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Breast cancers demonstrate substantial biological, clinical and etiological heterogeneity. We investigated breast cancer risk associations of eight susceptibility loci identified in GWAS and two putative susceptibility loci in candidate genes in relation to specific breast tumor subtypes. Subtypes were defined by five markers (ER, PR, HER2, CK5/6, EGFR) and other pathological and clinical features. Analyses included up to 30 040 invasive breast cancer cases and 53 692 controls from 31 studies within the Breast Cancer Association Consortium. We confirmed previous reports of stronger associations with ER+ than ER- tumors for six of the eight loci identified in GWAS: rs2981582 (10q26) (P-heterogeneity = 6.1 × 10(-18)), rs3803662 (16q12) (P = 3.7 × 10(-5)), rs13281615 (8q24) (P = 0.002), rs13387042 (2q35) (P = 0.006), rs4973768 (3p24) (P = 0.003) and rs6504950 (17q23) (P = 0.002). The two candidate loci, CASP8 (rs1045485, rs17468277) and TGFB1 (rs1982073), were most strongly related with the risk of PR negative tumors (P = 5.1 × 10(-6) and P = 4.1 × 10(-4), respectively), as previously suggested. Four of the eight loci identified in GWAS were associated with triple negative tumors (P ≤ 0.016): rs3803662 (16q12), rs889312 (5q11), rs3817198 (11p15) and rs13387042 (2q35); however, only two of them (16q12 and 2q35) were associated with tumors with the core basal phenotype (P ≤ 0.002). These analyses are consistent with different biological origins of breast cancers, and indicate that tumor stratification might help in the identification and characterization of novel risk factors for breast cancer subtypes. This may eventually result in further improvements in prevention, early detection and treatment.

Association studies in large series of breast cancer patients can be used to identify single-nucleotide polymorphisms (SNP) contributing to breast cancer susceptibility. Previous studies have suggested associations between variants in TP53 (R72P) and MDM2 (SNP309) and cancer risk. Data from molecular studies suggest a functional interaction between these genes. We therefore investigated the effect of TP53 R72P and MDM2 SNP309 on breast cancer risk and age at onset of breast cancer in a pooled series of 5,191 cases and 3,834 controls from the Breast Cancer Association Consortium (BCAC). Breast cancer risk was not found to be associated with the combined variant alleles [odds ratio (OR), 1.00; 95% confidence interval (95% CI), 0.81-1.23]. Estimated ORs were 1.01 (95% CI, 0.93-1.09) per MDM2 SNP309 allele and 0.98 (95% CI, 0.91-1.04) for TP53 R72P. Although we did find evidence for a 4-year earlier age at onset for carriers of both variant alleles in one of the breast cancer patient series of the BCAC (the German series), we were not able to confirm this effect in the pooled analysis. Even so, carriers of both variant alleles did not have different risk estimates for bilateral or estrogen receptor-positive breast cancer. In conclusion, in this large collaborative study, we did not find an association of MDM2 SNP309 and TP53 R72P, separately or in interaction, with breast cancer. This suggests that any effect of these two variants would be very small and possibly confined to subgroups that were not assessed in our present study.

The INT6 gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog of INT6 is located on chromosome region 8q22-q23. A processed INT6 pseudogene is located on chromosome 6q. INT6 is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affecting INT6. Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 of INT6. Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked to INT6.

The hereditary breast cancer gene BRCA1 previously has been localized to chromosome 17q21. We looked for evidence of involvement of this region of chromosome 17 in 130 sporadic breast cancers. Seventeen polymorphic sequence tagged site markers were examined in these tumors between the D17S250 and D17S579 loci to screen for deletions as measured by loss of heterozygosity. The smallest common region that was deleted occurred in the approximately 120-kilobase interval between the D17S846 and D17S746 loci within the BRCA1 region. Delineation of this commonly deleted area should accelerate attempts to identify the involved gene(s) and its relationship to BRCA1.

