A. Barletta

Heterogeneity of Intratumour Proliferative Activity in Primary Breast Cancer: Biological and Clinical Aspects

The present retrospective study was undertaken to verify whether the extent of intratumour proliferative activity variation or the method of quantifying tumour proliferative activity is related to biological characteristics and clinical outcome in a series of operable node-negative breast cancer patients. For tumour proliferative activity evaluation, the 3H-thymidine autoradiographic assay was used. After incubation of 3-8 samples from different areas of the equatorial section of each tumour for 1 h at 37 degrees C with 3H-thymidine, the following methods were used for evaluation of tumour cell labelling: mean tumour labelling index (LI), the highest labelling value from a specific area (LI-max), and the extent of intratumour labelling variation from several samples (LI-CV). LI-max was related to ER and PgR status, and linearly correlated with LI (c.c. = 0.92, P < 10(-6)) whereas LI-CV was independent of tumour size, grade ER and PgR status, but dependent on the number of tumour samples analysed for each tumour. After 5 years of median follow-up, disease-free survival was only related to tumour size (T1 versus T2: 84 versus 64%, P < 0.04 by log rank analysis) and different LI values (low versus high 3H-Tdr-LI:86 versus 61%, P < 0.03 by log rank analysis). LI-max and LI-CV values were not significantly related to clinical outcome. Cox multivariate analysis confirmed the independent prognostic value of LI and tumour size on disease-free survival.

Randomised Clinical Trial of Adjuvant Chemotherapy in Patients with Node-Negative, Fast-Proliferating Breast Cancer

SummaryA prospective randomised clinical trial in patients with node-negative, fast-proliferating breast cancer was initiated in January 1990 to verify the feasibility and reliability of a 3H-thymidine (3H-Tdr) autoradiographic assay in a prospective and consecutive series of node-negative patients and the therapeutic effect of adjuvant chemotherapy in patients with node-negative breast cancer and high tumour proliferative activity. Node-negative patients with a high 3H-Tdr Labelling Index (3H-Tdr-LI) were randomised to receive either no further treatment or combination chemotherapy consisting of fluorouracil, epirubicin plus cyclophosphamide (FEC) for 6 cycles. The autoradiographic assay was performed in 307 of 317 patients (97%) and was evaluable in 291 of 317 patients (92%). A total of 176 patients with a high 3H-Tdr-LI entered the clinical randomised study: 91 in the FEC arm and 85 in the control arm. Patient groups were fairly well balanced regarding the most important clinical and pathological characteristics. In total, 530 FEC cycles have been administered with an actual dose intensity of 90%. Patients receiving FEC demonstrated leucopenia in 35% of cases, alopecia in 70%, and loss of menses in premenopausal patients in an age-dependent manner. Patients are still being entered into the study.

Metastatic pattern and DNA ploidy in stage IV breast cancer at initial diagnosis: Relation to response and survival

Sixty-nine patients with metastatic breast cancer (MBC) at initial diagnosis were analyzed to verify if metastatic pattern and clinical outcome are related to DNA ploidy determined by flow cytometry (FCM). Characteristics of 55 fully evaluable patients were as follows: median age: 61 years; postmenopausal: 75%; bone-only metastases (BM): 60%; extra osseous-only metastases (EM): 40%. Overall response rates (CR + PR) obtained with different chemotherapies and/or hormonal therapies were 58% and 68% for patients with BM and EM, respectively. Sixty percent of specimens resulted aneuploid, and the mean coefficient of variation of the complete series was 5.1%. In the whole group of patients DNA ploidy of primary tumor did not predict the metastatic pattern and had no influence upon response to treatment, duration of response, time to progression, and overall survival. When analyses were carried out according to metastatic pattern, those patients with BM showed similar results. However, within the group with EM, those with diploid tumors presented a significantly better survival (median 18 vs 13 months, p = .04). FCM-DNA analysis seems to identify a subgroup of patients with poor prognosis constituted by those who had aneuploid primary tumors and metastases to extraosseous sites.

Mammographic aspect, cell kinetics and hormone receptor status of operable breast cancer

To investigate the relationships between the mammographic aspects (according to Broberg), proliferative activity and hormone receptor status, we studied 165 patients subjected to radical mastectomy for operable breast cancer. The highest percentage of estrogen receptor-positive cases was observed in mammograph class I (p < 0.02) while classes III and V were highest in progesterone receptor-positive cases. Proliferative activity (111 cases) was significantly lower in class I and IV with respect to all other classes (p < 0.03 and p < 0.01, respectively). This study suggests that certain mammographic signs can be related to the biological characteristics of breast cancer.

