Chiyomi Egawa

Association of centrosomal kinase STK15/BTAK mrna expression with chromosomal instability in human breast cancers

Over‐expression of a centrosomal serine/threonine kinase, STK15/BTAK, induces centrosome amplification, which results in chromosomal instability (CIN) in cell culture. In the present study, we investigated the correlation of STK15/BTAK mRNA expression with CIN and various clinicopathological factors in human breast cancer. STK15/BTAK mRNA levels were quantified by real‐time PCR, and CIN values were determined by FISH analysis of chromosomes 1, 11 and 17 using centromeric probes. STK15/BTAK mRNA levels (0.310 ± 0.413, mean ± SD, n = 47) in breast cancers were significantly (p < 0.01) higher than those in normal breast tissues (0.044 ± 0.029, n = 9). Furthermore, breast cancers were divided into 3 groups (low, intermediate and high) according to STK15/BTAK mRNA expression levels. CIN values of the low‐expression group (27.9 ± 12.6%, n = 18) were significantly (p < 0.01) higher than those of normal breast tissues (9.2 ± 2.6%, n = 6), and those of the high‐expression group (38.0 ± 12.7%, n = 14) were significantly (p < 0.05) higher than those of the low‐expression group. STK15/BTAK mRNA expression showed a significant (p < 0.05) correlation with high histological grade and negativity of estrogen and progesterone receptors. Our results demonstrate that STK15/BTAK mRNA is over‐expressed in the majority of breast cancers and its over‐expression is significantly associated with CIN, implicating STK15/BTAK in carcinogenesis through induction of CIN. STK15/BTAK mRNA levels might be useful as an indicator of poor prognosis and resistance to endocrine therapy.

Quantitative analysis of estrogen receptor‐β mRNA and its variants in human breast cancers

We have carried out a quantitative analysis of ER‐α and ER‐β mRNA expression in normal (n = 11) and breast cancer (n = 112) tissues using a real‐time (Taq‐Man) PCR assay. Expression of ER‐β mRNA variants has also been studied by triple‐primer PCR assay. ER‐α mRNA levels in normal breast tissues were significantly (p < 0.01) lower than those in ER‐positive breast cancers but not significantly different from those in ER‐negative breast cancers. However, ER‐β mRNA levels in normal breast tissues were significantly (p < 0.01) higher than those in ER‐positive and ER‐negative breast cancers. Proportions of ER‐β1 and ER‐β2 mRNA expression among total ER‐β mRNA expression were significantly higher and those of ER‐β5 and ER‐β5` mRNA were significantly lower in normal breast tissues than in ER‐positive and ER‐negative breast cancers. ER‐β mRNA levels and proportions of ER‐β mRNA variants did not show any significant correlation with age, tumor size, lymph node status and histological grade. Our results demonstrate that ER‐α mRNA is up‐regulated and ER‐β mRNA is down‐regulated during carcinogenesis of breast cancers. Changes in proportions of ER‐β mRNA variants are also implicated in this process. Int. J. Cancer 88:733–736, 2000.

Quantitative analysis of estrogen receptor‐α and ‐β messenger RNA expression in breast carcinoma by real‐time polymerase chain reaction

Estrogen action is mediated not only through a classic estrogen receptor (ER) (ER‐α) but also through a second ER (ER‐β) that has a structure and function similar to ER‐α. A correlation between ER‐β mRNA expression with ER and progesterone receptor (PR) protein levels as well as prognostic factors remains to be established in breast carcinoma.

Breast cancer risk associated with polymorphism in CYP19 in Japanese women

Screening of the entire coding and major promoter regions of the CYP19 gene identified two novel polymorphisms at codon 39 (Trp to Arg) and codon 408 (silent) in addition to those reported previously at codon 264 (Arg to Cys) and intron 4 [tetranucleotide (TTTA) simple tandem repeat]. A case‐control study was conducted in order to see whether or not these polymorphisms were associated with breast cancer risk in Japanese women. Homozygous and heterozygous carriers of the variant allele Arg at codon 39 showed a significantly decreased risk of breast cancer (OR=0.39, 95%C.I.=0.17–0.89). On the other hand, homozygous carriers of the allele with 10 or more TTTA repeats at intron 4 showed a trend toward an increase (OR=1.80, 95%C.I.=0.97–3.36) in breast cancer risk. Other polymorphisms were found not to be associated with breast cancer risk. These results suggest that the CYP19 polymorphisms at exon 39 and intron 4 would be useful for selecting Japanese women at a high risk of breast cancer. Int. J. Cancer 89:325–328, 2000.

