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Fine needle aspiration (FNA) samples were obtained from 176 breast tumors suspected of malignancy, which were then subjected to conventional cytological and fluorescence in situ hybridization (FISH) analyses using the centromeric probes for chromosomes 1, 11, and 17. Histological examination revealed 157 breast cancers and 19 benign diseases (ten fibroadenomas, six intraductal papillomas, one intracystic papilloma, and two ADH). Sensitivity, specificity, and diagnostic accuracy were 85.4% 94.7%, and 86.4%, respectively, for cytology and 90.4%, 100%, and 91.5%, respectively, for FISH. These results demonstrate that FISH diagnosis of FNA samples has a diagnostic accuracy comparable to that of conventional cytology.

Axillary lymphnode dissection (ALND) for breast cancer patients provides local control and information for the determination of the type of adjuvant therapy.The benefit of axillary surgery itself for survival is considered to be limitedto patients with positive nodes. Sentinel node biopsy is a recently developed, minimally invasive technique for precisely predicting axillary nodal status. As this technique has less morbidity and greater accuracy than ALND, it replaces ALND for patients with node negative breast cancer. In this report, we outline thecurrent status of sentinel node biopsy for breast cancer patients and introduceour preliminary results.

BACKGROUND: Four different endocrine therapeutic agents have been used in Japan since 1996. However, a consensus regarding proper use of these agents has notyet been established. Therefore, a questionaire survey of Japanese breast cancer authorities on endocrine therapy and a multi-institute survey to investigate the efficacy of a single first-line endocrine therapy for recurrent breast cancerwere conducted. MATERIALS AND PATIENTS: A total of 279 questionaires were sent to the Councilors of the Japanese Breast Cancer Society. The clinico-pathological data of 77 breast cancer patients who underwent a single first-line endocrine therapy were collected from five institutes. RESULTS: The response rate to this questionaire survey was 67.4%. The resultsshow that many authorities consider that: 1) both ER and PgR in primary tumors should be measured, 2) patient age, the disease-free interval and postoperative adjuvant therapy do not provide enough information for the selection of endocrine therapies, 3) antiestrogen and LH-RH agonists should be used as first-line endocrine therapies, 4) combined endocrine therapies, such as an antiestrogen plus an LH-RH agonist, should be used, 5) the optimal sequence of use of endocrine therapeutic agents is most controversial. The objective response rate to first-line endocrine therapies was 40.3% and the duration of response was over 15 months.The objective response rate to second-line endocrine therapies was 42.1%. A multiple regression analysis of predictive factors for the efficacy of first-line endocrine therapies indicated two factors, the disease-free interval and dominantsite of metastasis, to be significant. Conclusions: This questionaire survey suggests that clinical trials to investigate the optimal sequence of use of endocrine therapies and to clarify the usefulness of combined endocrine therapies should be conducted. Single first- or second-line endocrine therapies for recurrent breast cancer are effective and should be carried out by general clinicians.

BACKGROUND: To elucidate the cause of in-breast recurrence after breast conserving surgery, we analyzed the characteristics of resected specimens histopathologically, especially the surgical margin status. MATERIALS AND METHODS: 1) Pathological surgical margin positivity was reevaluated in terms of the distance from the resected surgical margin by pathologists from seven institutions in 486 cases with complete stepwise pathological examination. 2) We reviewed pathological specimens including surgical margins from 30 patients with in-breast recurrence for whom serial sections of resected primary breast cancer specimens were available and made comparisons of the time to in-breast recurrence. RESULTS: Cancer cells at the surgical margin were present at a rate of 4.1% on the surface and 15.2% within 5 mm on the areolar side of the surgical margin. Histopathologically, the reasons for local recurrence after breast conserving therapy included a positive surgical margin (21/30), lymphatic permeation (4/30), and others (5/30). The last category included cases with an inadequate margin diagnosis because of a biopsy scar. Disease-free intervals for the patients without postoperative radiotherapy decreased as the volume of cancer cell nests in thesurgical margin increased (P=0.06). On the other hand, this trend was not observed in the group with postoperative radiotherapy. CONCLUSION: Adequate materials are essential for accurate evaluation of surgical margin status. Quantitative evaluation of surgical margin status, apart fromwhether or not radiotherapy was performed, is important for estimating the riskand disease-free period to in-breast recurrence.

A 63-year-old man was referred to our institute for the treatment of squamous cell carcinoma of the upper lobe of his right lung. A right upper lobectomy of the lung was performed with a mediastinal lymph node dissection. The postoperative pathological examination of the dissected specimens revealed one of the superior mediastinal lymph nodes to be morbid with micrometastasis of occult thyroid cancer, while no node involvement was seen due to lung cancer. A right lobectomy of the thyroid gland with a modified radical neck dissection was done 4 years later after the confirmation of the absence of any recurrent sign of lung cancer. In the resected specimen, papillary thyroid microcarcinoma was observed with several intraglandular metastases and right regional lymph node involvement. Eight months later, a new primary lung cancer developed in the left lung, and a left upper lobectomy of the lung with a mediastinal lymph node dissection was performed. At that time, the absence of mediastinal lymph node metastasis from lung cancer or thyroid cancer was confirmed. Mediastinal lymph node involvement as the initial manifestation of occult thyroid cancer in surgical treatment for lung cancer is rare, but it is important to be aware of the possibility of incidentally detecting occult thyroid cancer in surgical dissections in this area for lung cancer. The appropriate surgical treatment should be determined while carefully considering the prognosis of the lung cancer as well as that of any coexisting malignancy.

