Jean Grenier

Pro-gastrin-releasing peptide, neuron specific enolase and chromogranin A as serum markers of small cell lung cancer

BACKGROUND Small cell lung cancer (SCLC) yields neuroendocrine properties. Pro-gastrin-releasing peptide (ProGRP), neuron specific enolase (NSE) and chromogranin A (CGA) are therefore putative serum markers in this disease. AIM To assess any difference in the sensitivity-specificity relationship between these neuroendocrine markers regarding various control populations and disease extent. METHOD A total of 146 patients were prospectively assessed clinically and biologically. Serum marker titrations were performed using commercial immunoradiometric assays (NSE, CGA) or ELISA (ProGRP). Areas under receiver operating characteristic curves (AUC-ROC) were calculated in order to assess the sensitivity-specificity relationship of each marker and to compare marker accuracy. Maximum Youden indices were used to determine marker thresholds able to produce the best overall diagnostic information. RESULTS Assessing the sensitivity in the SCLC population and the specificity in benign lung disease, ProGRP demonstrated the best sensitivity relationship in as much as its AUC-ROC was significantly greater than the ones calculated using NSE and CGA (respective values, 0.95, 0.89, 0.70; two-tailed Z-test <0.05). The ProGRP threshold value, which offered the best sensitivity-specificity relationship was 53 pg/ml corresponding to a 0.80 sensitivity and a 0.96 specificity. In addition, when specificity was assessed in NSCLC and again the sensitivity in the whole SCLC population, ProGRP continued to demonstrate a greater AUC-ROC in comparison with other markers. Using the 53 pg/ml threshold the specificity of this marker was excellent with no false positives in NSCLC. On the other hand, none of the markers were able to discriminate limited from extensive SCLC as suggested by the fact that AUC-ROC, constructed when sensitivity was defined as a positive test in extensive disease and specificity as a true negative test in limited disease, did not reach the upper left octant (AUC 0.65, 0.71 and 0.63 for ProGRP, NSE and CGA, respectively). CONCLUSION ProGRP yields the best sensitivity-specificity feature in SCLC, a result deserving further studies designed to evaluate the clinical applicability of this marker.

Progesterone Receptor Quantification as a Strong Prognostic Determinant in Postmenopausal Breast Cancer Women under Tamoxifen Therapy

There is now an emerging body of evidence that shows there is a relationship between the survival of breast cancer patients and the expression level of steroid receptors. The aim of this study was to determine the relationship existing between estrogen receptors (ER) and progesterone receptors (PR) cytosolic content and the prognosis of postmenopausal breast cancer women under tamoxifen therapy. Two hundred and nineteen postmenopausal patients, without neoadjuvant chemotherapy and treated postoperatively with tamoxifen for at least 2 years, were followed up in our Cancer Center. We used flexible regression modeling and log likelihood methods for determining optimum cut-off values for steroid receptors, which allows the separation of patients into significantly different categories in term of survival. For PR, 3 categories were defined (category 1: PR < 10, category 2: 10 ≤ PR < 60 and category 3: PR ≥60 fmol/mg P). Univariate analysis at 8 years indicated that significant differences in event-free survival (EFS) were found for tumor size (T) (p = 0.005), lymph node status (N) (p = 0.003), histological Scarff, Bloom and Richardson grade (p = 0.003), ER values divided into 5 categories (p = 0.02) and PR values divided into 3 categories (p = 1 · 10−5). Eight-year EFS rate for the 3 PR categories, adjusted for N, were 39, 66 and 81%, respectively. Multivariate Cox analysis indicated that only T, N and PR values were significant variables for EFS. Patients with PR values ≥60 present significantly greater EFS rates than patients with PR < 60 (p < 0.001). Our results show that the PR level in ER positive postmenopausal women is a strong prognostic marker in postmenopausal breast cancer women under tamoxifen therapy.

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.

Neoadjuvant Percutaneous 4-Hydroxytamoxifen Decreases Breast Tumoral Cell Proliferation: A Prospective Controlled Randomized Study Comparing Three Doses of 4-Hydroxytamoxifen Gel to Oral Tamoxifen

PURPOSE Two chemoprevention randomized studies using tamoxifen showed drug efficacy; however, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain a problem. 4 hydroxytamoxifen (4-OHT) is a very active metabolite of tamoxifen. This randomized study was designed to analyze if 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen. PATIENTS AND METHODS Fifty-five postmenopausal women with an invasive estrogen receptor-positive breast cancer were randomly assigned to receive (for 2 to 3 weeks) either 4-OHT gel (0.5, 1, or 2 mg/d) or oral tamoxifen (20 mg/d) or no treatment. Response was evaluated using proliferation markers (Ki-67, proliferating cell nuclear antigen) and apoptosis markers in tissue samples obtained by Tru-cut biopsy before treatment, and at surgery after treatment. RESULTS Administration of 4-OHT gel resulted in reductions in tumor tissue proliferation indexes (Ki-67 and PCNA), with approximate equivalence between the 1.0 mg/d or 2.0 mg/d 4-OHT dose, and oral tamoxifen, but had no effect on apoptotic markers. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the gel groups. No dose-related pattern was shown for estrogen or progesterone receptor levels, and topical 4-OHT gel appeared to be generally well tolerated. Hot flushes are as common in the two higher gel doses as with tamoxifen. CONCLUSION Percutaneous 4-OHT gel has a local impact on tumor proliferation. It could be tested in future prospective trials of chemoprevention or ductal carcinoma in situ adjuvant hormonotherapy.

