Takuji Iwase

One-step Nucleic Acid Amplification for Intraoperative Detection of Lymph Node Metastasis in Breast Cancer Patients

Purpose: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. Experimental Design: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry–based three-level histopathologic examination. The results from the two methods were then compared. Results: We established CK19 mRNA cutoff values of 2.5 × 102 and 5 × 103 copies/μL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. Conclusion: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.

Molecular Detection of Lymph Node Metastases in Breast Cancer Patients: Results of a Multicenter Trial Using the One-Step Nucleic Acid Amplification Assay

Purpose: Accurate assessment of metastasis in sentinel lymph nodes (SLN) of breast cancer is important but involves a heavy workload for the pathologist. We conducted a multicenter clinical trial in Japan to evaluate a new automated assay system for cytokeratin 19 mRNA, the one-step nucleic acid amplification (OSNA) assay (Sysmex), to detect lymph node metastasis of breast cancer. Experimental Design: Surgically obtained axillary lymph nodes were sectioned into four pieces, two of which were examined with the OSNA assay. The other two adjacent pieces were examined with H&E and immunohistochemical staining for cytokeratin 19. Serial sections at 0.2-mm intervals were used in trial 1 to determine the specificity of the OSNA assay, and three pairs of sections cut from the sliced surfaces of the pieces were used in trial 2 to compare the accuracy of the OSNA assay with that of a routine pathologic examination for SLNs in Japan. Results: In trial 1, the sensitivity and specificity were 95.0% [95% confidence interval (95% CI), 75.1-99.9%] and 97.1% (95% CI, 91.8-99.4%), respectively, for 124 axillary lymph nodes obtained from 34 patients. In trial 2, the agreement between findings of the assay and of the pathologic examination was 92.9% (95% CI, 90.1-95.1%) for 450 axillary lymph nodes obtained from 164 patients. Conclusion: The OSNA assay can detect lymph node metastasis as accurately as can conventional pathology and thus can be an effective addition to or alternative for rapid intraoperative examination of SLNs.

Clinical Importance of Estrogen Receptor-β Evaluation in Breast Cancer Patients Treated With Adjuvant Tamoxifen Therapy

PURPOSE The clinicopathologic importance of a second estrogen receptor (ER), ER-beta, in breast cancers has been intensely studied; however, there is still no real consensus regarding the clinical utility of an ER-beta assay, probably because of the lack of standardized methodology, the presence of several ER-beta isotypes (ER-beta1-5, and so on), and, more importantly, the lack of convincing data on whether the ER-beta status provides clinically useful information over what is already provided by the traditional ER-alpha/progesterone receptor (PR) assay. A large and systematic study is needed to address these important issues. PATIENTS AND METHODS Archival materials of 442 invasive breast cancers from women treated with adjuvant tamoxifen monotherapy and with a long follow-up period (median, 11.1 years) were subjected to immunohistochemical study using three commercially available anti-ER-beta antibodies that detect ER-beta1-3 (ER-betaN), ER-beta1, and ER-betacx (ER-beta2). RESULTS Positive staining for ER-betaN or ER-beta1 was associated with significantly better survival. By contrast, ER-betacx status did not influence survival. In multivariate analysis, ER-beta1 status emerged as an independent predictor of recurrence and mortality. ER-beta1 status was significantly associated with survival in postmenopausal, but not premenopausal, women. Importantly, ER-beta1 positivity was associated with significantly better survival in patients with ER-alpha-negative/PR-negative or ER-alpha-negative/PR-negative/human epidermal growth factor receptor 2-negative (triple-negative) tumors, which are widely believed to be hormone unresponsive, have poor prognosis, and require chemotherapy. CONCLUSION Immunohistochemical examination of ER-beta1 in addition to ER-alpha and PR is clinically important in patients with breast cancer treated with tamoxifen monotherapy. Further studies are needed to confirm our findings.

