Henri Pujol

Cathepsin D assay in primary breast cancer and lymph nodes: Relationship with c-myc, c-erb-B-2 and int-2 oncogene amplification and node invasiveness

In breast cancer, axillary lymph node invasiveness is the major prognostic factor in predicting relapse and metastasis. Nevertheless, since 30% of node-negative tumors also relapse, it is necessary to develop other independent prognostic factors. Oncogene amplification and the level of cathepsin D (cath-D), an acidic lysosomal protease produced and secreted in excess by breast cancer cells, have been proposed as additional prognostic factors. We have compared the cytosolic cath-D level and the amplification of three oncogenes: c-myc, neu-erb-B-2 and int-2 in 140 primary breast carcinomas and 64 axillary lymph nodes collected in 1987 and 1988 at the Cancer Center of Montpellier (Centre Paul Lamarque). None of the patients had previously received hormonal or chemotherapy. The cath-D concentration was measured with an immunoradiometric assay using monoclonal antibodies. DNA purified from the same samples was analyzed by a standard Southern blotting technique to estimate oncogene amplification. No correlation was found between the level of cath-D in the tumor and node invasiveness. Using a cut-off level of 60 pmol/mg protein, the status of cath-D was not correlated with neu-erb-B-2 and int-2 amplification and only correlated with c-myc amplification (P = 0.011). Both c-myc and cath-D are associated with cell proliferation, induced by estrogens in ER+ breast cancer, and constitutively produced in ER- breast cancer. The level of cath-D was significantly higher in the invaded lymph nodes (P = 0.04) than in the histologically non-invaded ones. Nevertheless, some non-invaded lymph nodes contained a high level of cath-D, as confirmed by immunoperoxidase staining. In conclusion, in breast cancer, a high cytosolic cath-D concentration is more frequent in tumors with c-myc amplification but is dissociated from neu-erb-B-2 or int-2 amplification, suggesting that the determination of these three markers will have an additional prognostic value.

A Prospective Study of the Prognostic Value of Cathepsin D Levels in Breast Cancer Cytosol

Background. Cathepsin D is a lysosomal protease overexpressed and abnormally secreted in most breast cancer cells. Several retrospective clinical studies have shown that cathepsin D is an independent prognostic factor in breast cancer that is associated with a higher risk of recurrence and a shorter overall survival.

Relationship between vitamin E and polyunsaturated fatty acids in breast cancer. Nutritional and metabolic aspects

Intake of vitamin E, total lipids, total cholesterol, and fatty acids were analyzed with the blood levels of vitamin E, total cholesterol, triglycerides, and the serum distribution of fatty acids in a hospital‐based population of 120 patients and 109 controls. In regard to nutritional intake, the only significant differences involve saturated and monounsaturated fatty acid consumption, which is more elevated in postmenopausal patients than in postmenopausal controls. Vitamin E and total cholesterol blood levels are significantly higher in patients than in controls, where the difference is that vitamin E is independent from cholesterol level in premenopausal women only. Fatty acid serum distribution is comparable in both samples, with the exception of arachidonic acid, which is significantly lower in premenopausal patients than in premenopausal controls. Two multivariate regression analyses of the plasma vitamin E levels of patients and controls were done with menopausal status and nutrients as independent variables for the first analysis, and with menopausal status and all blood analytes for the second one. The regression coefficients for total cholesterol and triglycerides are statistically significant for both samples, whereas a positive association between vitamin E plasma level and sunflower oil consumption and between vitamin E plasma level and serum linoleic acid distribution is significant for patients only. Furthermore, the multiple regression shows that, when adjusted for analyte variables, plasma vitamin E levels are higher in premenopausal than in postmenopausal patients. In addition, plasma lipid peroxidation, evaluated by malondialdehyde measurement, is shown to be significantly lower in patients than in controls. Malondialdehyde level is associated with a significant lower odds ratio (OR) after multivariate tertile analysis (OR for the highest tertile: 0.51;95% CI:0.29–0.89). Together, these findings are consistent with a picture of lower lipid peroxidation in patients than in controls.

