Molecular detection of lymph node metastasis in breast cancer patients treated with preoperative systemic chemotherapy: a prospective multicentre trial using the one-step nucleic acid amplification assay.

BACKGROUND: For patients with breast cancer treated with preoperative chemotherapy, residual tumour burden in lymph nodes is the strongest prognostic factor. However, conventional pathological examination has limitations that hinder the accurate and reproducible measurement. The one-step nucleic acid amplification (OSNA) assay is a novel molecular method for detecting nodal metastasis. In this prospective multicentre trial, we assessed the performance of the OSNA assay in detecting nodal metastasis after chemotherapy. METHODS: In total, 302 lymph nodes from 80 breast cancer patients who underwent axillary dissection after chemotherapy were analysed. Each node was cut into two or four slices. One piece or alternate pieces were evaluated by pathology, and the other(s) were examined using the OSNA assay. The results of the two methods were compared. Stromal fibrosis, histiocytic aggregates, and degenerated cancer cells were regarded as chemotherapy-induced histological changes. RESULTS: The overall accuracy, sensitivity, and specificity of the OSNA assay compared with the reference pathology were 91.1%, 88.3%, and 91.7%, respectively. Of the 302 lymph nodes, 66 (21.9%) exhibited chemotherapy-induced histology. For these nodes, the accuracy, sensitivity, and specificity were 90.9%, 88.9%, and 93.3%, respectively. CONCLUSION: The OSNA assay can detect the residual tumour burden as accurately as conventional pathology, although chemotherapy-induced histological changes are present.

Incidence and possible pathogenesis of sentinel node micrometastases in ductal carcinoma in situ of the breast detected using molecular whole lymph node assay.

BACKGROUND: The pathogenesis of lymph node metastases in preinvasive breast cancer ­ ductal carcinoma in situ (DCIS) ­ remains controversial. The one-step nucleic acid amplification (OSNA) assay is a novel molecular method that can assess a whole node and detect clinically relevant metastases. In this retrospective cohort study, we determined the performance of the OSNA assay in DCIS and the pathogenesis of node-positive DCIS. METHODS: The subjects consisted of 623 patients with DCIS who underwent sentinel lymph node (SN) biopsy. Of these, 2-mm-sectioned nodes were examined using frozen-section (FS) histology in 338 patients between 2007 and 2009, while 285 underwent OSNA whole node assays between 2009 and 2011. The SN-positivity rate was compared between cohorts, and the characteristics of OSNA-positive DCIS were investigated. RESULTS: The OSNA detected more cases of SN metastases than FS histology (12 out of 285, 4.2% vs 1 out of 338, 0.3%). Most of the metastases were micrometastases. The characteristics of high-risk DCIS (i.e., mass formation, size, grade, and comedo) and preoperative breast biopsy (i.e., methods or time to surgery) were not valid for OSNA assay­positive DCIS. CONCLUSION: The OSNA detects more SN metastases in DCIS than FS histology. Further examination of the primary tumours and follow-up of node-positive DCIS are needed to elucidate the pathogenesis.

Accurate staging of axillary lymph nodes from breast cancer patients using a novel molecular method.

BACKGROUND: The one-step nucleic acid amplification (OSNA) assay is a molecular-based lymph-node metastasis detection procedure that can assess a whole node and yields semi-quantitative results for the detection of clinically relevant nodal metastases. We aimed to determine the performance of the OSNA assay as an accurate nodal staging tool in comparison with routine histological examination. METHODS: Subjects comprised 183 consecutive patients with pT1-2 breast cancer who underwent axillary dissection after positive sentinel-node (SN) biopsy with the OSNA assay. Of these, for non-SN evaluation, 119 patients underwent OSNA assay evaluation, whereas 64 had single-section histology. We compared the detection rates of non-SN metastasis and upstaging rates from the SN stage according to the American Joint Committee on Cancer staging between the OSNA and histology cohorts. RESULTS: OSNA detected more cases of non-SN metastases than histology (OSNA 66/119, 55.5% vs histology 13/64, 20.3%; P<0.001), particularly micrometastases (36/119, 30.3% vs 1/64, 1.6%; P<0.001). Total upstaging rates were similar in both cohorts (20/119, 16.8% vs 9/64, 14.1%, P=0.79). CONCLUSION: OSNA detects a far greater proportion of non-SN micrometastases than routine histological examination. However, upstaging rates after axillary dissection were not significantly different between both cohorts. Follow-up of the OSNA cohort is required to determine its clinical relevance.

