Celecoxib, a cyclooxygenase-2 inhibitor, potentiates the chemotherapic effect of vinorelbine in the medullary thyroid cancer TT cell line.

We analyzed the in vitro effects of celecoxib, a COX-2 inhibitor, and determined if celecoxib can sensitize a human MTC-derived cell line (TT) to chemotherapeutics. We found that celecoxib induced apoptosis in TT cells and decreased drug efflux by reducing the expression of MDR-1 mRNA, which codes for the drug efflux pump P-gp. We also observed that TT cells were 10-fold more resistant to doxorubicin than to vinorelbine, mimicking what can be observed in clinical practice. In addition, we found that the combination of celecoxib and vinorelbine, but not doxorubicin, induced a significant reduction in cell viability and a significant increase in apoptosis. In conclusion, we showed that celecoxib was able to enhance the chemotherapeutic effect of vinorelbine. A clinical trial exploring the in vivo activities of celecoxib in MTC patients who cannot benefit from available treatments would be desirable, taking into account the possible risks of cardiovascular effects of this drug.

Beta-catenin activation is not involved in sporadic parathyroid carcinomas and adenomas.

Aberrant accumulation of beta-catenin has been found in various types of human tumors. The aim of this study was to evaluate whether Wnt/beta-catenin signaling is activated in parathyroid carcinomas and adenomas. We studied 154 parathyroid tumors (18 carcinomas (13 with distant metastases), six atypical adenomas, and 130 adenomas). Three normal parathyroid tissues were used as control. Direct sequencing of exon 3 of the CTNNB1 gene showed absence of stabilizing mutations in all the tumors. Immunostaining of beta-catenin was performed in all carcinomas and in 66 adenomas (including three atypical). Normal parathyroid showed a homogeneous distinct outer cell membrane staining in the majority of cells and no nuclear staining. A weak cytoplasmic staining was observed in one case. All tumors showed negative nuclear staining. With the exception of one carcinoma, which had a negative membrane staining, all other samples showed a membrane staining which was similar to that of the normal parathyroid. beta-Catenin expression was heterogeneous with a range of positive cells between 5 and 80%, independently of tumor type. Our results suggest that the Wnt/beta-catenin signaling pathway is not involved in the development of parathyroid carcinomas and adenomas.

Re-differentiation of thyroid carcinoma cell lines treated with 5-Aza-2'-deoxycytidine and retinoic acid.

We studied cell growth rate, mechanisms of growth inhibition, phenotype re-differentiation, expression of RARalpha, beta, gamma and differentiation thyroid genes before and after combined treatment with 5-Aza-CdR and RA (5-Aza/RA) of human thyroid carcinoma cell lines (FRO, WRO, TT). Furthermore, the activity and localization of the re-expressed sodium-iodide-symporter (NIS) protein was analyzed. After 5-Aza/RA treatment, all cell lines showed a significant reduction in cell growth. This was associated with apoptosis in the TT, with inhibition of cell proliferation in the WRO, and with cell cycle impairment in FRO and WRO. FRO and WRO treated with 5-Aza/RA lost the ability to grow in soft agar. FRO re-expressed thyroid transcription factor-1 and thyroglobulin, TT and WRO re-expressed PAX-8 and FRO and TT re-expressed RARbeta and NIS mRNA. Despite this expression, they were unable to take up iodine: a cytoplasmic localization of NIS protein was demonstrated in FRO. In conclusion, besides a significant reduction in cell growth rate and in the ability to grow in soft agar, treatment with 5-Aza/RA partially re-differentiated FRO and induced expression of NIS mRNA and protein in FRO and TT, but this treatment was unable to restore the functional activity of NIS, likely because it was located into the cytoplasm without reaching the plasma membrane.

Retinoic acid receptor beta2 re-expression and growth inhibition in thyroid carcinoma cell lines after 5-aza-2'-deoxycytidine treatment.

