Angelika Reiner

p53 protein expression, cell proliferation and steroid hormone receptors in ductal and lobular in situ carcinomas of the breast

p53 and c-erbB-2 expression, and their correlation with cell proliferation and steroid hormone receptors, were investigated in 121 carcinomas, 23 lobular in situ carcinomas (LCIS), 74 intraductal carcinomas (DCIS) and 24 minimal invasive carcinomas. DCIS were classified according to the EORTC classification. All markers were measured immunohistochemically on paraffin sections. None of the LCIS, 9 DCIS and 9 minimal invasive cancers showed nuclear positivity for p53. A strong association between histological type and p53 expression was found. Proliferation rates correlated with p53 expression. c-erbB-2 positivity was found in 1 LCIS, 27 DCIS and 12 minimal invasive cancers. There was a significant correlation between p53 expression and c-erbB-2. Both parameters were associated with high proliferation rate and negativity for steroid hormone receptor status. Nuclear pleomorphism could become a comparable prognostic marker in DCIS as it is for infiltrating carcinomas.

Histopathologic characterization of human breast cancer in correlation with estrogen receptor status. A comparison of immunocytochemical and biochemical analysis.

A detailed histopathologic analysis of 399 primary breast carcinomas was performed, and several morphologic features were correlated with the estrogen receptor (ER) status. In all cases ER status was determined immunocytochemically by estrogen receptor immunocytochemical assay (ER‐ICA). In 359 carcinomas ER status was also biochemically determined. Invasive lobular, mucoid, and tubular carcinomas rather than ductal carcinomas were ER‐positive more frequently in ER‐ICA. Medullary and papillary carcinomas had corresponding lower or higher ER positivity, respectively, by both methods. The correlation of histologic grade and its single factors with ER status was statistically significant by both methods. Lymphocytic reaction to tumor showed a significant inverse relationship to ER status by both methods. A statistically significant higher number of ER‐positive carcinomas in ER‐ICA and dextran‐coated charcoal assay (DCC) occurred when elastic tissue was present. Different associations were found between stromal content, tumor diameter, and ER status in DCC and ER‐ICA, respectively.

Quality assurance for detection of estrogen and progesterone receptors by immunohistochemistry in Austrian pathology laboratories.

Abstract.Steroid hormone receptors are important prognostic and predictive factors in breast carcinomas. Thus their determination is of essential importance. The aims of this study were to assess the quality of the immunohistochemical assays, and to assess the interlaboratory and interobserver variability performed by different laboratories in Austria. Ten unstained slides for interlaboratory variability evaluation and ten immunohistochemically prestained slides for interobserver variability evaluation from breast carcinomas known to show different degrees of steroid hormone receptor expressions were sent to 32 surgical pathology laboratories in Austria (participation rate 97%). The participants were requested to perform their in-house immunohistochemistry (IHC) technique for estrogen receptors (ERs) and progesterone receptors (PRs) on the unstained slides. All slides were evaluated by estimating percentage and intensity of stained nuclei semiquantitatively. From these data the Reiner, Remmele and the Allred scores were calculated. A less than 10% cut-off level was chosen as threshold for positive cases. Regarding the series of prestained slides, both sensitivity and specificity were very high (>96.88%); false-positive and -negative rates were low (<3.31%). Interobserver variability showed moderate multirater kappa values concerning the ER (Reiner score: kappa=0.57) and PR scores (Reiner score: kappa=0.53). The agreement among observers was better for negative cases than positive cases. In-house slides representing interlaboratory variability showed fair to moderate kappa values concerning the ER and PR scores (kappa for ER Reiner score=0.41; PR=0.32). In this slide series, sensitivity and specificity were high (>82.2%) and false-positive or -negative rates were low in ER cases (<3.03) and moderately low in PR cases (17.46%). These results demonstrate that variability is higher when participants use their own staining method. In more detailed analysis, the automated IHC techniques showed an advantage over manual techniques concerning interlaboratory variability. There exists no difference in reproducibility with respect to scoring systems for steroid hormone receptor estimation.

