Bone marrow micrometastases detected by RT-PCR for mammaglobin can be an alternative prognostic factor of breast cancer.

PURPOSE: To evaluate a new prognostic factor of breast cancer, bone marrow micrometastases which was detected by RT-PCR for mammaglobin, a sensitive molecular marker of breast cancer, was examined. MATERIALS AND METHODS: One hundred and eleven samples from stage I-III breast cancer patients were examined. Bone marrow micrometastases and clinicopathological parameters, which were age, tumor size, lymph node metastasis and status of the estrogen receptor, were evaluated for the prognostic factor by statistical analysis. RESULTS: Median follow-up time was 21.1 months. Thirty-three (29.7%) out of 111 samples were RT-PCR positive. Eight cases (24.2%) in this group showed recurrent lesions in the distant organs. Whereas six (7.7%) out of 78 RT-PCR negative patients had distant recurrences. In the premenoposal patients, and in the patients with axillary lymph node metastases, RT-PCR positive cases showed significantly higher distant recurrent rate. Bone marrow micrometastases, axillary nodal status, and estrogen receptor were independent prognostic factors for breast cancer by both univariate and multivariate analysis. CONCLUSIONS: Bone marrow micrometastases detected by RT-PCR for mammaglobin can be a useful predictive marker for early distant recurrence of breast cancer.

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy.

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.

Transareolar endoscopy-assisted partial mastectomy: a preliminary report of six cases.

Six patients with breast cancer in the upper inner quadrant underwent endoscopy-assisted partial mastectomy. The tumor was removed with a 2-cm-wide surgical margin through a periareolar semicircular incision using a special retractor and endoscope system for plastic surgery. Another small incision was made in the axilla for total lymph node dissection or sentinel lymph node biopsy. The average of total operation time in five patients who underwent partial mastectomy was 241 minutes (range, 190-315 minutes), and the average time for the procedure of partial mastectomy in six cases was 84 minutes (range, 69-113 minutes). The cosmetic outcome was excellent. Transareolar endoscopic partial mastectomy can be considered as an alternative surgery option and can offer great cosmetic advantage for patients with small cancers in the inner quadrants of the breast.

Clinical significance of micrometastases in axillary lymph nodes assessed by reverse transcription-polymerase chain reaction in breast cancer patients.

We evaluated the clinical significance of micrometastases in axillary lymph nodes (AxLNs) of breast cancer patients for prediction of prognosis. Archived formalin-fixed paraffin-embedded AxLN specimens from 129 node-negative breast cancer patients diagnosed by routine H&E staining between 1986 and 1990 were subjected to carcinoembryonic antigen-specific reverse transcription-PCR analysis. Micrometastases were detected in 40 of 129 (31.0%) node-negative breast cancer patients. After a median follow-up period of 105.6 months, log-rank test analysis indicated that 10-year disease-free and overall survival rates by Kaplan-Meier methods were significantly better in patients without micrometastases than in patients with micrometastases [disease-free survival, 87.6% versus 66.1% (P = 0.0008); overall survival, 93.7% versus 67.8% (P = 0.0024)]. The presence of micrometastases in AxLNs was revealed by multivariate analyses to be an independent and significant predictor of clinical outcome. The hazard ratio was 3.992 (95% confidence interval, 1.293-12.323; P = 0.0161) for relapse and 4.293 (95% confidence interval, 1.043-17.675; P = 0.0436) for cancer-related death. The molecular staging of AxLNs using reverse transcription-PCR is useful for prediction of clinical outcome in early-stage breast cancer patients and can provide a powerful and sensitive complement to routine histopathological analysis.

Overexpression of CDC25B phosphatase as a novel marker of poor prognosis of human colorectal carcinoma.