Technical note: the use of a physical activity monitor to estimate the eating time of cows in pasture.

Accurate eating time can be used as an index of forage dry matter intake in grazing cows. To develop a method for easily estimating the eating time of dairy cows in a pasture, 8 lactating Holstein cows were fitted with collars equipped with commercial uniaxial accelerometers; namely, the Kenz Lifecorder EX (LCEX; Suzuken Co. Ltd., Nagoya, Japan), and were allowed to graze in a pasture for 4, 8, or 20 h daily for 7 d. The LCEX device recorded the intensity of the physical activity categorized into 1 of 11 activity levels ranging among 0 (no movement), 0.5 (subtle) and from 1 to 9 (1, light; 9, vigorous intensity) every 4s during the experimental period. The activities of the animals were also video-recorded for 11h and were manually classified into 7 categories (eating, searching, ruminating, standing resting, lying resting, drinking, and walking) at 4-s intervals. According to the count distribution of the activity levels for the categorized activities, 94.4% of the counts involving eating activity ranged from activity level 1 to 7. On the other hand, most of the counts were activity level 0 or 0.5 when ruminating and resting activities were observed. No records of activity level 8 or 9 were found in any activities. When activity level 1 was used as a threshold for discriminating eating from the other activities, the lowest misclassification rate of 5.5% was observed. With a threshold of activity level 1, the eating times in pasture for cows grazing for 4, 8, and 20 h/d were 142.8, 290.6, and 438.4 min/d, respectively, and the proportions of the time spent in pasture that were made up of eating time were 0.66, 0.67, and 0.38, respectively [the proportion during daytime (8h of the 20-h grazing treatment) was 0.63 and that at nighttime (12h of the 20-h grazing treatment) was 0.23]. The use of the LCEX device allows for easy measurement of eating time and facilitates the determination of the pattern of eating activity in pasture for grazing cows.

Short communication: effect of grazing on the concentrations of total sialic acid and hexose in bovine milk.

Sialic acid, which is located at the terminal end of glycoconjugates, is believed to have important biological functions. Its concentration in bovine milk varies depending on lactation stage and season. However, it remains unclear whether dietary factors, especially fresh forage, affect the total sialic acid concentration in milk. The purpose of the present study was to investigate the effect of grazing on the concentrations of total sialic acid and hexose in bovine milk. Six healthy dairy cows were used in a crossover design (3 cows fed fresh forage and 3 cows fed grass silage) for 2 wk. Individual milk samples were collected at 2 consecutive milkings (morning and evening) at 0, 1, 3, 5, 8, 11, and 14 d of the experimental period, and 2 consecutive samples in each cow were combined on each sampling day in proportion of the morning and evening milk yields. No differences in body weight, milk yield, or milk composition were observed between the 2 groups during the experimental period. The hexose concentration in milk did not differ between these groups during the experimental period. Conversely, the total sialic acid concentration in the milk of each grazing cow significantly increased at 11 and 14 d of the experimental period compared with that at 0 d. In the grass silage group, the total sialic acid concentration at the end of the experimental period tended to be lower than that at 0 d, but the decrease was not significant. These results indicate that grazing management could have increased the concentration of sialoglycoconjugates in milk. This suggests that grazing may increase the biological function of milk because it is thought that sialic acid is significant in many ways.

Clinical features of 215 stage I ovarian tumors in Japanese women.

PURPOSE: Differences of the clinical features of Stage I borderline ovarian tumors and Stage I ovarian cancer need to be clarified. METHODS: We retrospectively investigated 215 patients with Stage I ovarian tumors (67 with borderline tumors and 148 with ovarian cancer) treated between 1988 and 2001. RESULTS: Only one patient with a borderline tumor developed recurrence, while recurrence was found in 20 patients with Stage I ovarian cancer. There was a significant difference in the recurrence rate between patients with Stage Ia or Ib ovarian cancer and those with Stage Ic cancer (p = 0.007). Clear cell adenocarcinoma showed a higher recurrence rate. Among our patients with recurrence, only five in whom the recurrent tumor could be surgically resected are currently alive and disease-free. CONCLUSIONS: This study confirmed the low aggressiveness of Stage I borderline ovarian tumors and high aggressiveness of Stage Ic ovarian cancer or clear cell adenocarcinoma. In patients with recurrence, surgical resection may improve survival.