It is possible to omit postoperative irradiation in a highly selected group of elderly breast cancer patients

The purpose of this study was the evaluation of the necessity of routinely applied postoperative radiotherapy in a highly selected patient-group after breast conserving surgery. Between 1983 and May 1994, 356 women over 60 years of age with Stage I or II breast cancer were treated by quadrantectomy and axillary dissection followed by either adjuvant irradiation or no radiotherapy. We have analysed our data retrospectively to investigate whether irradiation has any benefit in elderly patients with respect to locoregional recurrence rates. After a median follow-up of 60 months the multivariate model revealed lymph node status (p=0.002) as highly significant with regard to local recurrence free survival. We were not able to identify a positive effect of adjuvant irradiation in patients with negative lymph nodes and positive receptor status: both patient groups with or without irradiation had similar locoregional recurrence rates of 3%. In a subgroup of patients who were lymph node negative, receptor positive, and received adjuvant tamoxifen therapy, the local recurrence rates were as low as 2% in both groups. Concerning these results it may be possible to avoid the morbidity and potential psychological side effects of radiotherapy in breast cancer patients over 60 years of age treated by breast conserving surgery (T1, N0, positive hormone receptor, adjuvant tamoxifen) without increasing risk of locoregional recurrence. These data have to be confirmed in a prospectively randomized fashion.

Thymidine labeling index and Ki-67 growth fraction in breast cancer: Comparison and correlation with prognosis

SummaryIn situ determination of proliferative activity was performed on 184 consecutive primary invasive breast cancers. Methods used were monoclonal antibody Ki-67 in immunohistochemistry and thymidine labeling index. Tumor proliferation correlated between both methods (p = 0.0001). For thymidine labeling index and Ki-67, respectively, significant correlations existed with histologic tumour grade and steroid hormone receptors (Tumor grade: TLIp = 0.0001; Ki-67 p = 0.0001. ER-ICA: TLI = 0.0001; Ki-67 p = 0.014. PgR-ICA: TLIp = 0.0001; Ki-67 p = 0.0008).For thymidine labeling index a significant correlation was demonstrated for overall survival (p = 0.001) and recurrence free survival (p = 0.01). No statistical significance was observed for clinical outcome and Ki-67 (overall survival p = 0.18; recurrence free survival p = 0.1). None of the factors, TLI or Ki-67, was an independent prognostic factor as demonstrated by multivariate analysis.

Expression of epidermal growth factor receptors (EGFR) on breast carcinomas in relation to growth fractions, estrogen receptor status and morphological criteria. An immunohistochemical study.

Epidermal growth factor receptor (EGFR) expression was determined on 88 primary breast carcinomas immunohistochemically. These results were compared with growth fractions (Ki-67 immunoreactivity and Transferrin receptor (TrfR) expression), histologic tumor type, tumor grading, axillary lymph node status and estrogen receptor (ER) status. 60.2% were EGFR positive. Cytomorphology predominantly revealed a fine granular staining pattern. Sometimes a concentrated immunoreaction on the intercellular and basal oriented cell poles could be observed. EGFR expression in relation to growth fractions, grading, tumor diameter and lymph node status showed no correlation, suggesting that EGFR status seems to be independent to tumor growth and morphological prognostic parameters. ER status revealed an inverse correlation to EGFR expression (Kendalls tau: -0.22804, p = 0.012). In this context, it stands to reason to investigate further how far determination of EGFR expression justifies the existence of different subpopulations of breast cancer cells with respect to prognostic value.

Interphase cytogenetics reveals a high incidence of aneuploidy and intra-tumour heterogeneity in breast cancer

The occurrence of aberrations involving chromosomes 11 and 17 in malignant tissues of breast cancer patients has not yet been studied systematically. Using fluorescence in situ hybridisation (FISH) with centromere-specific probes, we determined chromosome 11 and 17 status in interphase nuclei from primary and/or metastatic breast cancer cells. In all cancerous specimens obtained from 30 patients, FISH identified cells with clonal chromosomal abnormalities, with aneuploidy rates ranging from 6% to 92% (median 59%). There was a gain of centromeric signals for chromosome 11, most likely corresponding to hyperploidy; aberrations of chromosome 17 in specimens from 26 patients (87%) were hyperploid as well; however, four cases (13%) showed loss of chromosome 17 centromeres. All specimens contained heterogeneous aneuploid cell populations with excessive gain of signals in some cases. The pattern of aneuploidy did not appear to correlate with tumour grade/stage and was comparable in primary tumours and corresponding metastatic axillary lymph nodes, indicative of genetic instability early in tumour development. Screening with a panel of FISH probes may lead to enhanced sensitivity and specificity in detecting malignant cells, as demonstrated in this study with effusions which could not be conclusively interpreted as being malignant by cytological criteria.

