Mutations in breast cancer.

The genetics of spontaneous breast cancer is reviewed. We have identified three regions of amplification and nine chromosomal arms with deletions in the genome. The significance and interrelations of these mutations is discussed with respect to the complex genetics of breast carcinoma. Recent work identifying a commonly deleted region between D17S846 and D17S746 is presented, which is approximately 0.5-1.0 Mb centromeric to the newly described BRCA1 gene candidate. Possible explanations for the different locations of our deleted region and the BRCA1 gene are presented.

Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas.

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

A physical map and candidate genes in the BRCA1 region on chromosome 17q12-21.

We have constructed a physical map of a 4 cM region on chromosome 17q12-21 that contains the hereditary breast and ovarian cancer gene BRCA1. The map comprises a contig of 137 overlapping yeast artificial chromosomes and P1 clones, onto which we have placed 112 PCR markers. We have localized more than 20 genes on this map, ten of which had not been mapped to the region previously, and have isolated 30 cDNA clones representing partial sequences of as yet unidentified genes. Two genes that lie within a narrow region defined by meiotic breakpoints in BRCA1 patients have been sequenced in breast cancer patients without revealing any deleterious mutations. These new reagents should facilitate the identification of BRCA1.

Evidence for involvement of BRCA1 in sporadic breast carcinomas.

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.

Identification of three regions on chromosome 17q in primary human breast carcinomas which are frequently deleted.

We have examined the long arm of chromosome 17 in sporadic breast carcinomas for the loss of heterozygosity (LOH) at 18 polymorphic loci. At least three distinct regions could be identified by the frequency of LOH and confirmed by high density deletion maps of individual tumor DNAs. A proximal region affected by LOH is located in a 22-cM region defined by D17S73 and NME1 and thus is similar in location to the region thought to contain the BRCA1 gene associated with familial breast and breast/ovarian cancer. The central region affected by LOH is bordered by the D17S86 and D17S21 loci and is estimated to be 28 cM in size. The third region is bordered by the D17S20 and D17S77 loci which are 11 cM apart. These results define three independent regions of chromosome 17q which are likely to contain tumor suppressor genes relevant to the etiology of sporadic breast carcinoma.

Two distinct regions involved in 1p deletion in human primary breast cancer.

Alteration of chromosome 1 is the most consistent cytogenetic abnormality found in human breast carcinoma. Cytogenetic studies have shown independent alterations on the two arms of chromosome 1, increased copy number of the long arm and loss of the short arm of chromosome 1. These deletions are thought to coincide with the location of tumor suppressor gene(s). We carried out deletion analysis of the 1p region by using restriction fragment length polymorphism markers mapping to the long (six markers) and short arm (22 markers). Thirty-five of the 74 (47.3%) human breast tumors tested showed somatic loss of heterozygosity at one or more loci on the short arm. Two commonly deleted regions, 1p13-p21 and 1p32-pter, were identified. The latter region is frequently involved in other types of tumors, suggesting that it harbors a common tumor suppressor gene. Our findings suggest that two tumor suppressor genes involved in the development of human breast carcinoma may occur on the short arm of the chromosome 1.

The role of protein kinase-C in gonadotropin-induced ovulation in the in vitro perfused rabbit ovary.

Tumor-promoting phorbol esters are believed to affect ovarian granulosa cell progesterone and prostaglandin (PG) production and possibly ovulation by activating protein kinase-C (PKC). The effects of phorbol esters and PKC inhibitors on ovulation, progesterone, and PG production were examined in an in vitro perfused rabbit ovary. The effect of tranexamic acid, an inhibitor of the conversion of plasminogen activator to plasmin, on phorbol ester-induced ovulation was also examined. Phorbol 12,13-dibutyrate (PdBU), a PKC stimulator, induced ovulation in a dose-related manner in the absence of gonadotropins (56%, 200 nM PdBU; 0%, 0 nM PdBU; P < 0.05). Perfusate progesterone levels were increased only after 600 nM PdBU treatment, and perfusate PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were increased in a dose-dependent fashion (P < 0.05). Staurosporine, a potent inhibitor of the catalytic domain of PKC, and calphostin-C, a specific inhibitor of the diacylglycerol-binding region, inhibited hCG-induced ovulation in a dose-related manner. Gonadotropin-induced ovulation decreased from 73% without staurosporine to 19% with 1.0 microM staurosporine (P < 0.01). Calphostin-C reduced ovulatory efficiency from 60% to 24% (P < 0.01). However, neither inhibitor decreased progesterone or PGF2 alpha production by ovaries exposed to hCG. hCG-induced oocyte maturation was also unaffected by exposure to either staurosporine or calphostin-C. Tranexamic acid reduced phorbol ester-induced ovulatory efficiency from 67% to 37% (P < 0.05). These findings demonstrate that the calcium-dependent PKC pathway is instrumental in gonadotropin-mediated follicular rupture in the rabbit. Although PGs may play an important role in ovulation, they do not appear to be directly responsible for PKC-mediated follicular rupture.