There is evidence to suggest that CDC25B phosphatase is an oncogenic protein. To elucidate the role of CDC25B in colorectal carcinoma, we examined the expression of CDC25B at the mRNA and protein levels. Reverse transcription-PCR assay indicated that CDC25B was overexpressed in tumor tissues relative to normal mucosa in 6 of 10 cases. Using immunohistochemistry, we identified high expression of CDC25B in 77 of 181 colorectal cases (43%). Univariate analysis showed that high expression was a significant predictor for poor prognosis compared with low expression (5-year survival rate; 59% versus 82%, respectively; P < 0.0001). Multivariate analysis indicated that CDC25B was an independent prognostic marker (risk ratio for death, 3.7; P < 0.0001) even after controlling for various factors such as lymph node metastasis, tumor size, degree of differentiation, and depth of invasion. Furthermore, the level of CDC25B expression clearly predicted the outcome of patients with Dukes' B and Dukes' C tumors. On the other hand, CDC25A mRNA was overexpressed in 9 of 10 colorectal cancer cases, and immunohistochemistry for CDC25A showed high expression in 52 of 111 cases (47%), but no significant correlation with prognosis. Our findings suggest that CDC25B is a novel independent prognostic marker of colorectal carcinoma and that it may be clinically useful for selecting patients who could benefit from adjuvant therapy.

Detection of microsatellite alterations in nipple discharge accompanied by breast cancer.

Nipple discharge in breast cancer cases was examined loss of heterozygosity (LOH). DNA samples were extracted from both supernatant and cell pellet components of the discharge, and examined for LOH at microsatellite markers, D11S1818, D11S2000, D16S402, D16S504, D16S518, D17S520, and D17S786. At least one LOH was found in either the supernatant or cell pellet in seven out of 10 patients (70%). Five of seven samples, which were cytologically negative, were LOH positive, and only one case, which was cytologically positive, showed no LOH on the markers examined. All three samples, which were judged 'negative' by CEA measurement (<400 ng/ml), were LOH positive. This method could be a useful novel diagnostic modality for nonpalpable breast cancer with nipple discharge.

Identification of a 7-cM region of frequent allelic loss on chromosome band 16p13.3 that is specifically associated with anaplastic thyroid carcinoma.

A total of 17 primary thyroid cancer specimens including seven anaplastic cancers, two papillary cancers adjacent to the anaplastic cancers, and eight papillary cancers were analyzed for loss of heterozygosity (LOH) on chromosome arm 16p. All tumors of anaplastic cancer showed LOHs at one or more loci, and a 7-cM region of the smallest deleted region was found on 16p13.3 between D16S423 and D16S406. This LOH was specifically found in the anaplastic cancer and not in the papillary thyroid cancer. Our present results suggest localization of the putative tumor suppressor gene on 16p13.3, which is likely to play an important role in the anaplastic transformation of thyroid cancer.

Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients.

The aim of this study was to search for specific and sensitive mRNA markers or a combination of markers for RT-PCR detection of micrometastases in axillary lymph nodes (LNs) from patients with breast cancer. LNs (n=177) from 17 patients were examined with Cytokeratin20 (CK20), melanoma-associated genes (MAGE1, MAGE3), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), mammaglobin (MGB1) and mammaglobin B (MGB2) as molecular markers. CK20, MAGE1 and MAGE3 were slightly positive in primary tumors and CEA, PSA, MGB1 and MGB2 were highly positive. MGB1 and MGB2 were 100% positive in HE-positive LNs while CEA and PSA were only 35.7% and 57.1% positive. MGB1 and MGB2 were also 30.1% and 17.8% positive in HE-negative nodes. Thus, MGB1 and MGB2 are specific and a combination of the two should be useful for detection of micrometastases in axillary LNs of breast cancer patients.

Prevention of cross-contamination during sampling procedure in molecular detection for cancer micrometastasis.

Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been widely employed as an ultra-sensitive method for detection of micrometastases in patients with various types of malignancies. Messenger RNA of a specific marker gene is a target for RT-PCR amplification to examine the presence of micrometastases in body fluids or tissues obtained from human. We developed the RT-PCR assay specific for rat beta-actin mRNA, which cannot detect human counterpart and assessed how much contamination of rat tissues can influence the result of RT-PCR assay and how to avoid the influence of the contamination in RT-PCR assay.

Selective augmentations of intratumoral 5-fluorouracil concentration by local immunotherapy with OK-432 and fibrinogen.