Regular consumption of green tea and the risk of breast cancer recurrence: follow-up study from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), Japan

Experimental studies suggest various features of anticancer activity of green tea including inhibitory effect of tumor invasion and metastasis. This study was conducted to examine the association between regular green tea consumption prior to diagnosis and subsequent risk of breast cancer recurrence. The Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC) was started in 1988, in which information on lifestyle has routinely been collected from all first-visit outpatients by questionnaire. A total of 1160 new surgical cases of female invasive breast cancers with HERPACC information diagnosed between June 1990 and August 1998 were followed up through December 1999, and the risk (hazard ratio: HR) of recurrence was assessed with reference to daily green tea consumption using a Cox proportional hazard model. During 5264 person-years of follow-up, 133 subjects (12%) were documented to suffer recurrence of breast cancer. A decreased HR for recurrence adjusted for stage was observed with consumption of three or more daily cups of green tea (HR=0.69, 95% confidence interval (95%CI)=0.47-1.00). Particularly in stage I, the HR was decreased statistically significantly (HR=0.43, 95%CI=0.22-0.84). A similar tendency was observed for stage II subjects, but was not present among more advanced stages. Although careful interpretation is needed, these results suggest the possibility that regular green tea consumption may be preventive against recurrence of breast cancer in early stage cases.

Intraoperative molecular assay for sentinel lymph node metastases in early stage breast cancer: a comparative analysis between one-step nucleic acid amplification whole node assay and routine frozen section histology.

Conventional histopathological examination is limited in measuring accurate total metastatic volume in a lymph node. Recently, a molecular‐based procedure to detect lymph node metastases, one‐step nucleic acid amplification (OSNA) assay, has been developed. OSNA assay can assess a whole lymph node and yields semiquantitative results. The authors compared the performance in intraoperative detection of sentinel lymph node metastases with OSNA assay using a whole lymph node versus routine frozen section (FS) histology with a 2 mm‐sectioned lymph node.

A case-control study of breast cancer among Japanese women: with special reference to family history and reproductive and dietary factors.

SummaryTo study the effects of family history and reproductive, anthropometric, and dietary factors on the risk of breast cancer among low risk populations, we conducted a hospital-based case-control study involving 908 patients with breast cancer and their matched controls, in Japan. A positive family history of breast cancer significantly increased the risk of breast cancer (odds ratio = 1.52, 95% confidence interval: 1.14–2.03). The risk further increased with increasing number of family members affected. Obesity, single marital status, fewer births, a late childbirth, and less consumption of green-yellow vegetables and dairy products were also associated with an increased risk of breast cancer. These associations were independent in multivariate analyses. There was no increase in risk associated with consumption of high fat foods. When analyzed by menopausal status, the association with family history of breast cancer, especially in the first degree of relatives, was more evident for premenopausal breast cancer. The associations with obesity and lower consumption of dairy products were more pronounced for postmenopausal breast cancer, while those with lower parity and single marital status were stronger for premenopausal breast cancer.

Clinicopathological analyses of triple negative breast cancer using surveillance data from the Registration Committee of the Japanese Breast Cancer Society

BackgroundTriple negative (TN) breast cancer is defined as a subtype that is negative for estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). To clarify the characteristics of TN breast cancer, surveillance data of the Registration Committee of the Japanese Breast Cancer Society were analyzed.MethodOf 14,748 cases registered in 2004, 11,705 (79.4%) were examined for ER, PgR, and HER2. Of these, the most prevalent (53.8%) was a hormone-responsive subtype with ER positive/PgR positive/HER2 negative, followed by TN subtype (15.5%).ResultsThe proportion of postmenopausal patients was relatively high in the TN subtype. This cancer was diagnosed at a slightly advanced stage and with more cases positive for lymph node metastases than other subtypes. Morphologically, the TN subtype was more frequently classified as solid-tubular carcinoma. Mucinous, tubular, or secretary carcinomas were frequently found in the hormone receptor positive/HER2 negative subtype, while squamous cell carcinoma, spindle cell carcinoma, and metaplastic carcinoma with bone/cartilage metaplasia were very frequently found in the TN group. Apocrine carcinoma was also found very frequently in the TN group. Selection of chemotherapy was not based on receptor subtypes, but was determined by the degree of tumor progression.ConclusionsAlthough TN types are similar to basal-like breast tumor, as determined by gene profiling, their diagnosis needs verification by determination of the level of epidermal growth factor receptor or cytokeratin 5/6 expression. TN type should be examined further for immunohistochemical features and analyzed for prognostic details in this cohort.