One of the etiologic factors involved in local recurrence after breast-conserving surgery may be malignant seeding of the wound during the lumpectomy procedure. A total of 340 patients with stage I and II breast cancer were entered into the study. Of these, 270 patients received breast-conserving surgery (BCS group), and the other 70 patients underwent mastectomy (control group). After resection, lavage cytology was performed at the surgical wound. There were 55 patients (20.4%) who showed positive lavage cytology in the BCS group. In the control group, there were only 3 patients (4.3%) with positive cytology. Positivity was significantly higher in the former group (p = 0.00064). Patients with evidence of cutting across cancer lesions showed significantly higher positive rates in lavage cytology (p < 0.00001). Positivity in lavage cytology was significantly higher in patients with positive surgical margins evaluated by frozen sections (p = 0.0017), touch cytology (p < 0.0001) and formalin-fixed, paraffin-embedded sections (lateral or medial margin; p = 0.0036, anterior and posterior margin: p = 0.0210). The positivity was also significantly higher in patients with an extensive intraductal component (p < 0.0001), and less than or equal to 50 (p = 0.0061) years of age. Multivariate analysis revealed that the highest relative risk factor for positive cytology was evidence of cutting across cancer lesions (relative risk = 8. 166; p < 0.00001).

A 10-center cooperative clinical study with a new formulation of epirubicin hydrochloride injectable solution (Epirubicin-RTU) was conducted in patients with breast cancer. One course of treatment consisted of one intravenous administration of Epirubicin-RTU at the dose of 60 mg/m2 followed by a 3-week drug-free interval and concomitant daily administration of oral cyclophosphamide at 100 mg/day during the period between Days 1 through 14. At least, two courses of treatment were given. Among 20 registered cases, all 20 cases were eligible and 16 cases completed the whole course of the study. In 16 completers, PR was observed in 5 cases, indicating the efficacy rate of 31.3% (5/16).. No local irritation was observed at the injection sites. Adverse reactions frequently observed were leukopenia, neutropenia, anorexia, alopecia, and nausea/vomiting, which were all reversible and tolerable. From the above results, Adverse reactions both locally and systemically were tolerable. Intravenous administration of Epirubicin-RTU was considered to be useful for the treatment of breast cancer.

Prognostic factors for distant metastases in patients with local recurrence after breast-conserving treatment (BCT) were studied. Fifty-six patients who developed local recurrence after BCT were recruited from 18 key hospitals/institutes in Japan. All 10 patients whose primary tumors were DCIS fared well without evidence of distant failure for a median follow-up period of 57 months (range 41-72) after the local recurrence. Inflammatory local recurrence was observed in 5 patients whose prognosis was grave: 3 with concomitant distant metastases and 1 developing them 7 months later. In the remaining 41 patients with noninflammatory local recurrence, various clinicopathological factors including age, disease-free interval, histology of the primary and recurrent tumors, axillary lymph node status, estrogen and progesterone receptor, immunohistochemical staining of erbB2 and p53 protein were evaluated as prognostic factors. Only the p53 immunostaining status of the primary tumor was found to be a significant prognostic indicator for distant metastases; distant disease-free survival at 5 years after the local recurrence was 92% for patients with p53-negative cancers and 51% for those with p53-positive cancers (p < 0.05).

The indications for a breast conserving operation in the treatment of ductal carcinoma in situ (DCIS) of the breast with clinical manifestations other than mammographically detected calcifications are controversial. A positive surgical margin has been suggested to be an improtant risk factor for local recurrence after a breast conserving operation. We attempted to clarify the frequency of positive surgical margins when performing breast conserving operations for DCIS, identify the risk factors for positive margins, and also to evaluate the short-term outcome. Between 1988 and 1992, 5571 breast cancer cases were surgically treated at the 7 institutions of the authors, of which 375 cases (6.7%) were histologically diagnosed as DCIS. The most frequent clinical manifestation was a tumor in 64% of the cases, followed by nipple discharge in 23% and calcification on mammography in 12%. Of these 375 cases, 242 cases were analyzed. Sixty-six cases had undergone a breast conserving operation. Axillary dissection was not performed in 29 cases. The median follow up period was 61.4 months. The initial surgical margin was positive in 29% of the cases. The most significant factor for a positive surgical margin was young age fllowed by large tumor size. There were four cases with local recurrence. Three recurrences developed in the same quadrant. All four cases remain alive after total mastectomy. There were no cases with distant metastasis or axillary recurrence. Breast cancerving operation for DCIS have shown satisfactory results to date, and when clear surgical margins can be obtained, this procedure, without axillary dissection, should be considered even for patients with clinical manifestations other than mammographically detected calcifications.

Urokinase-type plasminogen activator (u-PA) is a key protease in cancer invasion and metastasis. Recent studies demonstrated that u-PA, plasminogen activator inhibitor type-1 (PAI-1), and tissue-type plasminogen activator (t-PA) are prognostic factors in breast cancer. However, there have been no prospective studies of node-negative breast cancer on a multicenter basis. On the other hand, some patients, even those with node-negative breast cancer, developed recurrence, and only tumor size is available as a predicting factor in this group. Therefore, it is necessary to find other prognostic factors in node-negative breast cancer to determine suitable adjuvant therapies. Tissue samples in this prospective study were obtained from 130 patients with node-negative invasive breast cancer who underwent radical operation at four hospitals. The median follow-up was 52.6 months. u-PA, PAI-1, and t-PA antigen levels were assayed by ELISA kits using the cytosolic fractions of tumors. Patients with high u-PA, high PAI-1, or low t-PA had significantly higher relapse rates than did those with low u-PA, low PAI-1, or high t-PA, respectively, by the Kaplan-Meier method (P = 0.006, 0.032, and 0.028, respectively). Analyses of the combinations of both u-PA and PAI-1 or both u-PA and t-PA showed that the differences in relapse rate between the high- and low-risk groups were statistically very significant. In the univariate analysis, u-PA, PAI-1, t-PA, progesterone receptor, and tumor size (T3 versus T1) were significantly correlated with relapse. However, the multivariate analysis revealed that only u-PA (P = 0.023) was an independent prognostic factor. This study showed that u-PA was a new significant independent prognostic factor in node-negative breast cancer.