Accumulation of genetic alterations and progression of primary breast cancer.

In order to detect common regions of deletion, 219 breast tumors were examined for loss of heterozygosity at several loci on chromosomes 3p, 16q, and 17 by restriction fragment length polymorphism analysis. Allelic deletions of loci on chromosomes 3p, 13q, 16q, and 17, and amplification of the erbB2 oncogene, were analyzed and compared with histopathological and clinical features. Common regions of deletion were detected within chromosomal bands 3p13-14.3, 16q22-23, 17p13 (two separated loci), and 17q21. Concordant losses of alleles on chromosomes 3p, 13q, 16q, 17p, and 17q were observed. A significant association was detected between loss of heterozygosity on chromosomes 17p and 17q and amplification of the erbB2 oncogene (17p, P = 0.000721, by Fisher's exact test; 17q, P less than 0.001, chi 2 = 12.135). Furthermore, tumors showing highly malignant phenotypes had accumulated more genetic changes at the loci studied than those having less malignant phenotypes on the basis of histopathological classification, lymph node metastasis, and tumor size. These results suggested that accumulation of genetic alterations, including loss of function of tumor suppressor genes on chromosomes 3p, 13q, 16q, and 17, and amplification of the erbB2 oncogene, may contribute to tumor development and/or progression in primary breast cancer.

Allelotype of breast cancer: cumulative allele losses promote tumor progression in primary breast cancer.

Allele loss on a specific chromosome has implied the existence of a tumor suppressor gene such as the p53 gene and the RB gene. In order to determine which chromosome(s) carries a tumor suppressor gene(s) that contributes to tumor progression in primary breast cancer, we analyzed the loss of heterozygosity for each autosomal chromosome arm by using 39 restriction fragment length polymorphism markers including 25 variable numbers of tandem repeat probes. In 79 primary breast cancers, we found the frequent loss on the long arm of chromosome 13 (21%), the long arm of chromosome 16 (45%), and the short arm of chromosome 17 (56%). Interestingly, breast cancers in which loss of both chromosomes 13q and 17p was detected showed more malignant histopathological features, and a group of the tumors in which chromosome 16q loss was detected presented with frequent lymph node metastasis. Furthermore, the result of the deletion mapping on chromosome 17p implied the existence of a tumor suppressor gene distal to the p53 gene as well as the p53 gene itself for primary breast cancer. These results suggest that at least 4 tumor suppressor genes exist on chromosomes 13q, 16q, and 17p for primary breast cancer.

Correlation of loss of alleles on the short arms of chromosomes 11 and 17 with metastasis of primary breast cancer to lymph nodes.

To examine the role of loss of heterozygosity (LOH) during tumor development and/or progression, we looked for correlations between metastasis of breast cancer to a regional lymph node(s) and LOH of chromosomal arms 11p, 13q, 16q, 17p, and 17q, where frequent losses in primary tumors have been detected. No correlation between lymph node metastasis and LOH of chromosomes 13q, 16q, or 17q was observed. However, tumors showing LOH of chromosomes 11p (chi 2 = 10.82, P less than 0.01) and 17p (chi 2 = 6.78, P less than 0.01) revealed a significantly higher incidence of metastasis to a regional lymph node(s) than tumors without LOH on these chromosomal arms. Furthermore, only four of 30 (13%) patients with tumors that retained both 11p and 17p had metastasis to a regional lymph node(s), compared with 24 of 32 (75%) patients with tumors that had lost both 11p and 17p. Analysis of LOH with markers on chromosomes 11p and 17p in a large number of tumors indicated that the peritelomeric region of each of these chromosomal arms contains a tumor suppressor gene that may be associated with tumor progression, particularly metastasis to a regional lymph node(s).

Linkage regions between dermatan polysulfates and peptides.

Three different types of dermatan polysulfate peptides I, II and III, isolated from hagfish notochord, hagfish skin and shark skin, all contained serine, xylose and galactose in a molar ratio of about 1.2 : 1 : 2. After beta-elimination-reduction, dermatan polysulfate peptides II and III produced alanine and xylitol in amounts equivalent to the amount of the serine decrease. Accordingly, it was shown that these dermatan polysulfates were linked to peptides by O-glycosidic bond between xylose and serine, as in chondroitin sulfates and dermatan sulfate. However, dermatan polysulfate peptide I produced N-acetylgalactosaminitol, in addition to alanine and xylitol, in a molar ratio of about 2 : 3 : 1, although the increase of alanine was equivalent to the serine decrease. Consequently, it was concluded that the linkage region of dermatan polysulfate peptide I has two types of O-glycosidic bond: one between xylose and serine and the other between N-acetylgalactosamine and serine. This is the first finding of an N-acetylgalactosamine involved in the linkage region of glycosaminoglycans consisting of uronic acid as repeating constituents.

