008). The likelihood of breast cancer in the transplant cohort increased with C282Y allele dose (P(trend) = 0.010). These results were supported by the finding in a nontransplant cohort of a higher frequency of C282Y mutations in Caucasian (18.4%, P = 0.039) and African-American (8.5%, P = 0.005) women with breast cancer than race-specific national frequency estimates. A high prevalence of C282Y alleles in women with breast cancer with and without poor risk features suggests that altered iron metabolism in C282Y carriers may promote the development of breast cancer and/or more aggressive forms of the disease.

Glutathione S-transferases M1, T1, and P1 and breast cancer: a pooled analysis.

The glutathione S-transferase (GST) genes are involved in the metabolism of various carcinogens. Deletion polymorphisms in the genes GSTM1 and GSTT1 and a base transition polymorphism at codon 105 (Ile-->Val) in GSTP1 were investigated in relation to breast cancer risk. Tobacco smoking and reproductive factors were examined as potential effect modifiers. Individual data from seven case-control studies were pooled within the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens. To measure the effect of GSTs on breast cancer risk, odds ratios and 95% confidence intervals were computed adjusting for study center and age. The modifying effect was investigated by stratification on variables of smoking habits and reproductive history. A total of 2,048 cases with breast cancer and 1,969 controls were analyzed. The relative odds ratio (95% confidence interval) of breast cancer was 0.98 (0.86-1.12) with the GSTM1 null, 1.11 (0.87-1.41) with the GSTT1 null, 1.01 (0.79-1.28) with GSTP1 heterozygous mutants, and 0.93 (0.62-1.38) with GSTP1 homozygous mutants. Stratification by smoking or reproductive factors did not reveal a modifying effect of these variables, nor was there any association between GSTM1 and age at diagnosis of breast cancer. This is the largest study investigating susceptibility to breast cancer due to polymorphisms in the GST genes. The results conclusively show that single gene GST polymorphisms do not confer a substantial risk of breast cancer to its carriers. Furthermore, GSTs did not interact with smoking or reproductive history to modify cancer risk.

Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies.

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

A multistage genetic association study identifies breast cancer risk loci at 10q25 and 16q24.

BACKGROUND: Heritable risk for breast cancer includes an increasing number of common, low effect risk variants. We conducted a multistage genetic association study in a series of independent epidemiologic breast cancer study populations to identify novel breast cancer risk variants. METHODS: We tested 1,162 SNPs of greatest nominal significance from stage I of the Cancer Genetic Markers of Susceptibility breast cancer study (CGEMS; 1,145 cases, 1,142 controls) for evidence of replicated association with breast cancer in the Nashville Breast Cohort (NBC; 599 cases, 1,161 controls), the Collaborative Breast Cancer Study (CBCS; 1,552 cases, 1,185 controls), and BioVU Breast Cancer Study (BioVU; 1,172 cases, 1,172 controls). RESULTS: Among these SNPs, a series of validated breast cancer risk variants yielded expected associations in the study populations. In addition, we observed two previously unreported loci that were significantly associated with breast cancer risk in the CGEMS, NBC, and CBCS study populations and had a consistent, although not statistically significant, risk effect in the BioVU study population. These were rs1626678 at 10q25.3 near ENO4 and KIAA1598 (meta-analysis age-adjusted OR = 1.13 [1.07-1.20], P = 5.6 × 10(-5)), and rs8046508 at 16q23.1 in the eighth intron of WWOX (meta-analysis age-adjusted OR = 1.20 [1.10-1.31], P = 3.5 × 10(-5)). CONCLUSIONS: Our data supports the association of two novel loci, at 10q25.3 and 16q23.1, with risk of breast cancer. IMPACT: The expanding compendium of known breast cancer genetic risk variants holds increasing power for clinical risk prediction models of breast cancer, improving upon the Gail model.