PURPOSE: Pyrimidine nucleoside phosphorylase is an enzyme that converts 5'-deoxy-5-fluorouridine into its active metabolite, 5-fluorouracil. In colorectal cancer tissue pyrimidine nucleoside phosphorylase has been proven to be produced by macrophages in the cancer stroma despite presence of the cancer cells. We reported that local immunotherapy with OK-432 and fibrinogen induced aggregation of macrophages in the cancer stroma and enforced their pyrimidine nucleoside phosphorylase expression. Thus it was hypothesized that if colon cancer were treated with 5'-deoxy-5-fluorouridine, the 5-fluorouracil concentration in cancer tissues would be enhanced by local immunotherapy. The present study was conducted to investigate whether local immunotherapy for colon cancer could increase the intratumoral 5-fluorouracil concentration in patients given chemotherapy with 5'-deoxy-5-fluorouridine. METHODS: Twenty patients with resectable colorectal cancer were examined in this study. They were given 5'-deoxy-5-fluorouridine (600 mg/day) orally for seven days preoperatively. Nine randomly selected patients underwent intratumoral injection of OK-432 mixed with fibrinogen, which was performed on the third preoperative day (OK-432 and fibrinogen plus 5'-deoxy-5-fluorouridine group); eleven patients were given oral 5'-deoxy-5-fluorouridine only (5'deoxy-5-fluorouridine group). The 5-fluorouracil concentration in tumor tissue and normal colon mucosa tissue was measured, and the influence of the local immunotherapy was assessed. RESULTS: The 5-fluorouracil concentration in the cancer tissue was increased by the local immunotherapy, whereas that in the normal colon mucosa was not influenced. Thus, the influence of local immunotherapy was selective to the cancer tissue where the mixture of OK-432 and fibrinogen was injected. CONCLUSION: In patients with colorectal cancer, selective high 5-fluorouracil concentration in the cancer tissue could be achieved by a combination of 5'-deoxy-5-fluorouridine and local immunotherapy with a mixture of OK-432 and fibrinogen.

Expression of tumor suppressor gene p16(INK4) products in primary gastric cancer.

Recent studies have shown that the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) represents an indicator for patients' outcome in several human malignancies including gastric cancer. However, the clinicopathologic value of another class of CDK inhibitor, p16(INK4), has not been determined. In a retrospective study, we examined the expression of p16(INK4) by immunohistochemical assay of 80 samples of primary gastric cancers and their adjacent nonneoplastic mucosas. Less than 10% of non-tumor gastric mucosal cells were p16(INK4) positive, whereas the expression of p16(INK4) in gastric cancer cells varied widely from 0 to 100% (mean, 24.5%). The expression of p16(INK4) was not seen in 11.3% (9/80) of the cancer cases, but in 65% (52/80) this protein was even overexpressed when compared with the nonneoplastic mucosa. A clinicopathologic survey indicated that a low or no expression of p16(INK4) was associated with poorly differentiated carcinoma (p = 0.0133), but the level of expression did not correlate with other parameters including patients' prognosis or with the expression of the pRb protein. In an effort to explore the underlying mechanism for the p16(INK4)-negative cases, a prospective study was also performed on 20 cases of gastric cancer to compare the level of the p16(INK4) protein with the methylation status of the p16(INK4) promoter. Gastric cancer tissues with methylation expressed significantly lower levels of the p16(INK4) protein (p = 0.0013) and two of them lacked p16(INK4) expression altogether, whereas all the cancer tissues without methylation expressed it. These findings suggest that the p16(INK4) protein may be associated with differentiation of gastric cancer tissues and that methylation of the p16(INK4) promoter may, in part, account for the loss of p16(INK4) expression.

[A case report: effective trans-arterial neoadjuvant chemotherapy with docetaxel for a local advanced breast cancer patient].

Recently, there have been some reports about the effectiveness of docetaxel for breast cancer patients who had polychemotherapy previously in vein. We report here a case of a 47-year-old woman, who had been diagnosed as local advanced breast cancer. She was given trans-arterial chemotherapy with docetaxel after four series of CEF (cyclophosphamide, epirubicin, fluorouracil) therapy resulted in PD (progressive disease). Local disease was successfully controlled, and she could undergo standard radical mastectomy.

Intraoperative navigation for breast cancer surgery using 3D ultrasound images.

