Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines

Recent investigations have demonstrated p53 and Rb alterations in a subset of transitional cell carcinoma (TCC). Further genetic changes during tumor progression include overexpression of the c-myc gene in a significant number of mainly invasive bladder tumors. To study the possible interactions between these genes in TCC, urothelial cancer cell lines were chosen as an in vitro model. Expression and mutation of p53 was studied in 15 bladder cancer cell lines by immunocytochemistry, Western blot, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing of double stranded PCR products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniques. Rb protein expression was analyzed by Western blot. Twelve of 15 cell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb protein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in all cell lines and copy numbers were close to normal. Northern analysis demonstrated that all cell lines expressed c-myc mRNA but evidence for altered regulation was found in at least two cell lines. Our data suggest that amplification or translocation are not the underlying mechanism for c-myc overexpression in urothelial tumors. No correlation between loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. Thus, these cell lines may provide a suitable model for further analysis of molecular alterations in urothelial cancer.     

Ras oncogene point mutations in bladder cancer resistant to cisplatin

Summary Bladder tumors respond to cisplatin-based chemotherapy in 68% of cases, but only 30% will have a durable response. Recent studies have suggested that ras point mutations may produce cisplatin drug resistance in cell lines. To determine the role of ras point mutations in human tumors resistant to cisplatin, we evaluated ten tumors exposed to cisplatin and eight untreated bladder tumors for ras point mutations. Using polymerase chain reaction DNA amplification and allele-specific oligohybridization, we found that only one of the ten treated tumors and none of the untreated tumors harbored a ras point mutation. We conclude that ras point mutations occur infrequently in untreated bladder tumors and do not appear to correlate with cisplatin drug resistance in vivo.     

Neoplastic invasion of the connective stalks in transitional cell papillary tumors (TCPT) of the bladder

Summary Neoplastic invasion into the connective stalks of transitional cell papillary tumors of the bladder was assessed in TUR specimens from new cases. There were 21 G1 cases, none of which showed invasion of the connective tissue stalk; this compared with definite invasion in 19 of 77 G2 cases (25%) and in 16 of 17 G3 cases (94%). During a 5 year follow-up period more of the stalk invasive cases had an unfavorable course (fatal outcome or total cystectomy) than of the stalk non-invasive cases; this was statistically significant (p>0.01) for the whole group of patients and for the smaller group of patients with G2 tumors (p>0.05).     

Expression of p53 product in Chinese human bladder carcinoma

Summary Expression of p53 protein was examined in 67 cases of primary transitional cell carcinoma of the bladder and 6 normal controls using an immunohistochemical method on paraffin sections. Positive nuclear staining for p53 in malignant cells was found in 34 (51%) of the 67 cancer patients; no positive staining for p53 was detected in any of the normal controls or in the benign cells, including stromal and inflammatory cells, within the tumor tissue. There were 8 positive cases (33%) in 24 grade G1 tumors, 12 (48%) in 25 G2 tumors and 14 (78%) in 18 G3 tumors. p53 protein was detected positively in 14 (36%) of 39 superficial tumors (Tis-T1) and in 20 (71%) of 28 invasive tumors (T2–T4). Thus, positive staining for p53 was found more frequently in poorly differentiated tumors (chi-squared test: G3/G1+G2P<0.01) and in invasive tumors (chi-squared test: T2–T4/Tis-T1P<0.01). Expression of p53 was also closely associated with recurrence of tumors. Alterations in p53 expression may be of prognostic value in cases of bladder transitional cell carcinoma.     

Ki-ras point mutation in codon 12 and expression ofp53 in mucin-producing tumor of the pancreas