The treatment of both undifferentiated and de-differentiated thyroid tumors, which are unresponsive to radioiodine, represents one of the biggest challenges for thyroidologists. The aim of the present study was to investigate in vitro the methylation status of retinoic acid receptors (RAR)beta2 promoter and the effect of the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) on 5 human thyroid cancer cell lines. The methylation status of RARbeta2 promoter was analyzed by methylation-specific PCR. The effect of 5-Aza-CdR on cell growth and apoptosis was evaluated by cell counting, enzymelinked immunosorbent assay tests and fluorescence-activated cell sorting analysis, while the effect on the expression of RAR and thyroid-specific genes was measured by qualitative and quantitative RT-PCR. Methylation of RARbeta2 promoter was present only in ARO cells. 5-Aza-CdR determined growth inhibition in all cell lines, probably due to apoptosis in WRO, NPA, and ARO cells, and to inhibition of DNA synthesis in TT cells. Treatment with 5-Aza-CdR induced the expression of RARbeta mRNA in ARO and FRO cells, a slight increase of the expression of Tg, TPO and thyroid trancription factor 1 (TTF-1) mRNA and the new expression of low levels of NIS in TT cells. A significant increase of TTF-1 mRNA in FRO cells was also observed. In this study we demonstrated that RARbeta2 promoter was methylated in ARO cell line. However, the 5-Aza-CdR treatment induced RARbetamRNA expression not only in ARO but also in FRO and TT cell lines, whose RARbeta2 promoter was unmethylated. A significant reduction of cell growth, but not cell re-differentiation, was also observed after 5-Aza-CdR treatment.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo-micro-macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells.

The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5'-nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans.

Primary chemotherapy with gemcitabine, epirubicin and taxol (GET) in operable breast cancer: a phase II study.

This trial was conducted to assess the activity and tolerability of the gemcitabine, epirubicin, taxol triplet combination in patients with operable breast cancer. After core biopsy, 43 women with stage II-IIIA breast cancer were treated with gemcitabine 1000 mg m(-2) over 30 min on days 1 and 4, epirubicin 90 mg m(-2) as an intravenous bolus on day 1, and taxol 175 mg m(-2) as a 3-h infusion on day 1, every 21 days for four cycles. The primary end point was the percentage of pathological complete responses (pCR) in the breast; secondary end points were tolerability, clinical response rates, overall and progression-free survival, tumour biomarkers before and after primary chemotherapy (PCT). All patients were included in safety and survival analyses; 41 eligible patients were evaluated for response. The overall clinical response rate was 87.8% (95% CI 77.8-97.8), with 26.8% complete responses (95% CI 13.3-40.3). A pCR in the breast was observed in six patients (14.6%; 95% CI 3.8-25.4); 15 patients (36.6%; 95% CI 21.9-51.3) had negative axillary lymph nodes. Grade 4 neutropenia was observed in 67.4% of the patients; febrile neutropenia occurred in 1.9% of cycles (granulocyte colony-stimulating factor was used in 3.2% of the cycles to shorten the duration of neutropenia). A statistically significant difference between Mib-1 at baseline (> or =20% in 71.4% of the patients) and at definitive surgery (28.6%, P < 0.05) was observed. The gemcitabine, epirubicin, taxol regimen is active and well tolerated as PCT for operable breast cancer. This combination allows the administration of full doses of active agents with a low incidence of febrile neutropenia.

HER2 overexpression as a prognostic factor in metastatic breast cancer patients treated with high-dose chemotherapy and autologous stem cell support.