A case-control study was conducted on 143 case-control sets recruited from the 18 key hospitals/ institutes in Japan to identify risk factors for local recurrences and distant metastases after breast-conserving surgery in breast cancer patients: (1) positive surgical margin was a risk factor for local recurrences (relative risk (RR) = 16.70, p < 0.001) but not for distant metastases, (2) positive p53 immunostaining was a risk factor for both local recurrences (RR = 5.62, p = 0.003) and distant metastases (RR = 3.11, p = 0.034), (3) lymph node metastasis was a risk factor for distant metastases (RR = 9.28, p = 0.001) but not for local recurrences, (4) radiation therapy reduced local recurrences (RR = 0.13, p = 0.004) and (5) adjuvant chemotherapy (RR = 0.27, p = 0.120), endocrine therapy (RR = 0.21, p = 0.037), and chemoendocrine therapy (RR = 0.05, p = 0.013) reduced local recurrences.

pS2 protein expression is induced by estrogen through estrogen receptors (ER), and is known to be inhibited by antiestrogen in the breast cancer cell line MCF-7. The present study was undertaken to determine whether pS2 expression may be inhibited by the antiestrogen tamoxifen (TAM) human breast cancer. pS2 expression was immunohistochemically investigated in 22 patients treated preoperatively with TAM and in 45 without TAM treatment (control group). Immunostaining was considered positive when 10% of tumor cells showed cytoplasmic staining. We found that 94% of the pS2-positive tumors were ER-positive (ER+), and there was a significant association between the two proteins (p < 0.0001). In ER+ tumors, pS2 expression was seen in only 21% (3 of 14) of the TAM+ group, compared to 70% (16 of 23) of the control group. Statistical analysis of these data indicated a high likelihood (p = 0.006) of TAM treatment being associated with inhibition of pS2 expression in human breast cancers.

The main therapy for primary breast cancer is not surgery, but a systemic therapy involving administration of cytotoxic chemotherapy or the use of ablative or additive endocrine therapy to control disseminated micrometastasis. The results of randomized trials and meta-analysis show that CMF, the standard adjuvant chemotherapy, is effective regardless of axillar lymph node involvement or menopausal status. Effectiveness of adjuvant chemotherapy with an anthracyclin-based regimen remains controversial. The trial by CUBC and NSAS-BC comparing UFT, widely used in the management of patients with breast cancer in Japan, with CMF is on-going.

Breast conserving therapy (BCT) was conducted in 22.1% of breast cancer patients in 1994 in Japan and is being performed with increasing frequency. According to the data already published, 10-27% of patients treated with BCT had a positive surgical margin. The recurrence rate in the breast is 1-2% annually, The 5-year overall survival in patients mostly consisting of stage I is 90% or higher at the present time. A case-control analysis of a multicenter study revealed that significant risk factors for breast recurrence were a positive surgical margin and absence of radiation therapy. Progress in basic research and diagnosis has also been instrumental in improving treatment results.

Background: Amplification of keratin 19 mRNA (K19) by reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to be a sensitive method to detect occult breast cancer metastases in lymph nodes., Methods: Axillary lymph nodes were obtained from 126 patients with breast cancer, and metastases in these lymph nodes were studied by both histologic examination and K19 RT-PCR. The patients were categorized into 3 groups according to the results of these 2 examinations, i.e., patients with 1) both histologically and K19 RT-PCR negative lymph nodes (metastases negative group [n = 91]); 2) histologically negative but K19 RT-PCR positive lymph nodes (occult metastases positive group [ n = 15]); and 3) histologically positive lymph nodes (metastases positive group [n = 20])., Results: Various histologic parameters such as tumor size, histologic type, histologic grade, lymphatic invasion, vascular invasion, and estrogen receptor status were compared among these three groups. There were no significant differences among any of these histologic parameters between the metastases positive and occult metastases positive groups. Conversely, tumor size of the metastases positive (2.5 +/- 0.2 cm) and occult metastases positive (2.5 +/- 0.2 cm) groups was significantly (P < 0.05) greater than that of the metastases negative group (1.9 +/- 0.1 cm), and positivity of lymphatic vessel invasion in the former 2 groups (70% and 53%, respectively) was also significantly (P < 0.01) greater than that in the latter group (18%)., Conclusions: These results demonstrate that histologic characteristics of breast cancers with occult metastases are similar to those of breast cancers with histologically detectable metastases.

We measured pS2 protein in the serum of patients with adenocarcinoma and non-adenocarcinoma types of lung cancer, non-cancerous lung lesions, and the control sera. Although the serum pS2 protein level in patients with lung adenocarcinoma was significantly higher than that in patients with other diseases, as well in control samples, it had little clinical value as a screening tumor marker because the levels in control samples showed a wide range of variation. However, analysis according to the histological subtype of lung adenocarcinoma, ordinary and bronchioloalveolar, revealed a high serum level of pS2 protein in several patients with advanced stage disease in the former, and mucus-producing goblet cell subtypes in the latter, which showed strong pS2 protein expression in tissues, whose serum levels diminished to the control level after resection. Thus, the serum levels of pS2 protein may be a useful marker of tumor burden in selected patients with lung adenocarcinoma.

Phyllodes tumor of the breast is histologically unique in that it is composed of epithelial and stromal components. The histogenesis of this disease has remained largely unknown but recently we have obtained interesting information through clonal analysis, and have been able to show a difference in the histogenesis between phyllodes tumor and fibroadenoma. Elucidation of histogenesis would difinitely improve our understanding of the biological behavior of this disease and also help to establish the most appropriate treatment strategy. The pathology, histogenesis, diagnosis, and treatment of phyllodes tumor are reviewed in this paper. Our hypothesis on the histogenesis of this disease is also presented.

Keratin 19 mRNA reverse-transcriptase polymerase chain reaction (K 19 RT-PCR) was compared with histological examination in the detection of breast cancer micrometastases in axillary lymph nodes. Sixty-three axillary lymph nodes, which were obtained from 23 breast cancer patients, were bisected. One half was studied by conventional histological analysis of hematoxylin and eosin sections. Total RNA was extracted from the other half and subjected to K 19 RT-PCR. In all the ten lymph nodes, which were histologically metastasis-positive, K 19 mRNA was detected by RT-PCR. Of the 53 histologically negative lymph nodes, five (9%) lymph nodes were found to express K 19 mRNA, indicating the presence of micrometastases which could be detected by RT-PCR but not by a histological examination. These results demonstrate the usefulness of K 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes.