Mucin-producing tumors (MPTs) of the pancreas show a variety of clinical characteristics, including massive production of mucin in the pancreatic duct, dilatation of the main pancreatic duct, and a better prognosis than common invasive ductal carcinoma (IDC). These characteristics suggest that MPTs and IDCs have different cytomolecular backgrounds. The present study was designed to assess the differences in cytomolecular background between MPTs and IDCs, especially the differences in Ki-ras point mutation (PM) and wild and mutant typep53 expression. Cytomolecular backgrounds were compared in a 13 MPTs [8 carcinomas (MPCas) and 5 benign tumors (MPBTs)] and 36 IDCs. Cytomolecular studies included the evaluation of Ki-ras PM and the expression of Ki-ras p21, wild-typep53 (w-p53), and mutant-typep53 (m-p53). Ki-ras PM was assessed by the allele-specific oligonucleotide dot blot hybridization method, and the expression of p21 andp53 was assessed by an immunohistochemical staining method with monoclonal antibodies. Ki-ras PM was seen in 97% of IDCs and in 77% of MPTs (100% of MPCas and 40% of MPBTs), and MPBTs showed a significantly lower incidence of Ki-ras PM (versus IDC,P < 0.01). Guanine-Guanine-Thymine (GGT) to Guanine-Adenine-Thymine (GAT) mutation was seen in 55% of IDCs and 62% MPTs (87% of MPCas and 20% of MPBTs), and MPCas showed a significantly higher incidence of percent GAT mutation (versus IDC,P < 0.05). Ki-ras p21 was expressed in 43% of IDCs and in 31 % of MPTs (50% of MPCas and 20% of MPBTs). w-p53 and m-p53 were expressed in 51 % and 78% of IDCs and in 54% and 62% of MPTs (38% and 63% of MPCas and 20% and 60% of MPBTs), respectively. In MPBTs, hyperplasias showed higher rates of p21, w-p53, and m-p53 expression than cystadenomas. The study suggested that GAT mutation may be involved in the tumorigenesis of MPTs. It is suggested that MPBTs, especially hyperplasias, may be classified as low-grade malignancies like MPCas.     

Higher expression of oncoproteins c-myc,c-erbB-2/neu,PCNA, and p53 in metastasizing colorectal cancer than in nonmetastasizing tumors

Background: Expression of individual oncogenes may predict outcome in patients with metastatic colorectal cancer (CRC). We studied the oncogene profile in the tumors of patients with CRC and assessed their value as predictors of liver metastases. Methods: The oncoproteins c-myc, c-erbB-2/neu (c-neu), PCNA and p53, were measured by immunohistochemistry in sections of metastasizing human CRC (n=34) and their liver secondaries as well as in sections of nonmetastasizing CRC (n=25). Results: The metastasizing primary CRC expressed proliferating-cell nuclear antigen (PCNA), c-neu, and c-myc at significantly higher levels than the nonmetastasizing primary cancer. p53 was also overexpressed in the metastatic group compared with the nonmetastasizing CRC, but this difference was not significant. The frequency of expression of all these markers was similar in the metastasizing primary CRC and the liver secondaries from the same patients. There was no correlation between the expression of the individual markers and histological grade, DNA ploidy, and subsequent local recurrence and lung metastasis and survival. However, when both groups were assessed together, positive expression of c-myc was more likely to occur in poorly differentiated tumors, whereas PCNA expression increased with more advanced Dukes stages. Conclusion: These results suggest that the overexpression of c-myc, c-neu, PCNA, and p53 may occur in CRC that are likely to metastasise to the liver.     

Detection of a rare point mutation in Ki-ras of a human bladder cancer xenograft by polymerase chain reaction and direct sequencing

Summary This paper represents the first report of a codon 59 mutation in Ki-ras from a spontaneous human transitional cell carcinoma of the bladder. Point mutations have the potential to activate the ras genes if they occur in critical coding regions. These include the sequences of codons 12, 13, 59, 61 and 63. Mutations in codons 12, 13 and 61 have been reported in a wide variety of human cancers, including transitional cell carcinoma of the bladder. However mutations in codon 59 have been reported only in retroviral Ki-ras and as a result of in vitro mutagenesis experiments. We have used the polymerase chain reaction and direct sequencing to detect mutations of Ki-ras, and allele-specific restriction analysis to detect mutations of N-ras in xenografts and continuous cell lines established from bladder cancer biopsies of ten different patients as well as in direct biopsy specimens from five human bladder tumours. For studies of Ki-ras, a 139 bp fragment which spanned the critical codons 12 and 13 and a 128 bp fragment that spanned the sequences of codon 59, 61 and 63 were enzymatically amplified and then sequenced. No N-ras mutations were detected. A heterozygous mutation of Ki-ras at codon 59 GCA G/ACA was detected in one line. This mutation is being expressed and appears stable as it was detected over several xenograft passages and was present in paraffin-embedded tissue from the primary tumour of the patient. The biological significance of the mutation in bladder cancer is currently under study.     

