BRCA1 dysfunction in sporadic basal-like breast cancer.

Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age- and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P=0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P=0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (P<0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (P<0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer.

Nuclear expression of the c-erbB-4/HER-4 growth factor receptor in invasive breast cancers.

The prevalence and sites of expression of the c-erbB-4 receptor have been determined by immunocytochemical staining in a series of 178 human breast cancers. Most tumors displayed cytoplasmic staining of variable intensity. When compared with adjacent normal tissue, 32 cases (18%) showed lower than normal expression, and 13 (7%) showed greater than normal expression. Nuclear immunoreactivity, confirmed by two different antibodies, was present in 87 cancers (49%) but was found in normal adjacent breast epithelial cells in <5% of cases. There were no significant associations with cytoplasmic or membrane immunoreactivity, but cases showing nuclear expression in >25% of cells were associated with good histological grade, epidermal growth factor receptor expression, c-erbB-3 positivity, cripto, amphiregulin, and transforming growth factor-alpha overexpression.

A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells.

The helix-loop-helix protein Id-1 inhibits the activity of basic helix-loop-helix transcription factors, and is an important regulator of cell growth and tissue-specific differentiation. We have shown (P. Y. Desprez et al., Mol. Cell. Biol., 18: 4577-4588, 1998) that ectopic expression of Id-1 inhibits differentiation and stimulates the proliferation and invasiveness of mouse mammary epithelial cells, and that there is a correlation between the levels of Id-1 protein and the aggressiveness of several human breast cancer cell lines. Here, we show that aggressive and metastatic breast cancer cells express high levels of Id-1 mRNA because of a loss of serum-dependent regulation that is mediated by a 2.2-kb region of the human Id-1 promoter. Three lines of evidence suggest that unregulated Id-1 expression may be an important regulator of the aggressive phenotype of a subset of human breast cancer cells: (a) a constitutively expressed Id-1 cDNA, when introduced into a nonaggressive breast cancer cell line (T47D), conferred a more aggressive phenotype, as measured by growth and invasiveness; (b) Id-1 was an important mediator of the effects of sex steroid hormones on T47D cell proliferation. Estrogen stimulated proliferation and induced Id-1 expression, whereas progesterone inhibited proliferation and repressed Id-1 expression. Progesterone repressed Id-1 expression, at least in part by repressing transcription. Most importantly, an antisense oligonucleotide that reduced Id-1 protein levels reduced the ability of estrogen to stimulate cell proliferation, whereas constitutive Id-1 expression rendered cells refractory to growth inhibition by progesterone; and (c) using a limited number of breast cancer biopsies, we showed that Id-1 was more frequently expressed in infiltrating carcinomas compared with ductal carcinomas in situ. Our results suggest that Id-1 can control the malignant progression of breast cancer cells, particularly that mediated by sex steroid hormones. Moreover, Id-1 has the potential to serve as a marker for aggressive breast tumors.

The interaction of oestrogen receptor status and pathological features with adjuvant treatment in relation to survival in patients with operable breast cancer: a retrospective study of 2660 patients.

The oestrogen receptor (ER) status of 2660 patients with primary breast cancer has been related to the effect of different adjuvant systemic therapies on survival. However, as patients in the various treatment groups also had different prognostic features comparison between treatments was difficult. Over 90% of patients receiving tamoxifen (Tam) were postmenopausal compared with <20% of those receiving chemotherapy (CT). The latter had more positive nodes (85% vs 54%) and grade III tumours (54% vs 30%) than the Tam group. The combined CT and Tam group had similar characteristics to the CT alone group. The current reported increase in the proportion of women with ER+ tumours is explained by immunohistochemical analysis of ER and screening programmes. ER status was unrelated to survival in patients with small, low grade, node-negative tumours which was no different from that expected for age-matched women taken from the general population. The value of adjuvant treatment in these patients is therefore questionable. In those given any adjuvant treatment, survival of women with ER+ tumours was prolonged, with the greatest effect being seen in those receiving Tam. Patients with ER- tumours benefited from CT but the addition of Tam to CT improved survival only in those with ER+ tumours. ER status is now established as a major predictive factor for treatment selection in primary disease. Studies of prognostic and predictive markers may be invalidated by use of adjuvant therapy and selection criteria for different treatments. Survival will be influenced by both tumour biology and therapy. This important consideration must be remembered when analysing new markers, particularly in small studies.