Background: Parotid gland pleomorphic adenoma is histologically comprised of epithelial and mesenchymal elements. It remains to be established whether this neoplasm arises from epithelial and mesenchymal elements, or solely from the epithelial element., Methods: In an attempt to resolve this issue, we have conducted clonal analysis on five pleomorphic adenomas. The method for clonal analysis was based on the trinucleotide repeat polymorphism of the x-chromosome-linked androgen receptor gene and on random inactivation of this gene by methylation. The epithelial and mesenchymal elements were obtained separately from the paraffin sections of the pleomorphic adenomas using a microdissection technique and then subjected to clonal analysis., Results: Clonal analysis revealed that both epithelial and mesenchymal elements were monoclonal. In addition, the same allele of the androgen receptor gene was inactivated in both elements in every case., Conclusions: It is unlikely that the epithelial and mesenchymal elements of different origin happen to inactivate the same allele of the androgen receptor gene in all five tumors. Rather, it is more reasonable to consider that these two elements have a common single cell origin.

Usefulness of MUC1 mRNA and keratin 19 mRNA as a target of reverse-transcriptase polymerase chain reaction (RT-PCR) was compared in the detection of breast cancer micrometastases in axillary lymph nodes. RT-PCR amplification of MUC1 mRNA and keratin 19 mRNA was conducted using total RNA samples. RT-PCR products were stained with ethidium bromide and analyzed by agarose gel electrophoresis. Expression of both MUC1 mRNA and keratin 19 mRNA was detected by RT-PCR in a breast cancer cell line (MRK) and in all the 23 primary breast cancers but not in the control lymph nodes obtained from patients with benign diseases. A serial dilution study of MRK cells against normal lymph node cells has shown that detection sensitivity of MUC1 RT-PCR and keratin 19 RT-PCR were 1/10(5) and 1/10(6) (cancer/lymph node cells), respectively. Sixty-three axillary lymph nodes were obtained from 23 patients with primary breast cancer, and metastases in each lymph node were investigated by histological examination (hematoxylin and eosin sections) and RT-PCR method. In all 10 lymph nodes, which were histologically metastasis-positive, both MUC1 mRNA and keratin mRNA were detected by RT-PCR. Of the 53 histologically negative lymph nodes, 3 (6%) and 5 (9%) lymph nodes were found to express MUC1 mRNA and keratin 19 mRNA, respectively, indicating the presence of micrometastases which could be detected by RT-PCR but not by histological examination. These results demonstrate the usefulness of both MUC1 RT-PCR and keratin 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes, and also indicate the superiority of keratin 19 RT-PCR over MUC1 RT-PCR because of its higher detection sensitivity.

We examined the expression of ErbB-2 and pS2 proteins in 59 ductal carcinoma in situ (DCIS) of the breast, either pure DCIS or DCIS associated with invasive carcinoma, using immunohistochemical staining of paraffin-embedded sections. Positive staining for ErbB-2 and pS2 proteins was noted in 32% (19/59) and 46% (27/59) of DCIS, respectively. An inverse relationship between ErbB-2 and pS2 status in DCIS was observed (p < 0.01). From the viewpoint of histological subtype, the prevalence of ErbB-2 protein expression was significantly higher in the comedo subtype than the cribriform-micropapillary subtype. The prevalence of immunoreactivity for ErbB-2 in solid subtype was intermediate between those of the other two groups. In contrast, the prevalence of pS2 expression was significantly lower in the comedo subtype than in the cribriform-micropapillary subtype. Again, the prevalence of pS2 protein expression in the solid subtype was intermediate between those of the other two subtypes. Our results suggest that DCIS is biologically heterogeneous with regard to such marker substances. This has possible implications for management of these lesions.

Recently, a variety of new prognostic factors for breast cancer have been advocated, although most of these studies are still preliminary. Until the results of definitive studies to evaluate the clinical usefulness of these new factors are obtained, it is practically relevant to plan a treatment schedule of breast cancer patients through classical prognostic factors such as tumor size, axillary lymph node status, histological grade, and estrogen/progesterone receptor status.

Clonal analysis was conduced on a variety of benign and malignant human breast tumors using the method based on restriction fragment length polymorphism (RFLP) of the X 120 chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Breast carcinoma was shown to be monoclonal in origin, consistent with a somatic mutational theory. Precancerous lesions such as atypical ductal hyperplasia and multiple intraductal papilloma were also found to be monoclonal, indicating that certain genetic changes had been accumulated in these lesions. Solitary intraductal papilloma was found to be monoclonal. Since this tumor is composed of two types of cells, luminal epithelial cells and myoepithelial cells, it was suggested that the origin of solitary intraductal papilloma is a precursor cell which is capable of differentiating into both luminal and myoepithelial cells. The fact that fibroadenoma is polyclonal indicates that this tumor is not neoplasia but hyperplasia of a lobule. Epithelial component of phyllodes tumor was found to be polyclonal but stromal component was found to be monoclonal. Thus, phyllodes tumor is considered to be a neoplasm of stromal cells but not of epithelial cells.

There is no established method for assessing the prognosis of patients with breast cancer and metastasis confined initially to bone. The medical records of 82 patients with breast cancer nad metastasis confined initially to bone were reviewed. The following variables were analyzed at the time when bone metastasis was first diagnosed, to determine their relationship to length of survival: distribution of metastatic bone lesions on bone scan, presence of radiographic osteosclerosis in metastatic bone lesions, menstrual status, and disease-free interval. Univariate and multivariate analyses revealed that the distribution of metastatic bone lesions and the presence of radiographic osteosclerosis in these lesions were significant predictors of survival. Premenopausal or late postmenopausal status, and longer disease-free intervals (> or = 24 months) or no disease-free intervals (Stage IV breast cancer and metastasis confined to bone at the time of cancer diagnosis) showed a trend, although not statistically significant, toward longer survival. Distribution of metastatic bone lesions on bone scan and the presence of radiographic osteosclerosis in metastatic bone lesions should be considered prognostic variables for patients with breast cancer and metastasis confined initially to bone.