膀胱移行细胞癌中环氧化酶-2表达及其预后价值

目的探讨与膀胱移行细胞癌预后相关的新诊断方法。方法采用免疫组化SP法,检侧68例原发性膀胱移行细胞癌和10例正常膀胱组织标本环氧化酶-2(COX-2)的表达,结合临床资料,探讨其相互联系以及其表达与预后的关系。结果68例膀胱移行细胞癌组织中COX-2阳性表达率为63.2%(43/68),与病理分级、临床分期、血管浸润有关(P<0.05),而与淋巴结是否转移无显著性差异(P>0.05);对照组10例正常膀胱黏膜COX-2表达均为阴性;COX-2表达阳性生存超过5年者(54.1%)明显低于COX-2表达阴性者(91.3%)(P<0.05)。结论COX-2表达与膀胱移行细胞癌的恶性程度密切相关,检测COX-2表达对判断膀胱移行细胞癌预后有重要意义。     

Expression and prognostic value of proliferating cell nuclear antigen in transitional cell carcinoma of the urinary bladder

Forty-eight patients with transitional cell carcinima (TCC) of the bladder were investigated. Routine paraffin-embedded sections were stained with proliferating cell nuclear antigen (PCNA) monoclonal antibody in order to determine the growth fraction of the bladder tumors and to correlate this with tumor grade, stage, development of recurrence and survival rate during follow-up. PCNA positive staining was detected in 95.8% (46/48) of the tumors. The mean labeling index (LI) of superficial tumors (Ta-1,n = 28) was 12.58 ± 12.33%, and 34.55 ± 21.89% in invasive tumors (T2-4,n = 18). A similar correlation was found in association with tumor grade. The patients were followed up for a mean of 4.9 years (range 1–14 years). The mean PCNA Ll in nonrecurrent (n = 21) and simple recurrent (n = 7) superficial tumors was 11.29 ± 11.79% and 16.44 ± 14.05%, respectively, the difference not being statistically significant. To access survival, tumors with a PCNA LI above and below the median level (21%) were compared. Those patients (n = 19) with an index of > 21% (the mean of all the PCNA values) had a worse prognosis than those (n = 27) with an index of < 21%, a difference which is statistically significant. These results suggest that PCNA LI in bladder cancer may prove to be an objective and quantitative assay of biological aggressiveness and provide significant prognostic information, although it does not help the selection of patients at risk of simple recurrence in superficial tumors.     

膀胱移行细胞癌VEGF表达及与血管形成定量的关系

为了探讨膀胱移行细胞癌中血管内皮生长因子（ＶＥＧＦ）表达及其与血管形成定量关系，应用免疫组织化学方法，对６２例原发性膀胱移行细胞癌及８例正常膀胱组织中ＶＥＧＦ进行检测，并对其在３０例浸润性膀胱癌组织中表达与血管形成定量关系进行了研究。结果发现正常膀胱组织均为阴性反应，膀胱癌组织中ＶＥＧＦ蛋白阳性表达率为５６％。低分化和浸润性癌中ＶＥＧＦ蛋白阳性表达率明显高于高分化和表浅性癌组（Ｐ＜００５），浸润性膀胱癌组织中血管形成定量与ＶＥＧＦ表达密切相关（Ｐ＜００１）。结果提示：ＶＥＧＦ表达对膀胱癌生物学行为有重要影响。ＶＥＧＦ是膀胱癌发生发展过程中一个主要血管生成因子，ＶＥＧＦ蛋白表达和血管形成定量有可能成为预测膀胱癌转移和预后的指标     

Immunohistochemical analysis of p53 and ras p21 expression in colorectal adenomas and early carcinomas

To further investigate whether multiple genetic changes are involved in the development of colorectal cancer, we performed an immunohistochemical analysis of p53 and ras p21 protein expression in 139 specimens of colorectal adenoma with varying degrees of dysplasia, 57 specimens of early cancer with an adenomatous component, and 12 specimens of superficial early cancer without any adenomatous component. Positive p53 staining was found in 15% of the adenomas with moderate dysplasia and in 42% of the adenomas with severe dysplasia or intramucosal carcinoma (IMCA). Positive immunostaining of p53 was observed to be significantly correlated with the degree of dysplasia and the depth of invasion, as was the expression of ras p21. However, a closer correlation was observed with the increasing size of the adenomas. Furthermore, p53 staining was positive in 42% of the 12 superficial early cancer specimens, while ras staining was positive in only 1 specimen (8%). These results indicate that p53 gene overexpression may play some biological role in both the adenoma-to-carcinoma sequence and in de novo cancer development, whereas ras p21 expression may not be as involved in de novo cancer development as in the malignant conversion of colorectal adenomas.     