Somatic mutations and human breast cancer. A status report.

A systematic study of primary human breast tumor DNA demonstrated that three proto-oncogenes or regions of the genome (c-myc, int-2, and c-erbB2) are frequently amplified and that there is loss of heterozygosity (LOH) on chromosomes 1p(37%), 1q(20%), 3p(30%), 7(41%), 11p(20%), 13q(30%), 17p(49%), 17q(29%), and 18q(34%). Specific subsets of tumors can be defined based on the particular collection of mutations they contain. For instance, LOH on chromosomes 11p, 17p, and 18q frequently occurs in the same tumor. A search for putative tumor suppressor genes within the regions of the genome affected by LOH has been started. In a comprehensive molecular analysis of the p53 gene on chromosome 17p, 46% of the tumors contained a point mutation in the p53 gene.

Detection of loss of heterozygosity in tumor DNA samples by PCR.

We demonstrate that PCR amplification of human genomic DNA can be used for the detection of loss of heterozygosity (LOH) in tumor samples. A 250-bp fragment containing codon 72 of the human p53 gene was amplified, ThaI digested and electrophoresed. Tumor LOH is detectable both by ethidium bromide staining and autoradiography, despite 25% contamination with normal DNA. This technique provides a fast and reproducible alternative to conventional Southern blotting and has minimal sample requirements.

Somatic allelic deletion of nm23 in human cancer.

Tumor progression to the metastatic phenotype is accompanied in certain cell types by reduced expression of the nm23 gene. We have localized human nm23-H1 to chromosome 17 by somatic cell hybrid analysis. Regional localization in the CEPH database and in situ hybridization is reported. Somatic allelic deletion of nm23-H1 was observed in human breast, renal, colorectal, and lung carcinoma DNA samples, as compared to DNA from matched normal tissues. A homozygous deletion of nm23-H1 was observed in a lymph node metastasis of a colorectal carcinoma, indicating that nm23-H1 can be recessively inactivated. The data identify nm23-H1 as a novel, independent locus for allelic deletion in human cancer, a characteristic shared with previously described suppressor genes.

Loss of heterozygosity on chromosomes 17 and 18 in breast carcinoma: two additional regions identified.

The loss of heterozygosity (LOH) at specific regions of the human genome in tumor DNA is recognized as evidence for a tumor-suppressor gene located within the corresponding region of the homologous chromosome. Restriction fragment length polymorphism analysis of a panel of primary human breast tumor DNAs has led to the identification of two additional regions on chromosomes 17q and 18q that frequently are affected by LOH. Deletions of each of these regions have a significant correlation with clinical parameters that are associated with aggressive breast carcinomas. Previous restriction fragment length polymorphism analysis of this panel of tumors has uncovered several other frequently occurring mutations. LOH on chromosome 18q frequently occurs in tumors with concomitant LOH of loci on chromosomes 17p and 11p. Similarly, tumors having LOH on 17q also have LOH on chromosomes 1p and 3p. This suggests that certain combinations of mutations may collaborate in the development and malignant progression of breast carcinomas.

Antisperm antibodies.

Significant progress is being made in the study and treatment of infertility due to antisperm antibodies. Increased knowledge of the antigenicity of sperm and oocytes and continued study of the immunology of the male and female reproductive tracts should yield a greater understanding of the mechanisms of immunologic infertility. Such an understanding will permit novel and specific therapy for the infertile couple. Until such research is forthcoming, continued clinical research is imperative to increase the efficiency and success of treatment.