The effect of SK&F 96365 (1-(beta-[3-(4-methoxyphenyl)propoxyl]-4- methoxyphenethyl)-1H-imidazole hydrochloride), a recently synthesized inhibitor of receptor-mediated calcium entry, was investigated on human hematopoietic cell lines. We found that treatment of the T-cell leukemia line Jurkat with SK&F 96365 inhibited the Ca2+ influx triggered by antibodies against the CD3/TCR complex, while the inositol trisphosphate-dependent Ca2+ release from intracellular stores remained intact. A 50% inhibition of the Ca2+ influx was obtained with 5 microM SK&F 96365, while higher concentrations of the drug blocked the CD3-dependent Ca2+ influx completely. In addition to its blocking of the Ca2+ influx, treatment with SK&F 96365 was found to accumulate mitotic cells. The drug (5 microM) imposed a total cell cycle arrest in G2/M. The mitosis block could be reversed by removal of the inhibitor from the cultures, while elevation of intracellular or extracellular Ca2+ did not restore cell cycle progression. This suggests that the cell cycle block induced by SK&F 96365 is not directly related to its action as an inhibitor of receptor-mediated calcium entry. Our findings indicate that SK&F 96365, in addition to its ability to inhibit receptor-triggered Ca2+ influx, offers a new method for imposing a reversible mitosis arrest in hematopoietic cell lines.

Alu element-mediated polymerase chain reaction is a strategy for rapidly cloning and mapping human DNA markers from mixed DNA sources. A novel primer homologous to the 3' end of the human Alu repeat element provides the basis for preferential synthesis of human DNA fragments from human/rodent somatic cell hybrid DNA template. This approach has been used to isolate a series of new markers from chromosome 10. The Alu element-mediated PCR probes were regionally assigned on chromosome 10 by hybridization to Southern blots of Alu PCR-synthesized DNA derived from somatic cell hybrid template DNA. Alu element-mediated PCR is generally applicable and makes possible the analysis of complex genomes with a speed and sensitivity that has not been previously possible.

We have used X-ray irradiation and cell fusion to generate somatic cell hybrids containing fragments of human chromosome 10. Our experiments were directed towards isolating the region of the MEN2A gene in hybrids and to use those as the source of DNA for cloning and mapping new markers from near the MEN2A locus. A number of hybrid clones containing human sequences that are tightly linked to the MEN2A gene were identified. Some 25% of our hybrids, however, proved to contain more than one human chromosome 10-derived fragment or showed evidence of deletions and/or rearrangements. A detailed analysis of the human content of X-ray irradiation hybrids is required to assess the integrity and number of human fragments retained. Despite retention of multiple human-derived fragments, these hybrids will prove useful as cloning and mapping resources.

A cDNA corresponding to the beta-subunit of the human fibronectin receptor (beta-FNR) was used as a probe in Southern blot analysis of mouse/human somatic cell hybrid DNAs and in in situ hybridization to metaphase chromosomes. The beta-FNR cDNA detects sequences present on human chromosome 10 as well as recognizing homologous sequences in the genome of the mouse parent of the somatic cell hybrids. In situ hybridization refined the localization of human sequences reacting with the beta-FNR cDNA to 10p11.2. The A-1A5 monoclonal antibody which recognizes the beta-subunit of the fibronectin receptor on the cell surface was used to confirm that the sequences present on chromosome 10 correspond to those required for expression of beta-FNR.

Leu-M2 (Mac-120) antigen is a cell-surface marker present on a subpopulation of human blood monocytes and platelets. Its expression on monocytes and macrophages is necessary for their ability to present antigen to T lymphocytes. Only Leu-M2+ cells display the human Ia-like determinant, HLA-DS, which seems important in antigen presentation and stimulation in the autologous MLR. Expression of the Leu-M2/Mac-120 antigen was studied by indirect immunofluorescence analysis and flow cytofluorometry in mouse-human somatic cell hybrids segregating human chromosomes. In the mouse-human hybrid clones, expression of the Leu-M2/Mac-120 antigen was dependent on the presence of human chromosome 11. This was verified by fluorescence-activated cell sorting of heterogeneous hybrid cell populations into antigen-positive and-negative fractions. The Leu-M2 antigen co-segregated with chromosome 11. The gene controlling the expression of the Leu-M2/Mac-120 antigen thus is assigned to human chromosome 11.