Loss of c-met protooncogene in primary and metastatic sites of breast cancer

Background: It has been proposed that clones of tumor cells acquire higher metastatic potential as a result of specific genetic alterations. This study was designed to determine the role of the c-met protooncogene in systemic spread by comparing the loss of the c-met protooncogene between primary and metastatic breast carcinomas. Methods: Only patients who had not received chemotherapy or radiotherapy in the preceding 6 months were included in this study. Histologically proven malignant tissue was obtained from the primary tumor, involved nodes, and distant metastatic and recurrent tumors of patients with breast carcinomas. Allelic loss of the c-met protooncogene in tumor tissue was determined by Southern blotting using a polymerase chain reaction-generated 347-bp human met-H probe. Restriction digestion was performed usingTaq I andMsp I, with the patient's lymphocyte DNA as controls. Results: Of 52 patients, lymphocyte DNA from 36 patients was heterozygous for the c-met protooncogene (69% informative). Forty-six tumors from these 36 patients were analyzed. Four of 30 primary tumors (13%) showed allelic loss of c-met. Of the nine nodal metastases examined, three (33%) showed allelic loss of the c-met protooncogene. Of seven distant metastatic breast tumors or recurrent disease, two (29%) showed allelic loss (both in patients with skin metastasis in the chest wall). Conclusions: Allelic loss of the c-met protooncogene was detected in both primary (13%) and metastatic sites (31%) of breast cancer. Although a higher proportion of allelic loss of c-met was noted in nodal and distant/recurrent disease, the difference when compared with the primary tumor was not statistically significant. These findings indicate a limited role of the c-met protooncogene in breast cancer metastases.     

Microvessel density as a prognostic marker in bladder carcinoma: Correlation with tumor grade,stage and prognosis

Introduction: The aim of our study was toevaluate tumor angiogenesis as a prognosticmarker of transitional cell carcinoma of thebladder and to asses its relationship toestablished variables for survival and responseto therapy.Patients and method: Microvessel density(MVD), a measure of tumor angiogenesis, wereevaluated in 77 primary bladder cancers.Forty-three superficial carcinomas and 34invasive carcinomas were analysed. Tumorspecimens of all patients were obtained bytransurethral resection (TUR) and all thetumors were transitional cell carcinomas.Twenty-two patients with invasive bladdercancer have undergone M-VEC chemotheraphy. Thecorrelation between MVD and histopathologicalgrade, tumor stage and prognosis was evaluated.MVD was identified by immunostaining ofendothelial cells using anti-CD34 antibody. Forstatistical analysis Kruskal-Vallis,Mann-Whitney U and Fishers exact tests were used.Results: MVD was correlated with tumorgrade, stage and prognosis. Significantlyhigher MVD was determined in invasive tumorsthan superficial tumors (p < 0.05). MVDincreased with tumor grade and stage(p < 0.05). High MVD was correlated with therisk of clinical progression in bothsuperficial and invasive bladder carcinomas(p < 0.05, p < 0.001 respectively). Invasivetumors with remission after M-VEC chemotheraphyhad lower MVD than tumors with progressionafter M-VEC.Conclusion: These data demonstrate thatMVD in bladder carcinoma correlates with grade,stage and malignant potential of the tumor.Quantification of tumor angiogenesis may allowselection of the type of treatment for bladdercancer patients.     

Expression of mdm-2 and p53 protein in transitional cell carcinoma

Amplification of the mdm-2 gene and overexpression of the mdm-2 protein might inactivate p53 function, and may have prognostic relevance. The present paper investigated the immunohistochemical overexpression of the mdm-2 and p53 proteins in 25 biopsy specimens of transitional cell bladder carcinomas (10 pT1 and 15 pT2 or higher stages). Five cases (20%) showed strong mdm-2 protein immunoreactivity in more than 5% of the tumor cells; 14 cases (56%) showed p53 immunoreactivity in more than 20% of the cells, and were considered as overexpressing p53 protein. Four of the five cases with strong mdm-2 immunoreactivity did not show p53 overexpression, and 13 of the 14 cases with p53 overexpression did not show mdm-2 immunoreactivity. Our data are consistent with the hypothesis that p53 overaccumulation (and hence possible p53 gene mutation) or mdm-2 overexpression (and hence possible mdm-2 gene amplification) may mirror two different and possibly complementary gene alterations, which might finally interfere with the control of cell proliferation and apoptosis. In this perspective, evaluation of the combined mdm-2/p53 protein phenotype in human bladder carcinomas could have prognostic relevance and give us better prognostic information than evaluation of the p53 protein alone.     