Breast Cancer Risk Associated with CYP1A1 Genetic Polymorphisms in Japanese Women

Although several reports are available on the association between CYP1A1 polymorphisms and breast cancer risk in Caucasian women, it has never been reported in Japanese women. Since breast cancer incidence and clinicopathologic features of breast cancers are different between Japanese and Caucasian women, it is conceivable that the risk factors of breast cancer might also differ. In addition, a preliminary study has shown that the frequencies of the variant allele in the CYP1A1 gene are different among ethnic groups. Therefore, in the present study, we investigated the association of CYP1A1 polymorphisms with breast cancer risk in Japanese women. The association of two CYP1A1 polymorphisms, that is, 3′ noncoding region (6235(T/C)) and codon 462 (Ile/Val), with breast cancer risk was analyzed by a case‐control study (195 cases and 272 controls). Variant allele 6235C carriers at the 3′ noncoding region polymorphism showed a significantly (p < 0.01) reduced breast cancer risk (odds ratio 0.60; 95% CI 0.41–0.88) as compared with noncarriers, and variant allele 462Val carriers at the codon 462 polymorphism also showed a significantly (p < 0.05) reduced risk (odds ratio 0.66; 95% CI 0.45–0.96) as compared with noncarriers. The relationship between the genetic polymorphisms and clinicopathologic characteristics of breast cancers was also investigated. Variant allele 6235C carriers showed a significantly (p < 0.02) higher positivity of lymph node metastasis than noncarriers (54% versus 36%), and tumors measuring less than 2 cm were significantly (p < 0.03) more frequently observed in variant allele 462Val carriers than noncarriers (50% versus 33%). These results suggest that the CYP1A1 polymorphisms would be useful for predicting breast cancer risk as well as some tumor characteristics in Japanese women.

Mutational analysis of BARD1 in familial breast cancer patients in Japan.

Since BRCA1,which is a breast cancer susceptibility gene, form heterodimers with BARD1, it is speculated that BARD1 mutations might affect the function of BRCA1, contributing to breast carcinogenesis. Thus, in the present study, we have conducted mutational analysis of BARD1 in familial breast cancer patients (n=60) who were tested negative for both BRCA1 and BRCA2 germline mutations. We have been unable to show any clearly deleterious mutations but identified four missence mutations (Ser/Cys 241, Arg/Ser 378, Asn/Ser 470, Val/Met 507), one silent mutation (His/His 506), and one frameshift (in-frame) mutation (1139del21bp). Of these six mutations, one (Asn/Ser 470) was considered as a putative germline mutation since such a mutation was not observed in any of the healthy controls (n=152) but the other five mutations were considered as common genetic polymorphisms since they were frequently observed in the healthy controls. These genetic polymorphisms were further analyzed in their association with breast cancer risk by a case-control study using the population-based breast cancers (n=143) and healthy controls (n=155), which showed that carriers of the variant allele Met at codon 507 are significantly associated with the increased risk of breast cancer (adjusted odds ratio=2.05, 95% confidential interval=1.01-4.16) in postmenopausal women and that the other genetic polymorphisms are not associated with breast cancer risk. These results suggest that BARD1 mutations are responsible for, if any, a very small number of familial breast cancers. Genetic polymorphism of BARD1 (Val/Met 507) could be useful in the selection of postmenopausal women at a high risk for developing breast cancer.

Decreased expression of BRCA2 mRNA predicts favorable response to docetaxel in breast cancer

The clinical usefulness of BRCA1 and BRCA2 mRNA levels in tumor tissues in the prediction of response to docetaxel (DOC) treatment has been studied in breast‐cancer patients. Twenty‐five patients with locally advanced breast tumors (n = 13) or locally recurrent tumors (n = 12) underwent tumor biopsy and were treated with DOC (60 mg/m2 every 3 weeks). BRCA1 and BRCA2 mRNA levels in the tumors were determined by real‐time PCR, and the expression of 6 biological markers (P‐glycoprotein, p53, erbB2, BCL2, MIB1, estrogen receptor‐α) in the tumors was determined by immunohistochemistry. BRCA2 mRNA levels (0.547 ± 0.200, mean ± SE) of responders to DOC treatment were significantly (p < 0.05) lower than those of non‐responders (1.538 ± 0.358), but there was no significant difference in BRCA1 mRNA levels between responders (0.389 ± 0.081) and non‐responders (0.779 ± 0.172). Tumors were dichotomized into groups with high or low BRCA2 mRNA levels according to the cut‐off value of 0.13. The response rate (25%) of tumors with high BRCA2 mRNA levels was significantly (p < 0.01) lower than that (100%) of tumors with low BRCA2 mRNA levels. Positive predictive value, negative predictive value and diagnostic accuracy of the BRCA2 mRNA assay in the prediction of response to DOC were 100%, 75% and 80%, respectively. No significant difference was found between responders and non‐responders in the expression status of any of the other 6 biological markers. These results suggest that BRCA2 mRNA levels in tumor tissues might be clinically useful in the prediction of response to DOC treatment in breast‐cancer patients.