We retrospectively evaluated the predictive and prognostic role of HER2 expression in 44 metastatic breast cancer (MBC) patients treated with high-dose consolidation chemotherapy (HDCT) and autologous stem cell support after induction chemotherapy (IC) with six courses of epirubicin+paclitaxel (22 patients) or gemcitabine+epirubicin+paclitaxel (22 patients). HER2 expression was evaluated by an immunohistochemical method (Herceptest, Dako). A total of 13 patients (29.5%) showed a HER2 overexpression (score 3+). After IC, nine patients were in complete response (CR), 30 in partial response (PR), and five in stable disease (SD); after HDCT, 20 (45.5%) obtained a CR, and 23 were in PR, for a conversion rate of 48.5%. Conversion rate for HER2-positive patients was 87.5 vs 37% for HER2-negative patients (P=0.018). The median progression-free (PFS) and overall survivals (OS) were 17.6 (95% CI 13.2-22.0) and 44 (95% CI 25.9-62.3) months, respectively. Patients with HER2 overexpression experienced a significantly (P=0.0042) shorter median PFS (15.3 months, 95% CI 11.1-19.5) compared to HER2-negative patients (21.3 months, 95% CI 14.3-28.4). The median OS was 27.6 months (95% CI 4.5-50.7) in HER2-positive patients and 50.3 months (95% CI 38.7-62.0) in HER2-negative patients (P=0.345). These results indicate that HER2 overexpression predicts a worse outcome for patients with MBC treated with HDCT, despite the high CR rate obtained in this subset of patients.

Accelerated versus standard cyclophosphamide, epirubicin and 5-fluorouracil or cyclophosphamide, methotrexate and 5-fluorouracil: a randomized phase III trial in locally advanced breast cancer.

BACKGROUND: The purpose of this study was to evaluate the impact of a dose-dense primary chemotherapy on pathological response rate (pCR) in patients with locally advanced breast cancer (LABC) treated with combined modality therapy. PATIENTS AND METHODS: Stage IIIA/IIIB patients received three courses of induction chemotherapy (ICT) with cyclophosphamide, epirubicin and 5-fluorouracil (CEF) followed by local therapy (total mastectomy or segmental mastectomy with axillary nodes dissection) and adjuvant chemotherapy (ACT) with three courses of CEF alternated with three courses of cyclophosphamide, methotrexate, 5-fluorouracil (CMF). Patients were randomized to receive ICT and ACT every 3 weeks (arm A, 'standard treatment') or every 2 weeks with granulocyte-macrophage colony-stimulating factor (GM-CSF) support (arm B, 'dose-dense treatment'). In both arms radiotherapy was administered after the end of chemotherapy (in selected cases) and patients with hormonal receptor-positive tumors received tamoxifen for 5 years. RESULTS: A total of 150 patients were randomized (77 arm A and 73 arm B) and demographics were well balanced between the two arms. Compliance to treatment was excellent: 95% and 93% of patients in arms A and B, respectively, completed the treatment program with no modification or delay. Median duration of treatment (ICT+local+ACT) was 183 days (range 0-265) in arm A and 139 days (0-226) in arm B. The average relative dose intensity (ARDI) of chemotherapy was 1.3 with a 30% increase in the dose intensity in arm B in comparison with arm A. No difference in clinical [62%; 95% confidence interval (CI) 49% to 73.2%] and pathological response rates to ICT was observed between the two arms. Median follow-up was 5 years (range 1-96 months); median disease-free survivals were 4.8 years in arm A and 4.5 years in arm B. Median overall survival was 7.8 years in standard therapy: this figure has not yet been reached in the dose-dense treatment. CONCLUSIONS: In LABC a dose-dense regimen, while allowing a 30% increase in the dose intensity of chemotherapy, did not provide significant improvement in pathological response rates. However, accelerated chemotherapy reduced the duration of the combined-modality program (6.1 versus 4.6 months) with no additional toxicities.

Cyclins of phases G1, S and G2/M are overexpressed in aneuploid mammary carcinomas.

Expression of cyclins A, B1 and D1 in human breast cancer was analyzed using dual-parameter flow cytometry with simultaneous evaluation of the DNA content. The asynchronous MCF-7 breast adenocarcinoma cells were used to implement flow cytometry analysis and to analyze the cell cycle distribution of cyclins. The patterns of the cyclin expression were also analyzed in vivo in fresh tissue specimens of human breast carcinomas. The combined measurement of DNA and cyclins showed a higher cyclin expression in aneuploid (11.5 +/- 2.0%, 4.3 +/- 1.1%, and 19.5 +/- 3.4% positive cells for cyclins A, B, and D1, respectively) than in diploid carcinomas (3.9 +/- 1.2%, 1.1 +/- 0.4%, and 5.0 +/- 1.2% positive cells for cyclins A, B, and D1, respectively). A positive relationship was also found between cyclin A and D1 expression and H(3)-thymidine labeling index. In the in vitro model, the asynchronous growing MCF-7 cells showed a variable number of cells expressing cyclins in an unscheduled way, unrelated to the phase at which these cyclins are expressed in normal cells. A similar condition was also observed in tumors. In conclusion, the data showed a deregulated expression of cyclins in a transformed adenocarcinoma cell line and in breast tumors. Furthermore, overexpression of these proteins is related to the aneuploid and high proliferative activity of human mammary carcinomas.