In a patient with non-palpable breast carcinoma, c-erbB-2 gene amplification was detected by means of polymerase chain reaction (PCR) in the small number of breast carcinoma cells present in nipple discharge. Amplification of the c-erbB-2 gene is more frequent in carcinoma in situ than in invasive types. Detection by a PCR-based method may help diagnose non-palpable breast carcinoma with nipple discharge. Since this gene amplification is related to high proliferation, it might provide useful preoperative information regarding intraductal carcinoma of comedo type and predict responses to chemotherapy.

Since ER positivity in breast cancer is highest in the early stage of its natural history, hormonal treatment should be given as the initial treatment after both surgery and recurrence. Tamoxifen and medroxyprogesterone acetate have been commonly used as the standard hormonal treatment with a response rate of 20-30% and a median duration of response of 6-10 months. New agents for hormone therapy include tamoxifen analogues, LH-RH analogues and aromatase inhibitors. They are now on or have just cleared phase studies. LH-RH analogue is a unique drug that is active for premenopausal patients and should be used as the first-line therapy for them. Aromatase inhibitors lower serum estrogen levels by inhibiting conversion from andreogens to estrogens, thus inducing regression of ER-positive tumors. They are used as the second-line treatment after tamoxifen failure for postmenopausal patients. An increasing body of information has been accumulated as to how the hormonal treatments affect breast cancer at subcellular levels. New approaches controlling growth factors such as anti-angiogenesis are under way.

C-erbB-2 oncoprotein (ErbB-2 protein)and carcinoembryonic antigen (CEA)were simultaneously evaluated in the breast cyst fluid of 6 patients with intracystic cancer, 6 patients with intracystic papilloma, and 42 patients with gross cystic disease. A combination test, using 12 ng of ErbB-2 protein and 150 ng of CEA/ml of cyst fluid, respectively, as cutoff values resulted in a positive detection rate of 83%(5/6 cases) for intracystic breast cancer. Two of the 6 patients with intracystic papilloma and 2 of the 42 patients with gross cystic disease were positive. In addition, elevated levels of these markers in cyst fluid correlated well with the expression of these antigens in the corresponding tumor. Thus, it can be concluded that measurements of ErbB-2 protein and CEA aid in the diagnosis of intracystic breast cancer, and the assay of ErbB-2 protein is valuable for detecting tumors in which this protein is overexpressed.

Recently, it has been demonstrated that breast carcimoma is monoclonal and fibroadenoma is polyclonal in origin. In the present case report, this observation was successfully applied to a preoperative differential diagnosis of a fibroadenoma clinically and cytologically indistinguishable from carcimoma. Case report: A45-year old female presented for clinical examination with a history of breast lump. A firm tumor measuring 2 x 2 cm was palpable in the upper-outer quadrant of @the left breast. The margin of the tumor was partially ill-defined and its mobility was restricted. A tumor shadow with a partially ill-defined margin was revealed by mammography. Ultrasonographic examination showed and irregularly-shaped, hypo-echoic tumor accompanied by an acoustic shadow. Fine needle aspiration biopsy yielded positive cytology. Based on these results, the tumor was diagnosed as breast carcinoma. However, clonal analysis of fine needle aspirates showed a polyclonal pattern, strongly indicating that the tumor was not a carcinoma but benign disease; most probably fibroadenoma. Thus, an open surgical biopsy was performed. Histoglogical examination revealed that the tumor was indeed a fibroadenoma with epithelial hyperplasia. This case suggests the usefulness of clonal analysis of fine needle aspirates in differentiating fibroadenoma from carcinoma of the breast.

Clonality of solitary intraductal papillomas of the breast was analyzed using a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of such a small intraductal lesion as papilloma. Clonal analysis of DNA samples obtained from the nine solitary intraductal papillomas and adjacent normal breast tissues showed that all of the papillomas were monoclonal and all the normal breast tissues were polyclonal in origin. When DNA samples were obtained from four widely separated sites in the papillomas, clonal analysis showed that all were monoclonal and, in addition, the same allele of PGK gene was inactivated in each case. These results demonstrate that solitary intraductal papilloma arises as a single monoclonal tumor and extends along the ducts rather than occurring as multicentric monoclonal tumors and merging together subsequently. Immunohistochemical staining of smooth muscle alpha-actin, a marker protein of myoepithelial cells, revealed that solitary intraductal papilloma was composed of approximately equal mixtures of luminal epithelial and myoepithelial cells. Since solitary intraductal papillomas were shown to be monoclonal in origin, it was suggested that this disease originates from a common precursor that could differentiate into both luminal epithelial and myoepithelial cells.

Clonal analysis has been done using the method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Introduction of polymerase chain reaction (PCR) of this method has enabled clonal analysis of minute lesions. Utilizing this PCR-based method, clonality of a variety of human breast tumors (breast carcinoma, fibroadenoma, phyllodes tumor, intraductal papilloma, and atypical ductal hyperplasia) was studied in the present investigation in order to obtain valuable information on the histogenesis of these tumors.

Clonality of predominantly intraductal carcinoma (PIC) and precancerous lesions of the breast was analyzed by a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of small lesions. In order to eliminate the contamination by normal stromal cells, intraductal components were microdissected from the frozen sections of PIC under a dissection microscope. Clonal analysis of the intraductal components from seven PICs revealed that all were monoclonal in origin. In three PICs with intraductal spreading of carcinoma cells over nearly a whole breast gland, the intraductal components were collected from eight widely separated sites in each case. Clonal analysis of these samples showed that every sample was monoclonal and the same allele of the phosphoglycerokinase gene was consistently inactivated in each case. These results suggest that PIC arises as a single monoclonal carcinoma and spreads through the ducts over the gland rather than having multicentric origins. Clonality of precancerous lesions such as atypical ductal hyperplasia and intraductal papilloma arising in the terminal ducts was also studied. Intraductal components were microdissected from the paraffin sections of these lesions and subjected to clonal analysis. Both atypical ductal hyperplasia and intraductal papilloma were found to be monoclonal in origin, suggesting that certain genetic changes had already occurred in the precancerous lesions. A further study is needed to elucidate these genetic changes, which would greatly help our understanding of the mechanism of carcinogenesis.