Transforming growth factor B1 T29C polymorphism and breast cancer risk in Japanese women.

BackgroundA cohort study for Caucasians aged 65 years or older demonstrated a marked breast cancer risk reduction for those with theCC genotype oftransforming growth factor B1 (TGF B1) T29C polymorphism. This is a prevalent case-control study to examine the reported risk reduction for Japanese women.Patients and MethodsA total of 232 histologically diagnosed breast cancer patients who visited Aichi Cancer Center Hospital between June 1999 and March 2000 were enrolled. The controls were 172 female outpatients without cancer at the same hospital. DNA was extracted from peripheral blood, andTGF B1 genotype was determined by PCR-CTPP.ResultsThe genotype frequency was 23.7% forTT, 49.2% forTC, and 27.1% forCC among controls, and 28.9%, 46.1%, and 25.0%, respectively, among cases. Age-adjusted odds ratio (OR) relative to theTT genotype was 0.81 (95% confidence interval, 0.50-1.31) for theTC genotype and 0.77 (0.45-1.34) for theCC genotype. For premenopausal women, theCC genotype was significantly associated with reduced risk of breast cancer in comparison with theTT genotype (OR =0.45, 0.20-0.98). The association was not observed for postmenopausal women (OR = 1.40, 0.64-3.08).ConclusionThe present study showed risk reduction for Japanese premenopausal women with theCC genotype, but not for postmenopausal Japanese women.

Limited association between a catechol-O-methyltransferase (COMT) polymorphism and breast cancer risk in Japan.

AbstractBackground. Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy estradiol and 4-hydroxy estradiol. To date, three studies in Caucasians and one study in Chinese have been conducted to determine the association with breast cancer risk of a functional polymorphism (G-to-A, Val158Met) of this enzyme, but the results were inconsistent. In order to examine the impact of this polymorphism on breast cancer risk in Japan, a case-control study was conducted, at Aichi Cancer Center Hospital. Methods. The cases were 150 patients with histologically confirmed breast cancer who had been diagnosed within 4 years before enrollment at this hospital. The controls were 165 non-cancer patients, mainly from the gastroenterology and breast surgery clinics at the hospital. COMT-H (Val) is the wild-type allele, with high enzyme activity, while the COMT-L (Met) allele has low activity. Genotyping was conducted by a polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) method. Results. The allele frequency of COMT-L was 36.0% for cases and 33.0% for controls. Age-adjusted odds ratios relative to the COMT-HH genotype were 1.46 (95% confidence interval [CI], 0.90–2.36) for COMT-HL, and 0.99 (95% CI, 0.49–2.02) for the COMT-LL genotype. Significant odds ratios were not observed for any subgroup stratified by menopausal status, age at menarche, age at birth of first child, body mass index, and breast cancer history of mother and/or sister(s). Conclusion. The present study suggested that any association of the COMT polymorphism with breast cancer risk is limited in Japanese.

Association of genetic alterations on chromosome 17 and loss of hormone receptors in breast cancer.

To investigate possible relationships between genetic alterations and hormonal deregulation during breast cancer development and/or progression, we examined 616 primary breast cancers for loss of heterozygosity (LOH) at chromosomal regions 16q24, 17p13.3 and 17q21, and for amplifications of the ERBB2 and c-MYC loci. A comparison of oestrogen receptor (ER) and progesterone receptor (PgR) status in tumour cells with data concerning these genetic alterations revealed that LOH at 17q21 was significantly correlated with absence of oestrogen receptors (ER) (P < 0.0003) or progesterone receptors (PgR) (P < 0.0001), and with the absence of both (P < 0.0001). Similarly, a significant association was observed between amplification of ERBB2 and the absence of either ER or PgR. LOH at 17p13.3 was associated with the absence of PgR (P < 0.01). These data suggest a possible relationship between specific genetic changes on chromosome 17 and hormonal deregulation in the progression of breast cancer.