Congenital hypothyroidism with impaired thyroid response to thyrotropin (TSH) and absent circulating thyroglobulin: evidence for a new inactivating mutation of the TSH receptor gene.

Congenital hypothyroidism due to impaired thyroid response to TSH was originally described by Stanbury. A diagnosis of congenital hypothyroidism with thyroid unresponsiveness to TSH is accepted if the patient has congenital hypothyroidism, the thyroid gland is in the normal position in the neck, the size of the thyroid is either normal or atrophic, the serum TSH level is increased, the bioactivity of TSH is intact, and the response of the thyroid gland to TSH stimulation is decreased. In all originally described cases serum thyroglobulin was undetectable. We describe a 22-yr-old female patient who was severely hypothyroid and mentally retarded. Serum T4 and T3 concentrations were below the sensitivity of the methods, with elevated serum TSH levels. Serum thyroglobulin was undetectable. A normally shaped hypoplastic gland located in the appropriate anatomical position in the neck was found at scintiscan. The gland did not respond after administration of bovine TSH in terms of 131I uptake, serum thyroid hormones, and thyroglobulin secretion. A diagnosis of congenital hypothyroidism due to TSH unresponsiveness was formulated. Genetic analysis in the propositus showed a homozygous inactivating mutation of the TSH receptor that had not been previously described. The mutation consisted of the substitution of an isoleucine in place of a highly conserved threonine at position 477 in the first extracellular loop of the receptor (T477I). The brother, one sister of the father (whose DNA was not available), the mother of the propositus, one sister, and the brother were heterozygous for T477I. All the heterozygous persons were unaffected. After transfection in COS-7 cells, the mutant receptor displayed an extremely low expression at cell surface. At variance with cells transfected with the wild-type TSH receptor, cells transfected with the mutant T477I did not show constitutive activity for the adenylyl cyclase pathway. A dramatic reduction in the amount of cAMP accumulation after bovine TSH challenge was observed in cells transfected with the mutant T477I receptor. A structural defect in the mutant TSH receptor protein was probably responsible for the poor routing of the receptor to the cell membrane. This is the first time that a loss of function mutation of the TSH receptor is described in a patient with severe congenital hypothyroidism and absent circulating thyroglobulin due to TSH unresponsiveness and the first time that an inactivating mutation of the TSH receptor is described in the first extracellular loop.

Apomorphine has a potent antiproliferative effect on Chinese hamster ovary cells.

Apomorphine is a potent non selective agonist at the D1 and D2 dopamine receptors acting both pre- and post-synaptically. In this report we describe a novel function of apomorphine, independent from its dopaminergic activity. Apomorphine inhibits Chinese hamster ovary (CHO)-K1 cell proliferation in a dose-dependent manner. The EC50 of apomorphine-induced inhibition of CHO-K1 cell proliferation determined by cell counting was 3.24 +/- 0.07 microM. Remarkably, the dose-response curve obtained by measuring the incorporation of [3H]thymidine was practically identical to the previous one giving an EC50 of 3.52 +/- 0.04 microM. The dopaminergic antagonists SCH23390 and spiperone at a concentration of 10 microM (well beyond their Kd values for the dopamine D1- and D2-like receptors respectively) were not able to antagonize the effect of apomorphine on CHO-K1 cell proliferation. Apomorphine exerts its effect early during incubation; CHO-K1 cells exposed to apomorphine for a period as short as 1 h and then allowed to grow for three days were significantly reduced in number with respect to untreated control cells. After four hours of exposition to apomorphine (10 microM) the antiproliferative effect was similar to that seen when this compound was present in the bath for all three days. Concentrations of apomorphine higher than 10 microM induced cell death, and the colony was completely destroyed at 50 microM. Cytometric analyses showed a significant accumulation of CHO-K1 cells in the G2/M phase.