Background: It is often difficult to draw a firm conclusion as to whether the second breast cancer is primary or secondary (metastasis from the initial breast cancer) in a patient with metachronous bilateral breast cancer. In this study we have applied clonal analysis of breast cancer to distinguish whether the second breast cancer is primary or secondary., Methods: A 54-year-old woman underwent modified radical mastectomy of the right breast as a result of breast cancer. Five years later she had tumors in the right chest wall and left breast. Fine-needle aspiration cytologic examination revealed that both tumors were adenocarcinoma. To elucidate the origin of these tumors, clonal analysis was done on DNA samples prepared from cryostat sections of the initial right breast cancer and from fine-needle aspirates of the tumors in the right chest wall and left breast. The method for clonal analysis was based on restriction fragment length polymorphism of X chromosome-linked phosphoglycerokinase gene and on differential methylation of the gene caused by random inactivation of one of two X chromosomes., Results: Clonal analysis revealed that clonal origin of the right breast cancer was different from that of the left breast cancer and identical to that of the chest wall tumor. Therefore it was concluded that the left breast cancer was primary and the chest wall tumor was concluded that the left breast cancer was primary and the chest wall tumor was a recurrence of the initial breast cancer., Conclusions: Clonal analysis appears to be a useful method in discriminating a primary from a secondary cancer.

We examined the expression of pS2 protein in 48 invasive ductal breast carcinomas with an extensive intraductal component, using immunohistochemical staining of paraffin-embedded sections. The patients selected for this study would have met the criteria for breast-conserving surgery applied at our institute at present. The rate of pS2 expression in the intraductal lesion was significantly higher than that in the main invasive lesion. The incidence of pS2 protein expression in the latter lesions was very similar to that in invasive carcinoma without intraductal lesions. The pS2 positivity of the intraductal lesion was equal to or higher than that of the invasive lesion. Of intraductal lesions, those classified as non-comedo carcinomas frequently contained more pS2 protein than did comedo carcinomas.

Clonality of fibroadenoma and phyllodes tumor of the breast was analyzed by means of the polymerase chain reaction using small DNA samples prepared from cryostat sections. The method of clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase gene and on differential methylation of the gene. Specimens from 10 fibroadenomas and 5 phyllodes tumors heterozygous for the BstXI polymorphism of PGK gene were subjected to clonal analysis. It was found that fibroadenoma was polyclonal, but phyllodes tumor was made up of both monoclonal and polyclonal cell components. Since these tumors contained both epithelial and stromal components, clonality of each component was analyzed separately. Analysis of clonality of each cell component showed that both the epithelial and stromal cells were polyclonal in fibroadenoma and that the epithelial cells were polyclonal, but the stromal cells were monoclonal in phyllodes tumor. When DNA samples were prepared from widely separated sites of phyllodes tumors, every sample was found to contain a monoclonal stromal cell component. These results demonstrate that fibroadenoma is a hyperplastic lesion rather than a neoplasm, and that phyllodes tumor is a neoplasm of the stromal cells.

Background: The influence of tamoxifen treatment on transforming growth factor-alpha (TGF-alpha) levels in human breast cancer rarely has been studied in vivo., Methods: Postmenopausal patients with estrogen receptor (ER)-positive and progesterone receptor (PR)-positive primary breast cancer underwent two fine-needle aspiration biopsies (FNA) of the tumors. Between the two FNAs, 10 patients received no treatment (control group), and the other 10 patients received tamoxifen (20 mg/day) for 10 (8-12) days (TAM group). TGF-alpha levels in FNA samples were assayed by enzyme immunoassay., Results: No significant difference was found in TGF-alpha levels between the first and second FNA samples in the control group. On the other hand, in the TAM group, TGF-alpha levels in the second FNA samples (2.5 +/- 0.5; mean +/- SEM ng/mg.DNA) were significantly (P < 0.01) lower than those in the first (4.5 +/- 0.8). Studies on the influence of tamoxifen treatment on TGF-alpha levels in ER-negative and PR-negative breast cancer showed that TGF-alpha levels were not affected by tamoxifen treatment. Positivity of epidermal growth factor receptor (EGFR) was 60% in ER-negative and PR-negative breast cancer and 30% in ER-positive and PR-positive breast cancer., Conclusions: Tamoxifen downregulates TGF-alpha levels in ER-positive and PR-positive breast cancers through ER. The significance of TGF-alpha as an autocrine growth factor appears to be more important in ER-negative and PR-negative breast cancer with high EGFR positivity than in ER-positive and PR-positive breast cancer with low EGFR positivity.

The influence of a somatostatin analogue, SMS 201-995 (SMS), on the growth of an androgen-dependent mouse mammary tumor, Shionogi carcinoma 115 (SC115), was studied. Treatment of SC115 tumor-transplanted male mice with s.c. injections of SMS (0.04, 0.2, 1, and 5 micrograms twice a day) resulted in a dose-dependent inhibition of tumor growth. The growth-inhibitory effect of SMS reached its peak at a dose of 1 microgram twice a day. SMS was found not to elicit its growth-inhibitory effect through lowering plasma testosterone levels or down-regulating androgen receptor of SC115 tumors. Since specific binding sites for somatostatin were not observed in the membrane fractions of SC115 tumors and SMS did not inhibit the proliferation of primarily cultured SC115 tumor cells, a direct inhibitory mechanism of SMS on SC115 tumors was unlikely to be operative. Since SMS is a very potent inhibitor of growth hormone (GH) secretion, it was speculated that SMS might inhibit the growth of SC115 tumors indirectly through down-regulation of plasma GH levels. This possibility was evaluated by studying the influence of GH replacement on the growth of SC115 tumors grown in SMS-treated mice. GH replacement was done both in a male secretory pattern (intermittent injection, human GH 500 micrograms/kg twice a day) and in a female secretory pattern (continuous infusion, 1000 micrograms/kg/day). Intermittent injections of GH fully restored the growth of SC115 tumors in the SMS-treated mice to that in the normal controls but continuous infusion of GH was without effect. These results suggest that SMS inhibits the growth of SC115 tumors through suppression of GH secretion, and that the mode of GH administration is an important determinant of its action on SC115 tumor growth.