Phase I study of percutaneous 4-hydroxy-tamoxifen with analyses of 4-hydroxy-tamoxifen concentrations in breast cancer and normal breast tissue

Abstract4-OH-tamoxifen is an active metabolite of tamoxifen that is detectable in the serum and tumour tissue of patients treated by oral tamoxifen. As this metabolite penetrates through the skin, it is possible to compare percutaneous 4-OH-tamoxifen (4-OH-TAM) and oral tamoxifen treatments. We report herein a randomized study of percutaneous 4-OH-TAM versus oral tamoxifen in women with breast cancer. This pharmacology study was designed to compare the 4-OH-TAM concentration in breast cancer and normal breast tissue according to the route and dose used for adminstration of tamoxifen after a 3-week period prior to surgery and tissue sampling. Women were randomized into one of the five following groups: group I, oral tamoxifen given at 10 mg twice a day; group II, 4-OH-TAM delivered percutaneously at 0.5 mg day to both breast areas; group III, 4-OH-TAM applied percutaneously at 1 mg/day to both breast areas; group IV, 4-OH-TAM delivered percutaneously at 1 mg/day to a large cutaneous area excluding the breasts; and group V, 4-OH-TAM applied percutaneously at 2 mg/day to a large skin area excluding the breasts. 4-OH-TAM plasma and tissue concentrations were significantly higher in the oral tamoxifen group as compared with either the high- or the low-dose percutaneous 4-OH-TAM group. In group II, percutaneous 4-OH-TAM treatment resulted in tissue concentrations of 1,446 and 352 pg/g in tumour tissue and normal breast tissue, respectively. In group I these concentrations were as follows: tumour tissue, 12, 453 pg/g; and normal tissue, 10,214 pg/g. 4-OH-TAM concentrations in tumour tissue and normal breast tissue did not significantly differ in any group. In the oral group we observed classic effects on coagulation and lipid metabolism when pre- and post-treatment values of these biological variables were compared, whereas no difference was observed in the percutaneous group. Although precutaneous administration of 4-OH-TAM led to a low plasmatic concentration of this active metabolite, the breast tissue concentration remained lower than those observed after oral tamoxifen treatment. Therefore, at the doses described in this study, percutaneous 4-OH-TAM cannot be proposed as an alternative tamoxifen treatment.

Chest Wall Reconstruction for Radionecrosis After Breast Carcinoma Therapy

This study aimed at evaluating various reconstructive procedures for, chest wall radionecrosis after breast carcinoma therapy. Four different techniques were performed between 1973 and 1992 in 120 patients: latissimus dorsi musculocutaneous flap (LDF; n = 81); transposed omentum and split-thickness skin graft (TGO; n = 20); fasciocutaneous flap (FCF; n = 10), and transverse rectus abdominis musculocutaneous flap (TRAM; n = 9). Initial dose of irradiation ranged from 60 to 110 Gy. The average interval between initial treatment and reconstruction was 11 years. Local recurrence was suspected in 26 patients and was histologically proven after removal in 36 (30%). Surgical procedure results were analyzed by mean hospital stay (8 days for LDF vs. 52 days for TGO), early (13% LDF vs. 60% TGO) and late (7% LDF vs. 35% FCF) complications, second surgery (15% LDF vs. 53% FCF), and functional and cosmetic outcomes. In our experience, the LDF was the first-line flap. The TRAM was used to cover very large defects and when breast reconstruction was needed. When these flaps were impossible or dangerous, we performed a TGO. These three procedures have replaced FCF indications.

Variation of bcl-2 expression in breast ducts and lobules in relation to plasma progesterone levels: overexpression and absence of variation in fibroadenomas

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl‐2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl‐2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl‐2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean=10·1, quantitative immunochemistry score (QIC) arbitrary units (AU), n=19], than in normal lobules (mean=5·1 AU, n=43, P=7×10−5). bcl‐2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P=1·8×10−2 and P=1·7×10−2 respectively). This was further supported by a statistical link (P=5×10−3) between high levels of circulating progesterone and weak bcl‐2 staining in lobules and ducts. This progesterone‐dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl‐2 expression and circulating levels of oestradiol, and follicle‐stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl‐2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

Tamoxifen Treatment Increases the Concentration of 52K- Cathepsin D and Its Precursor in Breast Cancer Tissue

The pro‐cathepsin D of Mr 52,000 is regulated by estrogens via the estrogen receptor (RE) and is secreted by breast cancer cells in vitro. In an attempt to predict the hormone responsiveness of breast cancer in vivo, we have assayed total 52K cathepsin D and its precursor in the primary breast cancer cytosol of 36 patients treated before surgery with 30 mg of tamoxifen daily for 1 to 5 weeks (average, 3 weeks). Compared to a similar control population, total 52K cathepsin D was increased by tamoxifen (P = 0.02) but less so than its precursor (P < 0.001). Furthermore, 45% of the RE‐positive tumors from tamoxifen‐treated patients had a higher cathepsin D precursor concentration than the same type of tumor from control patients, or than RE‐negative tumors from tamoxifen‐treated patients. This 3‐week challenge test was probably too short to avoid partial estrogenic activity of tamoxifen (flare) and the authors infer that longer time of treatment would decrease rather than increase the concentration of cathepsin D in the RE‐responsive tumors. However, two cancers from patients with relapses after prolonged tamoxifen treatment (>6 months) also had high concentrations of 52K cathepsin D and its precursor. The authors conclude that the concentration of cathepsin D and its precursor in breast cancer cytosol can be increased by short‐term tamoxifen treatment, suggesting that these tumors are estrogen responsive.