A histopathological mapping study of the urinary bladder tumors induced byN-butyl-N-(4-hydroxybutyl)nitrosamine in dogs

Bladder tumors were induced byN-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in five Beagles and four mongrel dogs. The tumors were observed for long periods and the tumor progression was traced using histopathological mapping. The results indicated (1) that low-dose BBN over a long period induced multiple low-grade (G1-2) and low-stage (pTa-1) papillary tumors, resembling superficial bladder cancer in humans; (2) that high-dose BBN over a short period induced high-grade (G2-3) and high-stage (pT3b) nonpapillary tumors and carcinoma in situ (CIS) resembling invasive cancer and CIS in humans; (3) that beagle dogs required longer periods and higher total doses of BBN as compared with mongrel dogs; (4) that the tumors induced by low-dose BBN in beagles were observed without BBN as long as the animals lived, and neither increasing numbers of tumors nor malignant features such as deep infiltration and metastasis was observed; and (5) that low-dose BBN seems to induce mild dysplasia, which is followed by Brunn's nest-like proliferation in the lamina propria and nodular change, eventually leading to the development of papillary noninvasive transitional cell carcinoma (TCC); and that high-dose BBN seems to induce severe dysplasia which leads to CIS and nonpapillary invasive TCC. These results may contribute to clarifying the natural history of human bladder cancer.     

Evaluation of epidermal growth factor receptor, transforming growth factor alpha, epidermal growth factor and c-erbB2 in the progression of invasive bladder cancer

Introduction: Determination of the risk of invasive bladder tumors progressing is still imprecise due to the heterogeneous biological behavior of this neoplasm. The goals of this study were to evaluate the patterns of expression of the epidermal growth factor (EGF) system in invasive bladder cancer and to assess its prognostic value. Methods: This immunohistochemical study was performed using fresh frozen tumor samples and a panel of monoclonal antibodies on a series of 43 invasive bladder cancers treated by cystectomy. Results: EGF was detected in 45% of the tumors and did not correlate with survival from bladder cancer. Transforming growth factor alpha (TGF) was expressed by 60% of the tumors and correlated strongly with death from bladder cancer. Epidermal growth factor receptor (EGF-R) expression was seen in 86% of cases and had no prognostic significance. cerbB2 was expressed in 50% of cases and was inversely related to a poor prognosis. When EGF and TGF were both expressed, there was little or no expression of c-erbB2. Conclusion: The accumulation of several growth factors and the relevant receptor are necessary for the progression of invasive bladder cancers. They could be used as indicators of tumor aggressiveness.     

Detection of ras oncogenes by analysis of p21 proteins in human tumor cell lines

Summary To detect mutationally activated ras oncogenes, we analyzed electrophoretic mobilities of ras p21 proteins utilizing the fact that many ras oncogenes produce abnormal p21 proteins that migrate at SDS/polyacrylamide gel electrophoresis as a fast-moving or slow-moving species in comparison to a normal p21 depending on the kind of mutation. Of 18 human tumor cell lines analyzed, four (SW480, SW620 and SW403 colon cancers, and SW626 ovary cancer) produced p21 belonging to the slow-moving species, suggesting a point mutation within codon 12 of a member of the three ras genes, H-, Ki- and N-ras. Subsequent DNA transfection analysis using NIH/3T3 cells as recipients identified activated Ki-ras oncogenes in the same four but not in other 14 cell lines. Thus, the analysis of p21 might serve as a rapid primary method to screen a large number of tumor materials for the presence of certain types of mutationally activated ras oncogenes.     