A comparative immunohistochemical study of mammary and extramammary Paget's disease and superficial spreading melanoma, with particular emphasis on melanocytic markers.

A comparative immunohistochemical study was performed on Paget's disease of the nipple (PDN), extramammary Paget's disease (EMPD) and cutaneous superficial spreading melanoma (SSM) using antibodies to S100, NK1-C3 and HMB45, cytokeratin (CAM 5.2) and c-erb B2 oncoprotein (21N). Conventional histochemical stains for intracytoplasmic mucin and melanin were also done. Of the 20 cases of PDN, positivity was seen in 12 with S100, 16 with NK1-C3, none with HMB45, 20 with CAM 5.2 and 19 with 21N. All 5 cases of EMPD were CAM 5.2 positive and HMB45, S100 and 21N negative. Three EMPD were NK1-C3 positive. All 10 cases of SSM were S100, NK1-C3 and HMB45 positive and all were CAM5.2 and 21N negative. Mucin was demonstrable in 11 cases of PDN and all of EMPD but none of SSM. Melanin was seen in 2 PDN, 3 EMPD and all SSM cases. Identification of mucin and melanin, therefore, proved an unreliable means of distinguishing these diseases. Immunohistochemistry for cytokeratin and HMB45 appear to be the most specific markers in differentiating Paget's disease and SSM. Antibodies to c-erb B2 may also be valuable in this situation.

Oestrogen and progesterone receptor expression in mammary fibromatosis.

AIMS: To investigate the oestrogen and progesterone receptor concentrations expressed on mammary fibromatoses to determine their responsiveness to oestrogenic stimuli. METHODS: Six mammary fibromatoses were examined using immunohistochemistry for the presence of oestrogen and progesterone receptors using antibodies against the receptor proteins. Enzyme immunoassays (EIAs) using the same antibodies were also performed in four patients. Immunohistochemical staining for pS2 protein was also carried out as a measure of functional oestrogen receptors. RESULTS: Neither receptor nor pS2 protein was expressed using immunohistochemistry. Very low concentrations of both oestrogen and progesterone receptors were shown by EIA. CONCLUSION: These results indicate the absence of clinically important concentrations of oestrogen and progesterone receptors in breast fibromatoses and suggest that treatment directed against oestrogen is unlikely to be beneficial.

Comparison of three cell cycle associated antigens as markers of proliferative activity and prognosis in breast carcinoma.

The staining patterns obtained with two antibodies against proliferating cell nuclear antigen (PC10 and 19A2) and another cell cycle associated antibody (KiS1) were compared with each other and with a number of established prognostic markers of breast carcinoma. Although PC10 and 19A2 staining patterns were similar, only the latter was significantly associated with KiS1 antibody staining. These findings suggest that the two PCNA antibodies detect different epitopes. KiS1 was the only antibody to show an association with S phase fraction measured by flow cytometry (p < 0.001). It was also associated with histological grade (p = 0.003), oestrogen receptors (p = 0.045), and DNA index (p = 0.007). PC10 showed no association with any of the markers of prognosis, while 19A2 was associated with histological grade (p = 0.017) and oestrogen receptors (p = 0.043). The two PCNA antibodies do not seem to be of value in measuring proliferative activity nor do they seem to be associated with established markers of prognosis in breast cancer.

Breast cancer--from clinic to laboratory.

Breast cancer remains the most common cancer in women in the western world despite many years of intensive study. This update reviews some current research and its application to the management of patients with breast carcinoma. Specific topics include implementation of oestrogen-receptor immunohistochemistry in laboratories, the use of established and potential prognostic markers to predict clinical outcome and response to treatment, the Breast Screening Programme, and the current management of patients at different stages of the disease.

The challenges of integrating molecular imaging into the optimization of cancer therapy.

We review novel, in vivo and tissue-based imaging technologies that monitor and optimize cancer therapeutics. Recent advances in cancer treatment centre around the development of targeted therapies and personalisation of treatment regimes to individual tumour characteristics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response must address spatial heterogeneity of cancer within the body. A combination of different imaging modalities should be used to relate the effect of the drug to dosing regimen or effective drug concentration at the local site of action. Molecular imaging provides a functional and dynamic read-out of cancer therapeutics, from nanometre to whole body scale. At the whole body scale, an increase in the sensitivity and specificity of the imaging probe is required to localise (micro)metastatic foci and/or residual disease that are currently below the limit of detection. The use of image-guided endoscopic biopsy can produce tumour cells or tissues for nanoscopic analysis in a relatively patient-compliant manner, thereby linking clinical imaging to a more precise assessment of molecular mechanisms. This multimodality imaging approach (in combination with genetics/genomic information) could be used to bridge the gap between our knowledge of mechanisms underlying the processes of metastasis, tumour dormancy and routine clinical practice. Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing an early read-out of response or resistance.