Combination of consecutive low-dose cisplatin with bleomycin, vincristine, and mitomycin for recurrent cervical carcinoma.

PURPOSE: To evaluate the efficacy and toxicity of combination chemotherapy with bleomycin, vincristine, mitomycin, and consecutive low-dose (CLD) administration of cisplatin (CLD-BOMP) for patients with recurrent cervical carcinoma. PATIENTS AND METHODS: Ninety patients with recurrent cervical carcinoma and no prior chemotherapy were enrolled onto this study. The median age was 56 years. Eighty-seven of the 90 patients had received prior radiotherapy. The CLD-BOMP regimen was bleomycin 5 mg infused continuously days 1 through 7; vincristine 0.7 mg/m2 bolus day 7; mitomycin 7 mg/m2 bolus day 7; and cisplatin 10 mg/m2 infused over 4 hours days 1 through 7. The treatment was repeated at 3-week intervals. RESULTS: All 90 patients were assessable for response, toxicity, and survival. After a median of four cycles (range, two to 10 cycles), we observed objective responses in 68 patients (76%), with 25 (28%) complete responses (CRs) and 43 (48%) partial responses (PRs; 95% confidence interval (CI), 66 to 85; 18 to 38; 37 to 59, respectively). Median survival for all 90 patients was 24.3 months (range, 2.3 to 100 months). The median survival for patients who achieved CR, PR, no change (NC), and progressive disease (PD) were not reached (NR), 23.6, 8.2, and 6.4 months, respectively. The median progression-free survival for patients who achieved CR and PR were NR and 12.3 months, respectively. There was no significant nausea or vomiting, nephrotoxicity, or pulmonary toxicity, which was attributable to the CLD-cisplatin and the adequate dosing schedule of bleomycin. The reduced toxicities allowed this regimen to be administered at the projected dose-intensities. CONCLUSION: The CLD-BOMP regimen has significant antitumor activity with markedly reduced toxicity.

Unargued issues on the pathological assessment of response in primary systemic therapy for breast cancer.

PURPOSE: The role of primary systemic therapy (PST) in the treatment of operable breast cancer is currently under intensive investigation in the hope of allowing greater conservation of the breast, and emerging evidence suggests that induction of a pathological complete response (pCR) is at least, to some extent, predictive of long-term clinical response. In this review, we highlight the issues of pathologic evaluation after PST. METHODS: We performed a computer-assisted MEDLINE search, and additional references were found in the bibliographies of these articles. RESULTS: So far, several grading classifications are used to assess pathologic responses after PST, and pCR rates vary from 1% to 54.7% according to the PST regimens employed. However, the term "pCR" has not been applied in a consistent, standardized manner, and the pCR rates appear to depend not only on the differences in the definition of pCR, but also on the extent of tissue sampling and the techniques used for pathologic examination. So far, only limited information is available about the reliability and validity of the definition of pathologic responses. CONCLUSION: Assessment of pCR needs to be standardized, and each grading system should be verified for reliability and validity. As a lack of standard for tumor processing and evaluation may result in considerable fluctuation of pCR rates between trials, we should take into account the differences in the definition of pathologic response when comparing the results of PST.

Update results of FEC followed by docetaxel neoadjuvant trials for primary breast cancer.