The aim of this work was to develop an intraoperative image-guidance system for breast cancer surgery using three-dimensional (3D) ultrasound imaging. Using a 10-MHz annular array mechanical sector probe, ultrasound images were obtained from nine volunteer patients with breast cancer immediately before removal of the tumor in the operating room. A 3D tumor image was reconstructed using a workstation, then superimposed on the video image of the breast based on geometrical data. These data were obtained simultaneously by an optical 3D position sensor. The 3D images of the tumors were validated by the pathological data obtained after the surgery. In eight cases, the superimposed images were successfully obtained in approximately 15-20 min following scanning of the tumor. Scattered lesions around the main tumor were also visualized in the reconstructed tumor images, but artifacts of the ductal lesion caused by noise could not be eliminated in some cases. This system should be very effective in helping the surgeon to recognize the extent of a tumor within the breast itself and to determine the margin of surgical resection for breast conservation surgery.

Enhancement of the anti-tumor effect of 5'-deoxy-5-fluorouridine by transfection of thymidine phosphorylase gene into human colon cancer cells.

Thymidine phosphorylase (dThdPase) is an enzyme that converts 5'-deoxy-5-fluorouridine (5'DFUR) to the toxic substance 5-fluorouracil (5-FU); it is also known to be a platelet-derived endothelial cell growth factor. In order to investigate the feasibility of suicide gene therapy against colorectal cancer by means of the combination of 5'DFUR and the converting enzyme dThdPase, we transfected the dThdPase gene into the human colon cancer cell line SW480 and analyzed the growth pattern as well as the sensitivity to 5-FU or 5'DFUR of the dThdPase-transfected cells. The 50% inhibition (IC50) values of 5-FU against the SW480 parental cells, control vector-transfected cells SW480/V1, and dThdPase-transfected cells SW480/dThdPase were approximately 4.9, 6.3, and 2.9 microM, respectively. The IC50 of SW480/dThdPase was lower than that of SW480 or SW480/V1, although the differences were not statistically significant. The IC50 values of 5'DFUR for SW480, SW480/V1, and SW480/dThdPase were approximately 300, 330, and 3.2 microM, respectively. The sensitivity to 5'DFUR of SW480/dThdPase was increased by about 100-fold compared with that of SW480 or SW480/V1. With only 10% transfection efficacy, a high enough sensitivity to 5'DFUR was obtained to suppress the cell growth, indicating that a strong bystander effect was induced by this system. The in vivo growth of the s.c. transplanted SW480/dThdPase tumor in nude mice was significantly suppressed by i.p. injection of 5'DFUR compared with that in control mice that received phosphate-buffered saline (PBS) treatment. These results suggest that gene therapy using the combination of 5'DFUR and the dThdPase gene may be a useful approach for treatment of colon cancer.

Comparative genomic hybridization defines frequent loss on 16p in human anaplastic thyroid carcinoma.

Anaplastic thyroid cancer (ATC) is one of the malignant tumors with the poor prognosis that is thought to arise from well-differentiated thyroid cancer (DTC). To investigate the molecular mechanism of ATC, we studied genomic alterations of eight ATC cell lines and three DTC cell lines by means of the comparative genomic hybridization (CGH) method. Loss of 16p was observed in five of eight ATC cell lines (62. 5%), but none of the three DTC cell lines showed loss of this chromosome arm. It is notable that loss of 18q [7/8 of ATC (87.5%), 2/3 of DTC (67%)] and gain of 20q [5/8 of ATC (62.5%), 3/3 of DTC (100%)] were frequently seen in both histologic types. Our results suggest that there is a gene in 16p that is closely associated with transformation from well-differentiated thyroid cancer to anaplastic cancer.

Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus.