Coexpression of HER-2/neu and p53 is associated with a shorter disease-free survival in node-positive breast cancer patients

Breast cancer tissue was examined for overexpression of HER-2/neu and p53 oncogene proteins. Samples from 105 breast cancer patients were investigated by Western-blot analysis and their relationship to other established markers and clinical outcome was examined. In 21.0% of the cases HER-2/neu was overexpressed, and in 46.7% the p53 protein level was increased. Expression of these two oncogene products was closely correlated. Overexpression of both oncogenes was associated with larger tumour size and negative hormone receptor. The percentage of HER-2/neu and p53 overexpression was higher in node-positive patients, although statistical evaluation was not significant. While overexpression of HER-2/neu as well as p53 in node-positive patients was associated insignificantly with shorter disease-free survival, a significant difference could be documented when the disease-free survival of patients with overexpression of both oncogene proteins was compared to that of patients with no overexpression.

Prognostic Significance of Mutations in the p53 Gene, Particularly in the Zinc-binding Domains, in Lymph Node- and Steroid Receptor Positive Breast Cancer Patients

The aim of our study was to evaluate if p53 mutations, especially those in the L2/L3 domains of the p53 gene, add prognostic information for node-positive and steroid receptor positive breast cancer patients. Two hundred and five tumour samples from a randomised clinical trial of 596 lymph node- and steroid receptor positive breast cancer patients were included. All patients had been randomly allocated to receive 20 mg of adjuvant tamoxifen (TAM) daily for 2 years or TAM plus one cycle of low-dose, short-term chemotherapy. For detection of p53 mutations we used in vitro amplification by polymerase chain reaction and consecutively performed temperature gradient gel electrophoresis (PCR-TGGE) and direct sequencing. We found p53 mutations in 42/205 (20%) cases: 16/42 (38%) p53 mutations occurred within the L2/L3 domains of the p53 gene, and 26/42 (62%) outside the L2/L3 domains. p53 mutation served as a statistically significant parameter in predicting disease-free survival in univariate (P = 0.02) and multivariate (P = 0.009) analysis. For overall survival, no significant differences were observed. Patients with tumours that had p53 mutations within the L2/L3 domains of the gene showed no significant difference to those with mutations outside the L2/L3 domains for disease-free survival. For overall survival, mutations in the L2/L3 domains showed a marginally significant difference (P = 0.05) in multivariate analysis, but not in univariate analysis (P = 0.13). We conclude that mutation in the L2/L3 domains of the p53 gene is not an independent prognostic indicator of disease outcome for patients suffering from breast cancer with lymph node metastases and positive steroid receptors.

Transferrin receptor (TrfR) expression in breast carcinoma and its possible relationship to prognosis: an immunohistochemical study

TrfR, a primitive membrane protein was demonstrated by immunohistochemistry in 87,6% of 105 cases of breast carcinoma, predominantly on the cell surface and in a strong and rather uniform pattern. Sporadic staining in a patchy fashion was observed. No difference between individual tumour types was seen, neither in cytomorphological staining pattern nor in staining intensity. Exceptionally, mucoid carcinomas showed weaker intensity for receptor expression. Because of the heterogenous expression of TrfR within most of the tumours the extent of staining reaction was determined by semiquantitative grading (low, moderate, high). These results were compared with grade of anaplasia, tumour staging and nodal status of the axilla. The extent of immunoreactivity revealed significant correlation with grade of anaplasia, whereas no correlation was found with staging and status of axillary lymph nodes. Tumours with higher degree of malignancy (GII–GIII) showed a higher extent of staining. The presence of TrfR in a high degree of expression thus implies some prognostic value. Its quantitative determination can provide kinetic data on the neoplasm.