This study has been initiated to evaluate the safety, clinical and pathologic response as well as the relation of response (pCR or non-pCR) and survival (overall and relapse-free) of fluorouracil, epirubicin and cyclophosphamide (FEC) followed by docetaxel (DOC) as preoperative chemotherapy in patients with operable breast cancer. Japanese patients with primary breast cancer, Tlc-3N0M0 or T1-3NIM0, age 20-60, PS 0-1 were included in this study. Preoperative chemotherapy consisted of 4 cycles of FEC (500 mg/m(2), 100 mg/m(2), 500 mg/m(2)) every 3 weeks followed by 4 cycles of DOC (75 mg/m(2)) every 3 weeks. Since June 2002, 200 patients were enrolled in this study, and the time of this interim analysis, 80 patients were evaluable for safety and clinical efficacy. The overall clinical response rate was 71.4% (14% CR, 44% PR, 42% SD/PD), and the only G3,4 toxicities, neutropenia and febrile neutropenia were observed in 54% and 14% of patients, respectively. Eighty nine patients were evaluable for pathologic response by central review. Pathologic response was evaluated among invasive tumors on multiple cross-section specimens based on a modified version of the Japanese grading system for Japanese Breast Cancer Society. The pathologic response rate was 17%. In this ongoing trial, FEC followed by DOC was active and well tolerated.

Allelic losses of loci at 3p25.1, 8p22, 13q12, 17p13.3, and 22q13 correlate with postoperative recurrence in breast cancer.

We previously defined 18 chromosomal regions in which frequent allelic losses were observed in breast cancers (T. Sato et al., Cancer RES:, 50: 7184-7189, 1990; Y. Harada et al., Cancer (PHILA:), 74: 2281-2286, 1994; I. Ito et al., BR: J. Cancer, 71: 438-441, 1995; K. Tsukamoto et al., Cancer (PHILA:), 78: 1929-1934, 1996; S. Matsumoto et al., Genes Chromosomes Cancer, 20: 268-274, 1997; T. Yokota et al., JPN: J. Cancer RES:, 88: 959-964, 1997; K. Tsukamoto et al., Cancer (PHILA:), 82: 317-322, 1998; A. Iida et al., Genes Chromosomes Cancer, 21: 108-112, 1998; K. Fukino et al., Genes Chromosomes Cancer, 24: 345-350, 1999; T. Yokota et al., Cancer (PHILA:), 85: 447-452, 1999; Y. Utada et al., JPN: J. Cancer RES:, 91: 293-300, 2000). To identify specific allelic losses that might correlate with postoperative recurrence, we examined tumors from a cohort of 504 breast cancer patients, who were followed clinically for 5 years postoperatively, for allelic losses of 18 microsatellite markers. Patients whose tumors had lost an allele at 3p25.1, 8p22, 13q12, 17p13.3, or 22q13 had significantly higher risks of recurrence than those whose tumors retained both alleles at those loci; at 3p25.1, the 5-year recurrence rate was 27% among patients with losses versus 18% with retention (P = 0.0131); at 8p22, 27% versus 14% (P = 0.0129); at 13q12, 28% versus 15% (P = 0.0109); at 17p13.3, 27% versus 20% (P = 0.0482); and at 22q13, 29% versus 20% (P = 0.0477). These data indicate that loss of heterozygosity at any one of these five specific loci is a significant predictor of postoperative recurrence among patients who have undergone surgery for breast cancer. These allelic losses can serve as negative prognostic indicators to guide postoperative management of patients.

Allelic loss at 1p34-36 predicts poor prognosis in node-negative breast cancer.

Allelic losses of specific chromosomal regions in the DNA of tumor cells, which imply loss of tumor suppressor genes normally resident at those loci, may become useful postoperative prognostic indicators for breast cancers that have not yet metastasized to lymph nodes. To examine whether specific allelic losses might correlate with postoperative disease-free survival, we tested tumors from a cohort of 228 node-negative breast cancer patients for allelic losses at 18 microsatellite loci chosen to represent either a known tumor suppressor gene or a region where genetic alterations are frequent in breast tumors. We followed the patients clinically for 5 years or until death (if patient death occurred before completion of 5 years of follow-up). Patients whose tumors had lost an allele at 1p34-36 bore significantly higher risks of postoperative recurrence than those whose tumors retained both alleles of the markers in that region [the 5-year recurrence rate was 15% among patients with losses versus 2% among patients with retention (P = 0.001)]. Multivariate analysis demonstrated that allelic loss at 1p34-36 was an independent postoperative predictor of shorter disease-free survival (hazard ratio, 5.8; P = 0.0117). Thus, allelic losses at 1p34-36 in a tumor might have a potential to serve as a negative prognostic indicator to guide postoperative management of breast cancer patients, especially in the selection of high-risk women who will benefit from adjuvant chemotherapy and endocrine therapy.

Identification of a new commonly deleted region within a 2-cM interval of chromosome 11p11 in breast cancers.