Mutational analysis of the class I β-tubulin gene in human breast cancer

Non‐small cell lung cancers have a high incidence of somatic mutations of the β‐tubulin (class I) gene, suggesting involvement in the acquisition of resistance to taxanes, which exert their effects through binding to β‐tubulin. Since taxanes are often used in the treatment of breast cancer, we carried out a mutational analysis of the class I β‐tubulin (GenBank accession AF070600) gene in breast cancer. We paid special attention to the primer design so as not to amplify the pseudogenes. We identified 1 somatic mutation, codon 306 [Arg (CGC) to Cys (TGC)], and 2 genetic polymorphisms, codon 217 [Leu (CTG) to Leu (CTA)] and (C to T) at 57 bases downstream from exon 4. Our results suggest that acquisition of resistance to taxanes is unlikely to be explained by somatic mutations of the class I β‐tubulin gene in most breast cancers. In addition, the overestimation of the incidence of somatic mutations of the class I β‐tubulin gene due to the pseudogenes is discussed.

Genetic polymorphism in CYP17 and breast cancer risk in Japanese women.

A case-control study was conducted to investigate the association of two genetic polymorphisms (1931T/C and 1951G/A) in the promoter region of the CYP17 gene with breast cancer risk in Japanese women. No significant association was observed between CYP17 polymorphism(1951G/A) and breast cancer risk (odds ratio (OR) = 1.71, 95% confidence interval (CI): 0.28-1.84). In contrast, a significant increase in breast cancer risk (OR= 1.82. 95% CI: 1.07-3.12) was observed in CYP17(1931C/C) homozygotes compared with CYP17(1931T/C) heterozygotes and CYP17(1931T/T) homozygotes when women aged > or = 55 years were considered, but such a significant increase was not observed when women aged < or = 54 years were considered (OR = 0.96, 95% CI: 0.56-1.63). These results suggest that CYP17 polymorphism(1931T/C) would be useful in the selection of Japanese women at a high risk for developing breast cancer at the age of > or = 55 years.

Increased expression of BRCA1 mRNA predicts favorable response to anthracycline-containing chemotherapy in breast cancers

Clinical significance of BRCA1 and BRCA2 mRNA levels in tumor tissues as predictors of response to anthracycline-containing chemotherapy was studied in breast cancer patients. Fifty-one patients with locally advanced breast tumors (n = 43) or locally recurrent tumors (n = 8) underwent tumor biopsy and were treated with CE (cyclophosphamide (600 mg/m2) plus epirubicin (60 mg/m2), q3w). BRCA1 and BRCA2 mRNA levels in the tumors were determined by a real-time PCR assay, and the expression of p53 and erbB2 in tumors was determined by immunohistochemistry. BRCA1 mRNA levels (1.317 ± 0.277, mean ± S.E.) of responders were significantly (p < 0.05) higher than those (0.609 ± 0.097) of non-responders but there was no significant difference in BRCA2 mRNA levels between responders (1.590 ± 0.326) and non-responders (1.586 ± 0.410). Tumors were dichotomized into the BRCA1 mRNA levels high and low group according to the cut-off value of 0.55. Response rate (65%) of tumors with high BRCA1 mRNA levels was significantly (p < 0.05) higher than that (32%) of tumors with low BRCA1 mRNA levels. There was no significant association between response to CE and the expression of p53 or erbB2. Positive predictive value, negative predictive value, and diagnostic accuracy of BRCA1 mRNA determination in the prediction of response to CE were 65, 68, and 67%, respectively. These results suggest a possibility that BRCA1 mRNA levels in tumor tissues might be useful in the prediction of response to CE treatment in breast cancer patients.