Divided Latissimus Dorsi Musculocutaneous Flap for Chest Wall Radionecrosis

A 69-year-old woman with a history of mastectomy and radiotherapy for breast carcinoma demonstrated extensive anterior thoracic wall radionecrosis 22 years later. The latissimus dorsi musculocutaneous flap was chosen to repair two distant ulcerative defects. Chest wall coverage was performed by dividing the muscle into two separate musculocutaneous flaps. Results were particularly promising 6 months after surgery.

The role of diet history and biologic assays in the study of "diet and breast cancer".

Nutritional factors related to breast cancer were investigated by means of a hospital-based case-control study in Milan (Italy) and Montpellier (France). Liposoluble vitamins, cholesterol and triglycerides were measured in blood samples taken from interviewed subjects (319 cases and 344 controls). In addition serum zinc and copper was assessed in the Italian samples and serum fatty acids and malonyl-di-aldehyde in the French samples. A significant difference was found between cases and controls in total fat and cholesterol intake in both populations, and in saturated fatty acid and mono-unsaturated fatty acid consumption in the French samples. No difference emerged in liposoluble vitamin consumption in both popula-tiosn nor in zinc and copper consumption in the Italian samples. A statistically significant higher serum level of cholesterol and plasma level of vitamin E was observed in cases compared to controls in both populations. The difference in plasma vitamin E was confirmed after adjustment on total cholesterol and triglycerides. Similarly, zinc serum level was higher in Italian cases than in Italian controls, while malonyl-di-aldehyde was lower in French cases than French controls. A multivariate analysis was performed after classification of cases and controls according to quantile distribution of controls. Nutrient consumption and relevant blood markers were directly or partially correlated in both populations. All known risk factors plus age, serum total cholesterol and triglycerides were used as covariates. The odds ratio values for the highest quantiles are: Dietary cholesterol, OR = 1.9 (1.1-3.4); total dietary lipids, OR = 1.9 (1.0-3.4); plasma vitamin E, OR = 4.2 (1.9-9.0); serum zinc, OR=12.2 (5.4-27.7); serum malonyl-di-aldehyde, OR=0.56 (0.33-0.97).

Immunohistochemical study of P-glycoprotein distribution in lung cancer

Indirect immunoperoxidase was used to determine the reactivity of C219 (P-glycoCHEK C219, Centocor Diagnostics, Malvern, PA), a monoclonal antibody (Mab) with high affinity for an internal epitope of the P-glycoprotein encoded by the multidrug resistance (MDR1) gene, in 40 surgically resected primary lung tumours. C219 reactivity was qualitatively classified in seven small cell lung cancers (SCLC), 29 non small cell lung cancers (NSCLC), and four carcinoid tumours. Ploidy was analysed by means of static cytometry using a computer-assisted image processor following Feulgen staining of cytologic prints of 32/40 lung tumours. Indirect immunoperoxidase reactivities of Mabs S-L 11.14 and MOC-1 were also studied to characterize the expression of cluster 1 lung cancer antigens and hence to determine among the NSCLC those which expressed the neural cell adhesion molecule (NCAM). Eighteen (45%) lung tumours strongly expressed P-glycoprotein as an immunostaining of many islets of malignant cells or almost all malignant cells. In addition, 8/40 tumours (20%) showed a weak reactivity (few immunostained cells) and 14/40 (35%) no reactivity. There was no difference of reactivity when NSCLC were compared with SCLC. The expression of P-glycoprotein in NSCLC did not vary significantly when the stage of disease was considered. Among the 29 NSCLC, 10 (36%) expressed S-L 11.14 and MOC-1. The NCAM positive NSCLC did not show any difference of P-glycoprotein expression in comparison with NCAM negative ones. Finally, C219 immunoperoxidase reactivity did not significantly differ according to the ploidy status. In conclusion, the internal epitope of the P-glycoprotein encoded by the MDR1 gene is frequently expressed by lung tumours of any histological type. This expression is not higher in Stage III and IV lung cancers in comparison with Stage I and II ones, or in NSCLC in comparison with SCLC either. Thus, the C219 related epitope seems to have a weak implication in the lower chemosensitivity of both advanced stages and NSCLC.