Cyclin-D1 expression in node-positive (N+) and node-negative (N-) infiltrating human mammary carcinomas.

Cyclin-D1 (CD1) expression was analyzed in human mammary carcinomas by immunohistochemical (IHC) and flow-cytometry (FCM) methods: 52.5% and 50% of cases were strong expressors of CD1 by IHC and FCM analysis respectively. The percentage of CD1-positive cells was especially high in node-negative (N-) estrogen-receptor-positive (ER+) tumors, probably as a consequence of CD1 induction by estrogens in steroid-responsive tissues. However, CD1 expression was not related to ER positivity in node-positive tumors (N+). An interesting relationship between CD1 expression and H3-thymidine labelling index (H3Td-LI) was also found: CD1 and H3Td-LI were unrelated in N- tumors, while high CD1 expression was observed in N+ tumors with high DNA synthesis, as assessed by H3Td-LI. The combined measurement of DNA and CD1 showed that 27 specimens were aneuploid, 19 of them (19/27; 70%) strongly expressing CD1. Further studies are needed to clarify the role of CD1 in DNA abnormality of breast tumors. However, we cannot exclude that the CD1 may be differently de-regulated in the last phase of tumor progression, and that CD1 over-expression may contribute to the aneuploidy of mammary carcinomas.

Primary chemotherapy in locally advanced breast cancer (LABC): effects on tumour proliferative activity, bcl-2 expression and the relationship between tumour regression and biological markers.

The rate of tumour cell proliferation evaluated by the [3H]-thymidine labelling index ([3H]-dT-LI) is known to be an independent prognostic factor in patients with operable breast cancer and significantly predicts the response to chemotherapy in patients with advanced disease. In locally advanced breast cancer (LABG), we examined whether chemotherapy induced modifications in [3H]-dt-LI, and bcl-2 expression and their relationship with tumour regression and prognosis. 70 LABC patients received three courses of primary chemotherapy (FEC: 5-fluorouracil 600 mg/m2, epidoxorubicin 60 mg/m2, cyclophosphamide 600 mg/m2, followed by surgery and subsequent adjuvant chemotherapy consisting of three courses of FEC alternated with three courses of CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-fluorouracil 600 mg/m2). Tumour biological markers were evaluated on diagnostic biopsy, before primary chemotherapy and at surgery. Tumour cell proliferation was determined by [3H]-dT-LI, whilst bcl-2 expression was examined by immunohistochemical staining. The overall response rate to primary FEC was 74.3% (95% confidence interval 57.6-83.2%). The response rate correlated with high [3H]-dT-LI: 88% (29/33) of patients with high [3H]-dT-LI achieved an objective response compared with 62% (23/37) of patients with low [3H]-dT-LI (P = 0.014). The 3 patients achieving a pathological complete response after induction treatment had high proliferative tumours. The highest 2-year relapse free survival (66.6%) was observed in patients with low [3H]-dT-LI after primary chemotherapy. The median bcl-2 expression values before and after primary chemotherapy were 0% (range 0-80) and 30% (range 0-90), respectively (P = 0.03). Our data indicate that primary chemotherapy can modulate tumour cell kinetics and apoptosis-related genes. Pretreatment proliferative activity correlated with tumour response, whilst post-treatment [3H]-dT-LI correlated with relapse free survival.

The spectrum of 67-kD laminin receptor expression in breast carcinoma progression.