Expression of c-erbB-2 gene product in urinary bladder cancer

Expression of the c-erbB-2 gene product and the epidermal growth factor receptor (EGF-R) was investigated in 54 cases of human bladder cancer immunohistologically and by Western blot analysis. For detection of the c-erbB-2 product, two specific antibodies, a rabbit polyclonal antibody directed to the intracellular domain and a murine monoclonal antibody recognizing an epitope in the extracellular domain, were used. Seventeen cases of bladder cancer were stained by the anti-c-erbB-2 polyclonal antibody, while 20 cases were stained by the monoclonal antibody, with good correlation on both stainings (p less than 0.01). There were four c-erbB-2 positive cases in 26 G1 tumors, four in 15 G2 tumors, and nine in 13 G3 tumors. There were also eight erbB-2 positive cases in nine muscle-invasive tumors, nine of 45 superficial tumors, four of five with lymph node metastasis, and seven of 14 without metastasis, as revealed by staining with the polyclonal antibody. Thus, the c-erbB-2 gene product was more frequently expressed in high grade tumors (p less than 0.01), in high stage tumors (p less than 0.01), and nodal metastatic tumors (N.S. by Chi-square test). Twenty-two of the 54 tumors were stained by an anti-EGF-R monoclonal antibody, 528 IgG. The expression of EGF-R was independent of histological grading, tumor stage, and nodal status, and no correlation was observed between expression of the c-erbB-2 product and EGF-R. The c-erbB-2 product may be applicable as a tumor marker for evaluation of malignant potential, invasiveness, and probably metastatic potential of human bladder cancer.     

Cyclooxygenase-2 Expression Increases with the Stage and Grade in Transitional Cell Carcinoma of the Urinary Bladder

Objectives: Experimental models of carcinogenesis show that non-steroidal anti-inflammatory drugs (NSAIDs) increase apoptosis, inhibit angiogenesis and reduce metastases. A linkage between the activity of prostaglandin synthase enzyme cyclooxygenase-2 (COX-2), a known mediator of inflammation, and cancer angiogenesis is implicated. We investigated the expression of COX-2 in bladder cancer tissue specimens using immunohistochemistry. Methods: The immunohistochemical expression of COX-2 in bladder cancer was evaluated by scoring the intensity of immunoreactivity from 0 to 3. Further, the degree of COX-2 expression was correlated with the tumor grade and depth of invasion (T stage). Result: Fifty eight percent patients (n=22) had superficial bladder tumors, while 42% (n=16) were invasive bladder cancers. Overall, COX-2 immuno-positivity was seen in 84.2% (32/38) patients. COX-2 expression was positive in 76.4% (13/17) cases with pTa tumors, 100% (5/5) of pT1 tumors, 86.6% (13/15) of pT2 tumors and in 100% (1/1) of pT3 tumor. The higher stage tumors stained more intensely; this correlation wassignificant(p=0.01987; χ2=19.6977). With reference to the grade of tumors, a positive expression was seen in 81.25% (13/16) of the low-grade tumors and 89% (17/19) of the high-grade tumors. The differential COX-2 expression relative to the grade of tumor was found to be statistically significant (p=0.05; χ2=15.8612). Conclusion: The degree of COX-2 expression is significantly increased with advancing grade and T stage of disease (p < 0.05).     

Expression of pS2, c-erbB-2, and cathepsin D during the menstrual cycle in human breast cancers

Background: Many studies have addressed the effect of the timing of surgery for breast cancer relative to menstrual cycle phase, with conflicting results. Explanations for the possibility that survival could be altered by the appropriate timing of breast cancer surgery in humans remain speculative. Methods: We examined the expression of three estrogen related proteins (c-erbB-2, cathepsin D, pS2) in the breast tumors from 69 premenopausal women sampled in different phases of the menstrual cycle. Data on S-phase fraction and hormone receptor expression were also analyzed. Immunohistochemical assays were used to measure the proteins of interest. S-phase fraction was determined by flow cytometry. Analyses were performed based on fraction of cells staining positive for the protein, density of stain, and a histoscore that combined both fraction of positive cells and density. Results: We found no differences in c-erbB-2, cathepsin D, hormone receptor, or S-phase levels in tumors sampled in the follicular versus luteal phase, or perimenstrual versus periovulatory phase. The exception was pS2, which was expressed at greater levels during the luteal than during the follicular phase of the cycle (p<0.01); but there was no difference in pS2 expression when the patients were classified as periovulatory versus perimenstrual. Conclusions: Our findings do not support a variation in c-erbB-2, cathepsin D, S-phase fraction, or receptor expression as an explanation for the differences in breast cancer prognosis when surgery is timed by menstrual cycle phase. The finding that pS2 (an indicator of hormone sensitivity, and possibly better prognosis) is expressed at higher levels in tumor samples during the luteal phase suggests that the biologic profile of breast tumors may vary with the menstrual cycle and that these variations deserve further study.