Shunzo Kobayashi

Coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer

IntroductionMany laboratories are currently evaluating the usefulness of determination of HER2, p53, and Ki67 proliferation indices using immunohistochemical techniques in cancer. Although the available studies suggest that these factors might indeed be helpful in making treatment decisions in cancer patients, their clinical usefulness is still controversial.MethodsExpression of HER2, p53, and Ki67 was examined by immunohistochemistry in samples of breast tissue from 506 patients with invasive ductal carcinoma, obtained between 1981 and 1999 (median follow up period 82 months), and their significance for prognosis was analyzed.ResultsOf the 506 carcinoma tissue samples, 20.1%, 29.0%, and 53.6% were positive for HER2 over-expression, p53 protein accumulation, and Ki67 expression, respectively. Over-expression of HER2 significantly reduced disease free (P = 0.02) and overall survival (P = 0.005). Accumulation of p53 protein significantly decreased disease free (P = 0.01) and overall survival (P = 0.01). Patients with tumors that were positive for both HER2 and p53 relapsed and died within a significantly shorter period of time after surgery (P = 0.0001 and P < 0.0001, respectively). In multivariate analysis, patients with both HER2 and p53 positive tumors had considerably decreased overall survival (P = 0.04), as did patients with larger tumor size and positive lymph node status.ConclusionThe findings of the present study indicate that the coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer.

Suppression of ING1 expression in sporadic breast cancer

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 – 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

Clinical value of the wild-type estrogen receptor β expression in breast cancer

Abstract To estimate the clinical value of estrogen receptor (ER) β expression in breast cancer we used an immunohistochemical method to detect the wild-type ERβ in 88 primary breast cancers. We used a highly specific polyclonal antibody to the carboxyl terminus of wild-type ERβ. This antibody reacted with neither other variant forms of ERβ nor any part of ERα. Slides were evaluated on a scale representing the estimated proportion and intensity of positive-staining tumor cells. Positive staining could be seen in 52 (59.1%) of 88 breast cancers; 36 (40.9%) were negative. Although there was no correlation between ERβ staining and age, node status, tumor size, histological grade, or progesterone receptor (PgR)-enzyme immunoassay (EIA) status, we did observe a significant correlation with ERα-EIA (Fisher’s exact probability test: P =0.0169). Moreover, ERβ positive cases showed a better prognosis than negative cases in disease-free survival rate (Logrank test: P =0.0662, Breslow-Gehan-Wilcoxson test: P =0.0318). Our data demonstrated the possibility that wild-type ERβ protein expression could be used as a good prognostic indicator for breast cancer.

Production of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human breast carcinomas.

We examined production and tissue localization of matrix metalloproteinase (MMP)‐1 (tissue collagenase), MMP‐2 (gelatinase A), MMP‐3 (stromelysin‐1), MMP‐9 (gelatinase B), tissue inhibitors of metalloproteinase (TIMP)‐1 and TIMP‐2 in human breast carcinomas. In more than half of the cases, MMP‐1, MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were immunolocalized in carcinoma cells and MMP‐2 was on the carcinoma cell membranes as well, whereas MMP‐3 was positively stained in less than 15% of the cases. MMP‐1 staining in carcinoma cells was significantly higher in scirrhous carcinoma than in other types of carcinoma. MMP‐9 expression was remarkably higher in the carcinoma cases with lymphnode metastasis than in the non‐metastatic cases. MMP‐3 was mainly expressed in T‐lymphocytes infiltrated in the tumor stroma. Stromal fibroblasts were positive for all these MMPs except for MMP‐3. The TIMP‐1 levels released into the culture media by carcinoma tissues were significantly lower than those by fibroadenoma tissues, although there were no significant differences in the levels of MMP‐1, MMP‐2, MMP‐9 and TIMP‐2. Gelatin zymographical analyses showed that the activation rate of the zymogen of MMP‐2 (proMMP‐2) is significantly higher in the more advanced carcinoma group with lymphnode metastasis than in the metastasis‐negative and fibroadenoma groups. These data indicate that MMP‐1, MMP‐2 and MMP‐9 are highly expressed in human breast carcinoma tissue and suggest that activation of proMMP‐2 may be an indicator of lymphnode metastasis of the breast carcinoma.

Evaluation of oestrogen receptor β wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers

We addressed the clinicopathological significance of the oestrogen receptor (ER) beta protein, including an ERbeta variant, ERbetacx, in normal human breast and breast cancer. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that wild-type ERbeta (ERbetaw) mRNA expression was higher in normal than cancer tissues, and that ERbetacx mRNA was higher in cancer than in normal tissues. Immunohistochemistry of 22 normal breast tissues and 57 breast cancers was performed with three different ERbeta antibodies and one ERbetacx antibody. All normal breast samples showed staining with the three ERbeta antibodies, suggesting that ERbetaw might have a physiological role in oestrogen signalling in the normal breast. In breast cancer, expression of the ERbetaw protein correlated well with the expression of the ERalpha and progesterone receptor (PgR), as well as histological grade (HG), and tended to indicate a better prognosis than when ERbetaw was absent. Thirty-one (54%) breast cancer samples contained ERbetacx, whereas the corresponding tissue for normal breast samples stained positive in only two (9%).

Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer

IntroductionEndocrine therapy is the most important treatment option for women with hormone-receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and crosstalk between ER and other growth factor signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.MethodsUsing immunohistochemical techniques, we focused on the expression and phosphorylation of hormone receptors themselves and examined the phosphorylation of ER-α Ser118 and ER-α Ser167 and the expression of ER-α, ER-β1, ER-βcx/β2, progesterone receptor (PR), PRA, and PRB in the primary breast carcinomas of 75 patients with metastatic breast cancer who received first-line treatment with endocrine therapy after relapse.ResultsPhosphorylation of ER-α Ser118, but not Ser167, was positively associated with overexpression of HER2, and HER2-positive tumors showed resistance to endocrine therapy. The present study has shown for the first time that phosphorylation of ER-α Ser167, but not Ser118, and expression of PRA and PRB, as well as ER-α and PR in primary breast tumors are predictive of response to endocrine therapy, whereas expression of ER-β1 and ER-βcx/β2 did not affect response to the therapy. In addition, patients with either high phosphorylation of ER-α Ser167, or high expression of ER-α, PR, PRA, or PRB had a significantly longer survival after relapse.ConclusionThese data suggest that phosphorylation of ER-α Ser167 is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in metastatic breast cancer.

DNA methylation analysis at distal and proximal promoter regions of the oestrogen receptor gene in breast cancers

SummaryOestrogen receptor α (ER-α) gene has two specific promoters, distal (P0) and proximal (P1), which induce almost identical transcripts in size due to different splicing. We examined the methylation at both promoter regions of the ER-α gene using HpaII, a methylation-sensitive restriction enzyme, prior to polymerase chain reaction (PCR) amplification. To confirm the results of PCR-based methylation analysis, Southern hybridization was also performed. Twenty of 29 patients with ER-α-positive tumours and five of 27 with ER-α-negative tumours were unmethylated at the P1 promoter region of the ER-α gene. The incidence of methylation was highly negatively correlated with ER-α expression (P = 0.0002). A similarly negative correlation was observed at the P0 promoter region of the ER-α gene (P = 0.0154). Additionally, the tumours with the ER-α gene hypermethylated at both promoter regions had definitely negative ER-α values. It was suggested that this epigenetic change might control ER-α expression, and might play an important role in the loss of hormone-dependence in breast cancer.

Reproductive history and breast cancer risk

The fact that reproductive factors have significant influence on the risk of breast cancer is well known. Early age of first full-term birth is highly protective against late-onset breast cancers, but each pregnancy, including the first one, increases the risk of early-onset breast cancer. Estradiol and progesterone induce receptor activator of NF-kappa B ligand (RANKL) in estrogen receptor (ER)- and progesterone receptor (PgR)-positive luminal cells. RANKL then acts in a paracrine fashion on the membranous RANK of ER/PgR-negative epithelial stem cells of the breast. This reaction cascade is triggered by chorionic gonadotropin during the first trimester of pregnancy and results in the morphological and functional development of breast tissue. On the other hand, the administration of non-steroidal anti-inflammatory drugs in the early steps of weaning protects against tumor growth through reduction of the acute inflammatory reaction of post lactation remodeling of breast tissue. This is experimental evidence that may explain the short-term tumor-promoting effect of pregnancy. The protective effect of prolonged breast feeding may also be explained, at least in a part, by a reduced inflammatory reaction due to gradual weaning. Delay of first birth together with low parity and short duration of breast feeding are increasing social trends in developed countries. Therefore, breast cancer risk as a result of reproductive factors will not decrease in these countries in the foreseeable future. In this review, the significance of reproductive history with regard to the risk of breast cancers will be discussed, focusing on the age of first full-term birth and post lactation involution of the breast.

p53 protein accumulation predicts resistance to endocrine therapy and decreased post-relapse survival in metastatic breast cancer

IntroductionEndocrine therapy is the most important treatment option for women with hormone receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and cross-talk between ER and other growth factor-signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.Materials and methodsThe expression of HER2, p53, and Ki67 was examined by immunohistochemistry in primary breast tumour specimens from 73 metastatic breast cancer patients who received first-line treatment with endocrine therapy on relapse, and analysed to determine whether expression of these molecular markers affected the response to endocrine therapy.ResultsOf the 73 invasive ductal carcinomas, 12.3%, 21.9%, and 35.6% were positive for HER2 overexpression, p53 protein accumulation, and Ki67 expression, respectively. All patients received endocrine therapy as first-line treatment for metastatic breast cancer; 34 patients (46.6%) responded. Patients with primary breast tumours that had p53 protein accumulation and Ki67 expression showed significantly more resistance to endocrine therapy (P = 0.0049 and P = 0.024, respectively). There were also tendencies for HER2 overexpression to correlate with resistance to endocrine therapy, but this did not reach significance. p53 protein accumulation and HER2 overexpression significantly reduced post-relapse survival (P < 0.0001 and P = 0.001, respectively), and these factors were also statistically significant in a multivariate analysis.ConclusionThese data suggest that p53 protein accumulation is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in hormone receptor-positive metastatic breast cancer.

Microsatellite instability in sporadic human breast cancers

Human breast‐cancer specimens from 100 patients were analyzed for microsatellite instability (referred to as replication error; RER) at 12 genomic loci on 7 chromosomes, and results were correlated with clinicopathologic characteristics. In 42 of 100 breast‐cancer patients, we investigated whether RER was associated with the amplification of oncogenes and/or suppression of tumor‐suppressor genes. Of the 100 patients, 8 (8%) were RER‐positive at one or more chromosomal loci. The majority of RER‐positive patients had early‐stage disease with ER‐positive tumors, suggesting that RER occurs early in breast tumorigenesis. However, no significant correlation was observed between RER and oncogenes or tumor‐suppressor genes. Thus, the mechanism of RER in sporadic human breast cancer may be independent of the multi‐step carcinogenesis caused by the alterations of oncogenes and tumor‐suppressor genes.