Mammography and morphobiologic characteristics of human breast cancer

Aims A comparative analysis was performed to verify a possible correlation between mammographic features and morphobiologic characteristics of the tumor in a series of 176 invasive primary breast cancer patients. Methods Breast cancers were grouped according to mammographic features as follows: tumor mass with spiculated borders; tumor mass with well-circumscribed borders; tumor with density alteration of parenchyma with no clear borders; a cluster of micro-calcifications as the only sign of tumor presence; tumor without mammographic abnormality. The tumor tissue biologic characteristics investigated were: hormone receptor content, tumor proliferative activity, DNA content and cytohlstologic tumor-grade differentiation. Results Spiculated tumors showed a significantly higher percentage of estrogen-receptorpositive cases with respect to circumscribed tumors, independently of the patients menopausal status. Tumors with only microcalcifications were all from premenopausal patients and showed a significantly higher percentage of progesterone-receptor-positive cases (83 %). Tumor proliferative activity did not significantly differ in the 5 mammographic breast cancer groups; aneuploidy was less frequent in tumors with spiculated borders than in mammographic types (39 % vs 57 %; p = 0.05); percentages of G1-G2-G3 tumors did not differ significantly among the mammographic groups considered. Conclusions Certain relationships between mammographic features and biologic characteristics could be of potential clinical interest and stimulate more detailed studies on this issue.

Different assays for HER-2/neu . Evaluation in breast cancer

To evaluate different methodologic approaches for HER-2/neu analysis, we performed Southern, Northern, Western blot and histochemical assay on 112 samples from 86 primary tumors and 26 synchronous axillary metastatic lymph nodes of patients affected by operable breast cancer. Simultaneous statistical analysis of data obtained with the four methods (31 samples) showed that Western blot detected a higher percentage of alterations than the other assays (Cochran and Victor tests, 0.01 < p < 0.05). The same result was emphasized by pair analysis (McNemar, p < 0.05), which evaluated the assay data two by two. Immunohistochemical evaluations were more in accord with immunoprecipitation data when performed on frozen or Bouin-fixed, paraffin-embedded tissues than on formalin-fixed, paraffin-embedded tissues.

HER-2/neu gene in primary and local metastatic axillary lymph nodes in human breast tumors.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.

Expression of GST-mu transferase in breast cancer patients and healthy controls.

The expression of glutathione S-transferase mu was measured by a qualitative immunoenzymatic assay in the blood samples of 108 women (63 breast cancer patients and 45 healthy controls) in order to analyze the relationships of GST-mu phenotype and smoking habits with tumor characteristics of breast cancer patients, such as tumor extension, nodal status, hormone receptor status and DNA content by flow cytometry. GST-mu was expressed in 53/108 (49%) of cases without any significant difference between healthy or neoplastic subjects, smokers or non-smokers, pre or post-menopausal, younger or older subjects. Moreover, the percentages of the GST-mu phenotype did not differ significantly in patients with different ER and PgR tumor status, tumor extension or nodal status. By contrast, aneuploid DNA tumor content was shown to be significantly associated with GST-mu expression (24% and 76% GST-mu positive, respectively, in diploid and aneuploid cases; p < 0.003). The biological meaning of this association remains to be interpreted.

Nuclear grade and DNA ploidy in stage IV breast cancer with only visceral metastases at initial diagnosis.

Aims and background The presence of early metastases to distant sites in breast cancer patients is an infrequent event whose mechanisms are still not clear. The aim of this study was to evaluate the biologic and clinical role of DNA ploidy and cell nuclear grade of primary tumors in the metastatic process of a series of stage IV previously untreated breast cancer patients with only visceral metastases. Methods DNA flow cytometry analysis on paraffin-embedded material and cell nuclear grading of primary tumors was performed on a series of 50 breast cancer patients with only visceral metastases at the time of initial diagnosis. Results Aneuploidy was found in 28/46 (61%) of evaluable cases and was independent of site of involvement, clinical response, time to progression and overall survival of patients. Of the 46 cases evaluable for nuclear grade, 5 (11%), 16 (35%) and 25 (54%) were classified as G1 (well-differentiated) G2 and G3, respectively. Nuclear grade also was unrelated to response to therapy and overall survival, whereas time to progression was significantly longer in G1-2 than G3 tumors with the logrank test (P<0.03) and multivariate analysis. Conclusions Our results seem to stress the difficulty to individualize different prognostic subsets from a series of breast cancer patients with only visceral metastases at initial diagnosis according to DNA flow cytometry and nuclear grade.