Allelic loss has been observed on the short arm of chromosome 11 in a variety of human cancers. We have examined 184 breast cancers for allelic loss anywhere in chromosome 11p, using 15 well-spaced microsatellite markers. Allelic loss was observed in 86 cases (47%) and a new commonly deleted region 2-cM in length was identified at 11p11 between loci D11S986 and D11S1313, in addition to a 12-cM region of a common deletion at 11p15.5. A significant association was found between allelic loss on 11p15.5 and LOH on 11p11 and the loss of progesterone receptors.

Frequent multiplication of chromosome 1q in non-invasive and papillotubular carcinoma of the breast.

Carcinogenesis is considered to be a multistep process that may involve cumulative genetic alterations; one of these mechanisms, gain of chromosomal material, has the potential to activate tumor-promoting genes in breast carcinogenesis. Using 12 polymorphic microsatellite markers on the long arm of chromosome 1 (1q), we examined 130 sporadic breast carcinomas for abnormalities in the copy numbers of these loci in tumor cells using a differential PCR method. We also sought correlations between alterations on 1q and several clinicopathological parameters. At every locus examined, a 2-3-fold increase in copy number of an allele in tumor material was observed in one third of the tumors (46 of 130, 35%), indicating 'multiplication' of 1q. This multiplication involved the entire long arm in majority of those tumors (43 cases, 93%). The multiplication of 1q was observed more frequently in non-invasive ductal and papillotubular histological types than in solid-tubular and scirrhous types (13/25, 52% vs. 27/90, 30%) (P = 0.041). The predominant chromosomal alterations on 1q in breast carcinomas are found to be multiplications rather than losses. The multiplication represents polysomy of the entire region of 1q, and may confer a growth advantage during development and/or progression of non-invasive ductal and papillotubular histologic types of breast carcinomas.

Frequent multiplication of chromosomal region 8q24.1 associated with aggressive histologic types of breast cancers.

Carcinogenesis is considered to be a multi-step process that may involve cumulative genetic alterations. One such alteration, gain of chromosomal material, has the potential for activating genes that promote carcinogenesis in breast tissues. Using 14 polymorphic microsatellite markers on the long arm of chromosome 8 (8q), we examined 142 sporadic breast cancers for abnormalities in the copy-numbers of these loci. At each locus examined, a 2- to 3-fold increase in intensities of bands representing single alleles was observed in 57 (40%) of the tumors, indicating that 'multiplication' of the DNA sequence had occurred on 8q. A 16-cM region on 8q24.1 was commonly multiplied among the tumors with partial multiplications. Multiplication on 8q24.1 was observed more frequently in invasive solid-tubular or scirrhous tumors (48/92, 52%) than in less aggressive histologic types (7/25, 28%, P = 0.031). Thus, multiplication of tumor-promoting gene(s) located on 8q24.1 may play a role in the development and/or progression of a substantial proportion of primary breast cancers, particularly those of the invasive histology.

Experience of quadrantectomy with axillary dissection without radiotherapy sustained by serial pathological examination for stage I breast cancer.

Breast conserving treatment usually consists of lumpectomy and axillary dissection followed by a limited dose of irradiation so that no significant side-effects occur. However, the precision of lumpectomy depends on the surgical maneuver and pathological evaluation performed at each institution. For this reason, post-operative irradiation to the preserved breast and for the occult carcinoma in the same breast is absolutely mandatory, and effectively becomes a routine step. In 1986, we started to adopt the new breast-conservation method of quadranectomy with axillary dissection for restricted stage I breast cancer without using radiotherapy, at the Cancer Institute Hospital, Tokyo, Japan. As an alternative to irradiation to ensure safety, we chose to administer an elaborate pathological examination on serial sections. The pathological proof has saved troublesome post-operative irradiation, and the results have shown this method to be safe and clear-cut compared to the traditional breast-conserving treatment cited in the literature. From July 1986 to December 1994, we performed 321 cases of quadranectomy and axillary dissection (Q+Ax). If the detailed pathological examination of 5-mm serial sections revealed the stump to be negative, we did not treat the preserved breast with radiotherapy. Out of 321 cases, 247 were analyzed as being stump-negative and of these 235 did not receive radiotherapy at all. During a 5 year 4 month observation period, we have not yet encountered any local recurrence. However, we have experienced 4 cases (1.70%) in which a second cancer developed in the conserved breast. The annual incidence rate was 0.32%. These results are the best so far compared to other published world reports.