To estimate cell kinetics of intraductal carcinoma present outside the invasive ductal carcinoma of the breast, an argyrophil stain for proteins associated with nucleolar organizer regions (Ag-NOR) was applied on paraffin sections of 28 breast cancers with an extensive intraductal component. The patients chosen for study were comparable to those eligible for breast-conserving surgery in our institute. The mean numbers of Ag-NOR in the nucleus were: normal duct 1.2 +/- 0.2 (mean +/- SD), normal lobule 3.1 +/- 1.2, main tumor 10.7 +/- 4.1 and intraductal lesion 7.7 +/- 2.6. A significant difference of Ag-NOR numbers was observed not only between the normal epithelium and main tumor (p < 0.01) but also between the main tumor and intraductal lesion (p < 0.01). As for intersite variation in individual cases, the Ag-NOR number of the intraductal lesion was lower than that of the main tumor in 25 out of 28 cases (89%). These results demonstrate that an intraductal lesion is less aggressive than the main tumor.

The expression of nucleoside diphosphate (NDP) kinase/nm23 has been reported to be inversely related to metastasizing potential of experimental cells and human breast cancer. In the present study, levels of NDP kinase/nm23 gene product in curatively resected human pancreatic adenocarcinomas were examined immunohistochemically using anti-NDP kinase antibody. Immunoreactivity for NDP kinase varied between tumors. Of 31 pancreatic tumors examined, 17 (55%; positive staining group) showed strong immunoreactivity for the NDP kinase, while 14 (45%; negative staining group) showed low or no immunoreactivity. Positive staining was associated with higher incidence of lymph node metastasis (13/17; 77%) and perineural invasion (13/17; 77%) than negative staining (5/14, 36%, P < 0.03; 4/14, 29%, P < 0.01, respectively). Positive staining was also associated with shorter overall survival and relapse-free survival than negative staining (P < 0.01, P < 0.01, respectively). No significant difference in age, sex, size, location of tumor, serum carcinoembryonic antigen (CEA) level, or histological type was found between the two groups. These results showed that, in contrast to the reports on breast cancer, NDP kinase/nm23 expression in human pancreatic cancer is positively associated with lymph node metastasis or perineural invasion and with poor prognosis. These, together with other previous reports, suggest that NDP kinase may play an important role in cancer progression or aggressiveness by altering its expression in a tissue-specific manner.

We have previously reported carcinoembryonic antigen (CEA) measurement in nipple discharge to be a useful adjunct in the diagnosis of non-palpable breast cancer. We have now developed a "microdot-immunobinding assay" using a specially constructed device to screen efficiently large numbers of patients with nipple discharge for non-palpable breast cancer. The method is as follows: a sample of nipple discharge is placed on a solid phase monoclonal anti-CEA antibody and, if CEA is present in the discharge, it will be detected by a second monoclonal anti-CEA antibody conjugated with alkaline phosphatase. The use of bromochloroindolyl phosphate as a chromogen results in a stable color reaction that can be semiquantitatively analyzed with the naked eye. CEA levels determined by this microdot assay correlated well with those determined using the earlier Elmotec assay. To determine the accuracy of the method, a collaborative study involving 11 institutes in Japan was organized. The CEA levels in nipple discharges from 77 patients undergoing surgery, 44 of whom were diagnosed as having breast cancer, were assayed. The results were that 17 of the 23 patients with palpable breast cancer, and 16 of the 21 patients with non-palpable breast cancer exhibited CEA values > 400 ng/ml, a cut-off value determined in a previous study. The overall accuracy (78%) of this test for diagnosing non-palpable breast cancer was higher than that obtained from ductography or cytology. The system may thus be of use in the screening of early breast cancer.

Clonality of human breast cancer was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of one of two X-chromosomes by methylation in females. All the 20 breast cancer samples analyzed by the PCR-based method were monoclonal in origin and adjacent normal breast tissues were polyclonal. When DNA samples were prepared from widely separated sites of cancers, every sample was found to be monoclonal, always exhibiting inactivation of the same X-chromosome in each tumor. The study on sensitivity showed that the PCR-based method for clonal analysis can detect the presence of monoclonal cells against a polyclonal background when the monoclonal cell population is 50% or more. These results demonstrate that clonal analysis by means of PCR offers a good method for studying the clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign (polyclonal) and malignant (monoclonal) breast lesions.

Among the 93 surgical specimens of stage I breast cancer treated by breast preserving operation, surgical margins were less than 10mm in 23 (25%) cases because of intraductal spread. Such tumors with extensive intraductal component, however, were frequently positive for estrogen receptor (ER). Possible risk factors for local failure have been studied in an attempt to exclude high-risk patients not suitable for breast preserving operation. The results so far obtained suggest that biological markers such as c-erb B-2 or PS2 proteins may provide useful informations to select patients who are most likely to recur. In addition, adjuvant endocrine therapy may be recommended for the patients treated by breast preserving operation.

A phase II study of 5'-deoxy-5-fluorouridine (5'-DFUR) tablets for breast cancer was done at 31 institutions in Japan. Forty-five patients were registered and 44 of them were eligible for the study. Of the 40 patients whose results could be evaluated, 11 (28%) responded (four complete responses and seven partial responses). Side effects, such as diarrhea, anorexia, leukocytopenia, and liver dysfunction were observed in 24 of the 44 patients. The side effects were mild and transient. 5'-DFUR is a promising drug for breast cancer treatment, and its tablet form makes chemotherapy easier for the patient.

Six to 12 cycles of CMF is regarded as the standard regimen for adjuvant chemotherapy for breast cancer, producing 10-20% increase in survival for premenopausal and node-positive patients. Tamoxifen combined with CMF has induced an additional reduction in recurrence rate only in postmenopausal patients. Oral administration of 5-FU or its derivatives combined with tamoxifen for as long as 1-2 years is the regimen most widely used in Japan for considerable improvement in survival. But it needs further evaluation in comparison with CMF, and in ultimate duration of treatment and better combination with other drugs. Adjuvant chemotherapy for node-negative patients is advocated in the U.S., where the recurrence rate is as high as 30%. Only 10% of Japanese n0 patients undergo recurrence. Therefore, adjuvant therapy should be given only to high-risk patients.