PIP: The blood testis barrier, a specialized inner Sertoli's cell attachment, is listed among modulators of antigenicity. Antigen mediated mechanisms include suppressor T-lymphocytes which partially mediate the normal state of immunologic unresponsiveness toward sperm autoantigens. The cervix is a site of sperm filtration, and uterine fluid has significant concentrations of IgG and IgA. The postcoital test (PCT) screens for sperm antibodies. In a study, 25% of women in 172 infertile couples had antisperm antibodies in their cervical mucus, and 12.7% had antisperm antibodies in their sera. 64% of 66 couples had adequate PCT in which there was no male autoimmunity to sperm. The PCT was 26% in 122 couples with untreated male autoimmunity. Increased phagocytosis of sperm by macrophage of the female reproductive tract is another mechanism, as is impaired interaction with zona pellucida when antibodies occupy sperm receptor sites blocking sperm binding. In a study of men with antisperm antibodies, 18 of 23 (78%) had poor egg penetration compared to 5 (16%) of 30 normal men. The risk of spontaneous abortion is higher in women whose male partner has antisperm antibodies. Cytotoxic sperm antibodies were found in 47% of husbands of women who miscarried habitually. Diagnostic tests for immunological infertility include the Kibrick or gelatin agglutination test (GAT), the Franklin Dukes test or tray slide agglutination test (TSAT), the microtray agglutination test (MAT), the Isojima or sperm immobilizing test (SIT), the mixed antiglobulin reaction test (MAR), and the immunobead test (IMB). In the sera of 140 subfertile men, antisperm antibodies were found in 2.3% by the Kibrick test, 4.6% by the Franklin Dukes test, 18.6% by the Isojima test, and 15.2% by the MAR test. Therapy of infertility includes the condom method, intrauterine insemination, immunosuppressive therapy with corticosteroids, in vitro fertilization, and donor insemination.

Effect of low-dose oral contraceptive on gonadotropins, androgens, and sex hormone binding globulin in nonhirsute women.

The effectiveness of a low-dose oral contraceptive (OC) in suppressing plasma levels of gonadotropins, ovarian, and adrenal androgens and stimulating sex hormone-binding globulin (SHBG) was evaluated prospectively in nonhirsute women. Thirty-three women ingested 35 micrograms of ethinyl estradiol and 1 mg of norethindrone beginning within day 1 to 5 of the menstrual cycle. Baseline levels of luteinizing hormone, follicle-stimulating hormone, total testosterone (T), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), and SHBG were obtained before ingestion of the OC and repeated after 3, 6 and, 9 months of OC use on day 1 to 5 of the OC "cycle". A significant suppression of gonadotropin levels is seen in nonhirsute women. Sex hormone binding globulin is consistently stimulated by the low-dose OC. A significant suppression of T and DHEAS is observed. No change was seen in levels of A. The demonstrated effects become evident at 3 months and are maintained at 6 and 9 months.

Frequent alteration of the DF3 tumor-associated antigen gene in primary human breast carcinomas.

The gene for the human DF3 breast carcinoma-associated antigen contains a conserved (G + C)-rich 60-base pair tandem repeat and maps to chromosome 1q21-24. In the present study we isolated and characterized 1220 base pairs of nonrepetitive adjacent sequences. Multiple alleles were identified by fragment size. Signal intensity of hybrids with the tandem and unique sequence probes indicated that allelic variation is due to different numbers of repeats. Probes for both the tandem and the unique sequences were used to study the DF3 locus in human breast tumor DNAs. Seventy of 110 breast tumor DNAs were informative at the DF3 locus. Of these, 20 (29%) showed a loss of heterozygosity, while eight (11%) had an increased copy number of one allele. In some cases, the loss of heterozygosity or increased copy number did not extend to other markers on chromosome 1q or 1p. These data indicate that the chromosomal region around the DF3 locus is affected by mutations at high frequency.

Failure of tubal closure following laser salpingostomy for ampullary tubal ectopic pregnancy.

A patient with recurrent ectopic pregnancy is described. The first ectopic gestation was treated by laparoscopic linear laser salpingostomy of the right fallopian tube. Her hCG became negative and a hysterosalpingogram demonstrated right tubal patency. She conceived again after Pergonal ovulation induction, but had a recurrent right ectopic pregnancy. At laparotomy, the pregnancy was extruding through the unhealed incision of her prior linear salpingostomy. This complication of conservative management of ectopic pregnancy has important potential clinical significance.