Futoshi Akiyama

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.

Mapping of a new target region of allelic loss to a 2-cM interval at 22q13.1 in primary breast cancer.

Allelic losses on chromosome arm 22q are frequently observed in human meningiomas and in carcinomas of the colon, ovary, and breast. Among 140 primary breast cancers we examined for loss of heterozygosity (LOH) at 16 polymorphic loci on the long arm of chromosome 22, 56 (40%) showed LOH for at least one locus. Eleven of these tumors had retained heterozygosity for markers proximal to the NF2 locus but showed LOH for markers distal to NF2. Deletion mapping indicated a new common region of deletion, 2‐cM in extent, at q13.1 between Interleukin 2 receptor β (IL2RB) and D22S279. Our results raise the possibility that one or more tumor suppressor genes associated with breast cancer may exist at 22q13.1. Comparison of these results with clinicohistological data indicated that allelic losses on 22q tend to occur more frequently in tumors of malignant histological types. Genes Chromosomes Cancer 21:108–112, 1998.

Microdissection Is Essential for Gene Expression Profiling of Clinically Resected Cancer Tissues

The gene expression array method enables us to achieve expression profiling with thousands of genes. Clinically resected bulk cancer tissues, however, contain not only cancer cells but also stromal cells, which may affect gene expression profiling and hamper accurate analysis of the cancer cells per se. Therefore, a procedure for dissecting specific cells, such as laser capture microdissection, is neededfor the clinical application of a gene expression array. There has been no study actually comparing 2 gene expression profiles, one obtained using RNA extractedfrom cancer cells by laser capture microdissection and one obtained using RNA extractedfrom bulk cancer tissues. Wefirst demonstrated the difference in expression patterns between them, without any amplification procedures. In addition, differential expression analysis between tumor and nontumor tissue yielded quite different patterns between the 2 methods. We conclude that microdissection is essential for gene expression profiling of clinical specimens.

Identification of Rad51 alteration in patients with bilateral breast cancer

AbstractThe human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response path-way associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the Rad51 gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6. This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150, which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation.

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.

Histopathological criteria for assessment of therapeutic response in breast cancer (2007 version)

In preoperative drug therapy (chemo-, endocrine, molecular-targeting, and other therapies) and/or radiotherapy for breast cancer, various degrees of histological changes of cancer tissue occur depending on the sensitivity of cancer cells to the particular treatment, kinds of therapeutic agents, dosage of drugs, method of administration, kind of combination, type and dosage of isotope, method of irradiation, duration of treatment, interval from the last treatment, and resection of cancer tissue. Histopathological criteria for assessment of response to neoadjuvant therapy for breast cancer have been established according to the degree of histological change of cancer tissue as described below. Histological diagnosis should be confirmed before treatment using biopsy materials, and it is important to compare the histopathological findings of cancer tissues before and after treatment for the assessment of response to therapy. Pathological response should be evaluated only from histological changes in the invasive area, and the presence of residual ductal components should be described. If only ductal components remain, the pathological response is evaluated as Grade 3.

Allelic Loss on Chromosome 1p Is Associated with Progression and Lymph Node Metastasis of Primary Breast Carcinoma

Frequent allelic losses on the short arm of chromosome 1 have been observed in a wide variety of human tumors. Cytogenetic and molecular genetic studies in breast carcinomas have shown frequent alterations on chromosome 1, involving loss of 1p or gain of 1q.

Duct endoscopy and endoscopic biopsy in the evaluation of nipple discharge

SummaryMicrodochectomy is usually performed on patients with nipple discharge caused by intraductal proliferative lesions, such as intraductal papilloma and carcinoma. But this operation often sacrifices large amounts of normal mammary gland even when the lesion is a benign intraductal papilloma a few millimeters in diameter. We have developed duct endoscopy for the mammary duct system, and have reliably performed biopsies for intraductal proliferative lesions intraductally. From June 1989 to April 1990, we examined 22 cases by duct endoscopy, and performed endoscopic biopsy in 16 cases. The method of endoscopic biopsy is as follows. First, a bougie is inserted, without anesthesia other than Xylocaine jelly, into the orifice of the duct to enlarge it. Second, the outer cylinder and the inner needle are inserted; then the inner needle is removed, and the endoscope is inserted. After examination, the outer cylinder is moved up to the lesion to be biopsied and the endoscope is taken out. Then a sample is taken into the outer cylinder by aspiration. We diagnosed 10 cases of benign lesion and 5 cases of malignant lesion by cytological and/or histological examination. In conclusion, endoscopic biopsy, aided by duct endoscopy, is a useful and harmless diagnostic procedure in the evaluation of nipple discharge.