From 1977 to 1987, 23 patients with isolated chest wall recurrence, excluding sternal metastasis, from breast cancer underwent full thickness chest wall resection. The 5-year survival rate after chest wall resection was 48% but the 5-year relapse-free survival rate was 26%. Mediastinal metastasis was proved histologically at the time of chest wall resection in 7 patients, and survival period with mediastinal involvement was significantly (P less than 0.01) shorter than that with no mediastinal involvement (n = 16). In 17 patients with a long disease-free interval (DFI greater than or equal to 24 months), survival was longer than in 6 patients with a short DFI (less than 24 months). For the selected patients without mediastinal involvement and long DFI, surgical treatment for chest wall recurrence of breast cancer should play a significant role in improving the quality of life, and even in prolonging the survival rate.

Over the past 10 years, we have witnessed a variety of potential prognostic factors of breast cancer including proliferative rate, ploidy, growth factor receptors, oncogenes and cathepsin D production. Some of these variables seem to predict the prognosis of the patients but the available data are conflicting and call for carefully conducted quality-control studies to analyze intra- and interlaboratory variations. In this review, we provided a framework from which prognostic factor information can be used directly to make treatment decisions.

Monoclonal antibodies against pancreas cancer-associated antigen (PCAAp) were produced by established hybridoma cells. Two monoclonal antibodies, 3F1 and 3B6, were selected and these two monoclonal antibodies were found immunohistologically to react strongly with cancer cells and intraductal mucin-like products in well-differentiated pancreatic cancer tissues, but weakly, if at all, with gastric, colorectal and other cancers and at all not with normal adult or fetal pancreatic tissue. PCAAp is usually expressed in normal colonic mucosa (PCAAc), but the two monoclonal antibodies scarcely reacted with normal colonic mucosa. A sandwich enzyme immunoassay was developed to measure circulating PCAAp. Thirty-two normal subjects and 271 patients comprised of 210 with malignant disease and 61 with benign disease were surveyed. The cut-off value of PCAAp levels in the 32 normal subjects was 2.06 micrograms/ml, being the mean + 2SD, while PCAAp levels of more than this were observed in 72 per cent of the patients with pancreatic cancers, 65 per cent of those with bile duct cancers, 60 per cent of those with hepatomas, 0-30 per cent of those with other malignant diseases, and 10 per cent of those with benign hepatobiliary diseases. The susceptibility of isolated PCAAp and PCAAc to various enzymes and chemical reagents were also studied. The results of this study suggest that the assay for PCAAp might be useful in the diagnosis of pancreatic cancer.

To elucidate the etiology of the thrombogenic effects of high-dose medroxyprogesterone acetate (MPA) in the treatment of breast cancer, hematologic parameters were sequentially assessed in 12 patients receiving MPA 800 mg p.o. daily for 6 months as adjuvant hormone therapy after mastectomy. The results were as follows. (1) Coagulation system: levels of factor VII and fibrinogen decreased significantly, whereas factors II and IX increased significantly, with a shortened activated partial thromboplastin time. (2) Fibrinolytic system: plasminogen and alpha 2-plasmin inhibitor-plasmin complex increased, whereas fibrinogen degradation products remained low. (3) Anticoagulation system: antithrombin III increased significantly. (4) These changes were most marked after 2 or 4 weeks of MPA treatment, and returned to the pretreatment level one month after discontinuation of treatment. (5) No patients in this study developed thromboembolic disease during or after MPA administration. These results indicate that MPA may induce a hypercoagulable state, but this state does not directly lead to the development of thrombosis.

Secondary prevention, detection and treatment at an early stage, may be the only means of controlling breast cancer. This is rational behind screening for breast cancer. We have previously reported that CEA measurement in nipple discharge is a useful adjunct in the diagnosis of nonpalpable breast cancer. As an extension, a dot-immunobinding assay was developed to screen a large number of patients with nipple discharge for nonpalpable breast cancer. This article is a review of the current status of CEA assay in nipple discharge for mass screening of breast cancer. False positive and negative cases will be also described.

We describe a breast cancer with ectopic production of amylase, found in the patient's serum, urine and in the tumour. Clinically, serum amylase levels reflected both the progression of the disease and regression induced by various therapies. Using agarose gel electrophoresis and a wheat protein inhibitor assay, the predominant serum amylase appeared to be identical to pancreatic-type isoenzyme. However, the action mode analysis using a new fluorogenic substrate revealed that the serum contained non-salivary, non-pancreatic amylase. The tumour had microscopic features of invasive ductal carcinoma with some argyrophilic differentiation. The component cells stained positively for amylase, and ultrastructurally numerous secretory granules were seen.

As a second line therapy after failure to previous therapies, a combination therapy with MPA 1,200 mg po and 5'DFUR 1,200 mg po daily was given to 31 patients with recurrent breast cancer. At a median follow up period of 18 months, the overall response rate was 42%. The response rates for bone and visceral lesions were still good for the second line therapy. Patients previously exposed to tamoxifen (24 patients), 5-FU or its derivatives (21) and/or adriamycin (18) had response rates of 42%, 33%, 33%, respectively. The median duration of response in responders was 10 months. The overall median survival for the entire series was 9 months after start of the treatment. Thirteen (81%) of 16 patients with bone lesions were relieved from their bone pain. It is of special interest that the pain relief was also obtained in 7 out of 10 NC/PD patients with bone lesions, resulting in much improvement of their performance status. Side effects included obesity 52%, edema of the leg 35%, diarrhea 16% and so on. One patient developed venous thrombosis of her lower extremities and 4 were suspected to have the same condition. Fifty-five % of the patients underwent dose reduction of MPA at the 5th month of treatment in a median. This combination therapy is useful for recurrent disease even in late stages, so long as close observation is made for the occurrence of thrombosis.