Tumor progression and oxidant-antioxidant status

Severity of prognosis factors in breast cancer cases was found to be associated with an increase in plasma vitamin E and a decrease in plasma malondialdehyde (peroxidability index). The first aim of this study was to determine whether this association is also present in other cancers. Measurements were taken before therapy on 129 patients with various carcinomas. Cholesterol was also investigated, as vitamin E is closely related to this analyte. Patients were classified by tumor size (T < or = 5 cm and T > 5 cm) and by invasion status, assessed by the presence of nodes and/or metastasis. The vitamin E/total cholesterol concentration ratio was higher and the cholesterol and malondialdehyde concentrations were significantly lower in the plasma of patients with large tumors or in whom nodes and/or metastasis were present, whatever the site. The multivariate analysis performed to measure the association of these analyte concentrations with tumor progression showed that the presence of nodes and/or metastases was inversely associated with a low vitamin E/total cholesterol ratio (OR, 0.5; CI, 0.3-1.1) and, directly associated with low plasma concentrations of cholesterol and malondialdehyde (OR, 3.0; CI, 1.3-6.8 and OR, 2.8; CI, 1.2-6.7 respectively). The same types of associations were identified with large tumors, but were less strong. Together these findings supported an alteration of lipid parameters related to the oxidant-antioxidant status in cancer patients. This alteration appears to be associated with tumor growth and progression in patients with various cancer sites.

Oxidant-antioxidant status in relation to survival among breast cancer patients.

The role of plasma oxidant‐antioxidant status in survival after breast cancer surgery was investigated in a cohort of patients (n = 363) hospitalized in Southern France between 1989 and 1992. The median follow‐up was 8 years after surgery for histologically confirmed breast cancer. Plasma analyses were performed after diagnosis and before surgery and adjuvant therapy. We found an inverse relationship between plasma lipoperoxides (MDA) and tumor size at diagnosis, together with higher lipoperoxide levels in node‐negative tumors than in node‐positive ones (TNM). The longitudinal approach revealed an increased risk of recurrence for patients with plasma lipoperoxides in the highest tertile of the sample (RR = 2.1, 95%CI 1.1–4.0). In addition, the risk of recurrence increased (RR = 1.7, 95%CI 1.0–3.0), after adjustment for the known prognostic factors (TNM), for patients with plasma lipid‐adjusted vitamin E levels of over 22 μmol/l. The risk of breast cancer death was twice as great for patients with plasma lipid‐adjusted vitamin E levels above this value. Excesses of plasma lipoperoxides and vitamin E appear to be factors in poor prognosis for breast cancer‐specific survival (OVS) and disease‐free survival (DFS), respectively, independent of tumor characteristics at diagnosis. Several hypotheses are advanced to explain the possible role of plasma vitamin E as a factor in poor prognosis for survival.

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.

A Prospective Prognostic Study of the Hormonal Milieu at the Time of Surgery in Premenopausal Breast Carcinoma

Despite numerous studies, the influence of timing at surgery in relation to the menstrual cycle on the prognosis of breast carcinoma is still controversial. Most studies are retrospective, and the reliability of the menstrual history data is limited by the lack of hormonal assessment at the time of surgery. The authors prospectively studied the influence of the menstrual cycle phase as determined by circulating hormones at the time of surgery on the outcome of breast carcinoma.

Analysis of the potential contribution of estrogen receptor (ER) β in ER cytosolic assay of breast cancer

Estrogen receptor (ER) content is the most useful parameter for predicting hormone response therapy in breast cancer. Assays available for detecting ER in breast tumor cytosol are ligand‐binding assay (LBA), which detects both ERα and ERβ, and the enzymatic immunoassay (EIA), in which monoclonal antibodies are directed against ERα. As shown in several studies, the 2 assays correlate and both are used routinely. However, some discrepancies between the 2 assays were found and explanations remain controversial. We evaluated ERα and ERβ mRNA coexpression in breast tumors in order to study whether the presence of ERβ could account for differences between LBA and EIA in the determination of ER protein level. Using HeLa cell lines transfected with either ERα or ERβ, we confirmed that EIA, using H222 and D547 monoclonal antibodies, recognizes only ERα expression, whereas LBA detects both isoforms. In 119 breast tumor cytosols, the correlation between ER‐EIA and ER‐LBA was high (r = 0.72), although some discrepancies were found. When analyzing ER mRNA expression of samples with higher LBA values, no overexpression of ERβ mRNA relatively to ERα mRNA were observed. There was a difference in ERβ/ERα ratio between ER‐negative and ER‐positive samples, with a 10‐fold increased median ratio in ER‐negative samples (p = 0.01). We thus confirmed that the major form of ER in breast cancer is the ERα at both the protein and mRNA levels. Moreover, our data do not support the hypothesis that ERβ expression could explain differences between LBA and EIA in the determination of ER protein level.

NASBA: a novel approach to assess hormonal receptors and ERBB2 status in breast cancer.

Abstract In human breast cancer, estrogen receptor-α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERα status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.