Expression of p27kip1 in breast cancer and its prognostic significance.

p27kip1 is a member of the KIP/CIP family of cyclin-dependent kinase inhibitors and is a negative cell-cycle regulator that is thought to play a role in tumour suppression. Reduced levels of this protein have been observed in a number of human cancers. However, evidence is conflicting as to whether p27kip1 has a role to play in breast cancer, including predicting behaviour and prognosis. The present investigation aimed to provide a definitive study of 830 breast cancer cases with median patient follow-up of 104 months to determine the true prognostic significance, if any. Immunohistochemical analysis of tissue microarrays and three scoring methods were used to assess p27kip1 expression. Univariate analysis showed a significant relationship between reduced p27kip1 expression and increasing tumour grade, nuclear pleomorphism, mitosis, and decreasing tubule formation (all p<0.001). Significant associations between reduced p27, negative oestrogen receptor status, and ductal/no special type tumours were also observed. Survival analysis demonstrated that patients with tumours with high p27kip1 levels had an improved survival compared with those with cancers with low expression. On multivariate analysis, when compared with existing factors, p27kip1 was not, however, an independent prognostic factor. It is concluded that the inverse relationship between p27kip1 levels and histological grade and individual grade components suggests a role for p27kip1 in both cell proliferation and differentiation, but is not clinically useful.

Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres.

Accurate diagnostic assessment of HER-2 is essential for the appropriate application of the humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) to the treatment of patients with metastatic breast cancer. The diagnostic test needs to be applicable to archival, fixed tissue removed at excision, in many cases several years earlier. We compared the assessment of HER-2 by immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH) in 426 breast carcinomas from patients being considered for trastuzumab therapy. The tumours were tested in three reference centres having been sent in from 37 hospitals. Only 2/270 (0.7%) IHC 0/1+ tumours were FISH positive. Six of 102 (5.9%) IHC 3+ tumours were FISH negative. Five of the six had between 1.75 and 2.0 HER-2 gene copies per chromosome 17 and the sixth had multiple copies of chromosome 17. Thirteen per cent of tumours were IHC 2+ and overall 48% of these were FISH positive but this proportion varied markedly between the centres. Sixty IHC-stained slides selected to be enriched with 2+ cases were circulated between the three laboratories and scored. There were 20 cases in which there was some discordance in scoring. Consideration of the FISH score in these cases led to concordance in the designation of positivity/negativity in 19 of these 20 cases. These data support an algorithm in which FISH testing is restricted to IHC 2+ tumours in reference centres. The results may not extrapolate to laboratories with less experience or using different methodologies.

E-cadherin as a prognostic indicator in primary breast cancer.

Epithelial cadherin (E-CD) is a member of the cadherin family of cell adhesion molecules and has been implicated as an invasion suppressor molecule in vitro and in vivo. We analysed 174 breast tumours from the Nottingham/Tenovus Breast Cancer Series immunohistochemically for E-CD expression using the mouse monoclonal antibody HECD-1 (Zymed Laboratories Inc.). In normal epithelial cells E-CD was strongly expressed at cell-cell boundaries. 66% of the breast cancers examined had reduced intensity of E-CD expression with 74% having significant reductions in the proportion of E-CD-positive tumour cells. Using a combined intensity/proportion score, significant associations were found between E-CD expression and tumour type (P

An immunohistochemical examination of the expression of E-cadherin, alpha- and beta/gamma-catenins, and alpha2- and beta1-integrins in invasive breast cancer.

This study examines the expression of the cell-cell adhesion molecules E-cadherin and its associated proteins, the catenins and the matrix-cell adhesion molecules beta1- and alpha2-integrins, in primary invasive breast carcinoma. Expression was assessed immunohistochemically on frozen sections by semi-quantitative scoring of the intensity and proportion of immunoreactivity in 55 cases. Associations with each other and with other histological and prognostic features and survival were sought. There was a significant association between loss of E-cadherin expression and loss of alpha- and beta/gamma-catenin immunostaining. In 20 per cent of cases, membranous immunoreactivity with E-cadherin antibody was absent. Absent cytoplasmic expression of alpha- and beta/gamma-catenins was seen in 24 and 22 per cent of breast cancers, respectively. The intensity of reactivity with E-cadherin showed a significant association with histological grade (p=0.002) and tumour type (p<0.001). Lobular carcinomas frequently showed loss of expression of E-cadherin, as reported elsewhere; loss of catenin expression was also found in these tumours. alpha-catenin intensity also showed a relationship with grade (p=0.008) and with oestrogen receptor (ER) status (p=0.006). beta/gamma-catenin expression was not associated with other known prognostic factors. Forty-nine per cent and 42 per cent of cases showed no membrane immunostaining with beta1- and alpha2-integrin, respectively, and co-ordinated loss of beta1- and alpha2-integrin expression was found. Both beta1- and alpha2-integrin expression were associated with histological grade (p=0.003 and p=0.031, respectively) and beta1 immunoreactivity with tumour type (p=0.010). None of the variables examined showed a statistically significant association with tumour size or lymph node stage, or with overall survival, although a trend was seen (p=0.087) towards poorer survival of patients with tumours with absent or weak expression of beta1-integrin. The expression of these markers is of biological interest, but appears to be of little additional use in predicting clinical behaviour.

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations.

We have assessed the multiple biological variables on breast carcinoma FNA specimens using a Cytoblock technique. The growth fraction (MIB1), oestrogen receptor (ER), progesterone receptor (PR), p53 mutant protein, c-erbB-2, epidermal growth factor receptor (EGFR), NCRC11/epithelial membrane antigen (EMA) and DNA plopidy were examined. Objective quantification using image analysis (CAS 200) was applied as appropriate. Fifty cases were examined in this preliminary study. Excellent correlation between the Cytoblock preparations and parallel tissue sections was seen. Of the cancers, 81% were aneuploid with only 19% diploid in character, but 67% of the carcinomas were of histological grade 3. The mean nuclear area staining with MIB1 was 31.3% and with ER was 26.7%. Twenty-four percent (24.1%) of the nuclear area showed immunoreactivity with PR. Significant EGFR and EMA, respectively. A significant association between histological grade of the resected tumours and both MIB1 (P = 0.04) and EGFR (P = 0.02) expression in the Cytoblock samples was seen. p53 (P = 0.03) and EGFR (P = 0.01) immunoreactivity showed an association with tumour size. EGFR (P = 0.04) immunostaining also showed a relationship with the lymph node status of the patient. The technique is, we believe, a useful one for the assessment of multiple variables on breast cytology specimens; these preliminary data suggest that some of these may be useful in predicting prognosis in breast cancer patients.

Intracellular gastrin in human gastrointestinal tumor cells.

Flow cytometry and immunohistochemical analyses of the human gastric adenocarcinoma cell line MKN45G identified an intracellular peptide recognized by an anti-gastrin-17 (G17) antiserum but not by an anti-cholecystokinin-specific antiserum. Staining was not associated with the parental line MKN45, of which MKN45G is a clonal variant. The MKN45G cell line had elevated in vitro growth in serum-free medium in which the proliferation of MKN45G cells but not MKN45 cells was reduced to 58% of the control value by treatment with a rabbit anti-G17 antiserum. This inhibition of proliferation was reversed by preabsorbing the antiserum with excess G17. Disaggregated primary human gastric and colorectal tumors were screened for gastrin immunoreactivity by flow cytometry, and 6 of 28 colorectal and 8 of 22 gastric tumors had greater than 20% positively staining cells.