Laminin is a glycoprotein of the basement membrane (BM), involved in a variety of normal and pathological cellular events including tumour invasion and metastasis. Cells bind laminin through different types of receptor. The 67-kD laminin receptor (67LR) is a cell-surface protein which binds laminin with high affinity. 67LR expression has been shown to increase in neoplastic cells, compared with normal tissues, and 67LR seems to play an important role during the first steps of neoplastic progression. In this study, 67LR expression was analysed during the morphological phases of breast cancer progression from normal tissue to invasive carcinoma. A total of 506 formalin-fixed, paraffin-embedded normal breast structures and lesions were stained by immunohistochemistry usign the MLuC5 monoclonal antibody, which is specific for 67LR. The results show that in normal breast and in any kind of breast lesion, myoepithelial and endothelial cells express 67LR. While 67LR is not seen in the epithelium of normal breast, cysts, adenosis, and benign tumours, it is expressed in the epithelial cells of several hyperplasias and carcinomas in situ, both ductal and lobular, as well as in all invasive carcinomas. The 67LR-positive cell subpopulation expands from hyperplastic lesions to invasive carcinoma, suggesting that 67LR could be related to the induction and progression of breast cancer.

NM23 gene expression in human breast carcinomas: loss of correlation with cell proliferation in the advanced phase of tumor progression.

NM23 is a protein associated with tumor progression, expressed in all tissues and in human tumors. Reduced expression of NM23.H1 is related to high incidence of lymph node and distant metastasis or to poor prognosis of the patient in several human malignant tumors. In this study we analyze NM23 expression in non-neoplastic mammary tissues surrounding the tumoral lesions, in human mammary carcinomas and in lymph node metastasis. Our analysis shows that NM23.H1 expression is lower in the mammary cells surrounding the tumor than in the tumor itself. In the primary tumors we observed a negative trend between degree of local invasion and level of NM23.H1 expression. A further decrease of NM23.H1 was detected in the invasive tumors that metastasized to axillary lymph nodes and in the metastasis. NM23.H2 was always more highly expressed than NM23.H1, and reduced expression of NM23.H1 but not NM23.H2 was concordant with the presence of lymph node metastasis or local invasiveness of the primary tumor. A positive correlation between NM23.H1 mRNA content and cell growth rate of breast tumor cells has been confirmed. However, this trend was not maintained in cancer cells from tumors that metastasized to axillary lymph nodes and in metastatic cells; in these 2 situations the NM23.H1 mRNA content varied without any relationship to the proliferative rate of the cells. In addition, in comparison with the initial tumor, the metastatic cell population showed a strong decrease of NM23.H1 expression and increased proliferative activity.

Tumor uptake and mitotic activity pattern of 5-[125I]iodo-2'- deoxyuridine after intravesical infusion in patients with bladder cancer.

UNLABELLED: In patients with bladder cancer, little is known about diffusion in the tumor mass of 5-iodo-2'-deoxyuridine (IUdR) administered intraluminally, although previous studies based on external scanning have shown promising tumor-targeting properties of IUdR instilled intravesically. This study compared the pattern of IUdR uptake by bladder cancer cells with the actual distribution of mitotic activity, as evaluated by incubation of ex vivo tumor specimens with tritiated thymidine. METHODS: The [125I]IUdR (2-13 MBq) was instilled over 1-3 hr in the bladder of four patients with bladder cancer scheduled for ablative surgery. Twenty-four hours later, surgical samples were assayed for radioactivity and processed for microautoradiography, while fresh tumor specimens were fragmented, incubated with [3H]thymidine and further processed for microautoradiography. The diffusion of labeled IUdR across the bladder wall was evaluated by blood sampling. RESULTS: Tumor incorporation of [125I]IUdR 24 hr after intravesical instillation was 0.002%-0.05% ID/g, while the average tumor-to-normal bladder ratio was about 20. Microautoradiography showed that [125I]IUdR incorporation was confined to tumor cells in the most superficial layers of the bladder, while incubation of the tumor fragments with [3H]thymidine demonstrated the presence of diffuse mitotic activity also in the deeper tumor mass. Diffusion of labeled IUdR in the general circulation was minimal. CONCLUSION: Poor diffusion in the tumor mass makes *IUdR unsuitable for intracavitary therapy of bladder cancer, but the role of such an approach in the postsurgical "sterilization" of cancer remnants floating in the bladder lumen after partial cystectomy should be explored.