BACKGROUND: Heat shock proteins (HSPs) first were defined as proteins induced by heat shock and other environmental and pathophysiologic stresses and are implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancer. METHODS: Immunohistochemical study for HSP 27 and HSP 70 was performed on buffered formalin fixed, paraffin embedded sections of 102 esophageal squamous cell carcinoma specimens using monoclonal anti-HSP 27 antibody and anti-HSP 70 antibody. RESULTS: Normal squamous cells expressed both HSP 27 and HSP 70 with the exception of the basal layer. In cancerous tissue, expression of HSP 27 was evaluated as positive (+) (39 cases; 38%), reduced (+/-) (53 cases; 52%), or negative (-) (10 cases; 10%) and expression of HSP 70 was evaluated as (+) (14 cases; 14%), (+/-) (57 cases; 56%), or (-) (31 cases; 30%). There was a strong correlation between the expression of HSP 27 and HSP 70 (P < 0.0001). When compared with clinicopathologic features, expression of both HSP 27 and HSP 70 correlated negatively with lymph node metastases (P < 0.05), but not with depth of invasion or histologic grade. The reduction of the HSPs was associated significantly with poor postoperative survival (P < 0.0001). In addition, multivariate analysis revealed that HSP 27 (-) was the strongest prognostic factor among the clinicopathologic features. CONCLUSIONS: This study suggests that the expression of HSP 27 and HSP 70 frequently is reduced in patients with esophageal squamous cell carcinoma and therefore should be considered an independent prognostic factor of this disease.

Molecular characteristics of poorly differentiated adenocarcinoma and signet-ring-cell carcinoma of colorectum.

In a series of 45 poorly differentiated adenocarcinomas (por) and 7 signet-ring-cell carcinomas (sig) of the colorectum, K-ras gene mutation, p53 immunostaining and microsatellite instability (MSI) were analyzed for a comparison with 46 cases of colorectal carcinomas of the well or moderately differentiated type (well/mod). In addition, the mutations of simple repeated sequences in the transforming-growth-factor-beta type-II receptor (T beta R-II) gene and the BAX gene were analyzed as possible targets for DNA replication errors. Mutation of the K-ras gene in the por, sig and well/mod specimens was detected in, respectively, 22%, 11% and 48%, positive immunostaining for p53 in 41.8%, 28.6% and 60.3%, and MSI in 36%, 30% and 4%. Frameshift mutation of the T beta R-II gene was detected in 27.5% of the por and none of the sig specimens, while corresponding figures for mutation of the BAX gene were 15.7% and 0%. Significant differences between the por and well/mod tumors were found in the occurrence of K-ras mutation at codons 12 and 13, and MSI. Clinicopathologically, the tumor status of por with MSI was found to significantly correlate with the tumor's location in the proximal colon. In cases without MSI and sig, no frameshift mutation of either the T beta R-II or the BAX gene was found. These results suggest that poorly differentiated and signet-ring-cell carcinomas have a genetic background different from that of well or moderately differentiated carcinomas of the colorectum, and that DNA-replication error is at least partly involved in the carcinogenesis of these specific types of colorectal carcinomas.

Mammaglobin B as a novel marker for detection of breast cancer micrometastases in axillary lymph nodes by reverse transcription-polymerase chain reaction.

A novel reverse transcription-polymerase chain reaction (RT-PCR) assay using mammaglobin B gene was developed for detection of breast cancer micrometastases in axillary lymph nodes. Fourteen primary breast cancers and 56 axillary lymph nodes from six patients with primary breast cancer and 15 control lymph nodes from non-cancer bearing patients were subjected to this assay. The transcript of mammaglobin B gene was detected in none of the control lymph nodes, but in all of the 14 primary breast cancers. Eleven out of the 56 lymph nodes from the patients, which were shown to be positive by histological examination, were also proven positive by this assay. On the other hand, fourteen of the 45 (31%) histologically negative lymph nodes were also shown to express mammaglobin B mRNA, which suggested the presence of micrometastases in these lymph nodes. RT-PCR using mammaglobin B gene could therefore be a useful tool for detection of micrometastases of breast cancer.

Transaxillary endoscopic partial mastectomy for comparatively early-stage breast cancer. An early experience.

For optimal cosmetic results in breast cancer surgery, we developed an alternative operation using endoscopy to remove tumors through a small single incision remote from the breast itself. The tumor is endoscopically resected with electrocautery and low constant suction through a short incision in the axilla, 5 cm long, after which axillary dissection is performed through the same wound in the usual manner, under visualization by the naked eye. Primary reconstruction of the breast is performed with the remaining mammary gland and a subcutaneous fat flap on the serratus muscle. Seven patients underwent this surgery. The postoperative shape of the breasts was judged to be cosmetically satisfactory, and the surgical wounds were not visible from the front in all cases. This operation has the same therapeutic oncological effect as a standard lumpectomy and, in addition, offers a greater cosmetic advantage in cases of early breast cancer.