Different patterns of inflammation and prognosis in invasive carcinoma of the breast.

AIM: Inflammation in carcinoma of the breast may represent an immune response to the tumour, but there is evidence that this response is impaired. Inflammation may also stimulate tumour growth by releasing proteolytic enzymes and angiogenic factors. Prognostic studies have produced conflicting results, but most investigators have not evaluated the different patterns of inflammation. The aim of this study was to test the hypothesis that moderate or marked diffuse inflammation is associated with a better prognosis. We also tested the 'danger model', which suggests that necrosis is necessary for an effective immune response. METHODS AND RESULTS: On multivariate analysis of women with stage 1 and 2 tumours (n = 679, median follow-up of 9.8 years), survival was independently associated with diffuse inflammation (relative risk 0.43, 95% confidence interval 0.24, 0.77, P =0.005) in addition to histological grade, axillary lymph node status, tumour size and oestrogen receptor status. The presence or absence of tumour necrosis did not have a clear effect on the relationship between survival and diffuse inflammation. CONCLUSIONS: Moderate or marked diffuse inflammation in breast cancer is associated with a better prognosis, suggesting that the immune effects of the inflammation predominate over the protumour effects.

Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast.

AIMS: Cytokeratin (CK) 14, a myoepithelial marker, is also expressed in a proportion of breast carcinomas. There is evidence that these tumours show a differing metastatic pattern and clinical outcome from other invasive ductal carcinomas (IDCs) and may need different management. Currently, they are not identified in routine practice and no morphological guidelines exist to aid their identification. The aim of this study was to analyse the histological features of CK14+ IDC. METHODS AND RESULTS: A detailed histological review of 453 grade 3 IDCs revealed 88 (19.4%) that expressed CK14. Assessment was made independently by two pathologists using a standardized 'tick-box' proforma covering grade, architectural and cytological features. The results were analysed using logistic regression to identify features that predicted for basal phenotype. Concordance between the two pathologists was fair to good for most parameters (kappa 0.4-0.6). On multiple logistic regression, the basal phenotype was highly significantly associated with the presence of a central scar (P = 0.005), tumour necrosis (P < 0.0001), presence of spindle cells (P = 0.006) or squamous metaplasia (P < 0.0001), high total mitotic count (> 40 per 10 high-power field) (P = 0.0002) and high nuclear-cytoplasmic ratio (P = 0.0002). CONCLUSIONS: Specific morphological features are strongly associated with basal-like breast carcinoma. These could be used in routine diagnostic practice to identify this important subset of tumours.

Cyclin D1 in breast cancer.

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over-expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c-erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over-expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over-expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.

S100 protein in human mammary tissue--immunoreactivity in breast carcinoma, including Paget's disease of the nipple, and value as a marker of myoepithelial cells.

The expression of S100 protein, as assessed by immunohistochemistry, has been evaluated in 101 mammary carcinomas of various histological types, including Paget's disease of the nipple. S100 immunoreactivity was seen in 44 of 101 primary carcinomas, including in situ lesions. It was present in all histological types, with the exception of mucoid carcinoma. In the 33 cases with associated Paget's disease of the nipple, S100 expression was seen in the Paget's cells in six cases. S100 immunoreactivity has been suggested as a marker of myoepithelial cells, but in our hands staining of these cells is less consistent using the S100 antibody than with antibodies to actin. Furthermore, S100 protein is also expressed by some luminal epithelial cells. Therefore, in contrast to actin immunoreactivity, S100 immunoreactivity is not a reliable means of differentiating between luminal epithelial and myoepithelial cells. The possibility that staining with antibody to S100 protein may be affected by methods of fixation and immunohistochemical technique is discussed.

Cyclin-dependent kinase inhibitor p27Kip1 expression and interaction with other cell cycle-associated proteins in mammary carcinoma.

p27, cyclin D1, and retinoblastoma (Rb) protein have been demonstrated using immunohistochemistry in 189 cases of primary breast carcinoma with long-term follow-up. There was a statistically significant association between the expression of p27 and both cyclin D1 and the retinoblastoma gene product (pRb), corresponding to their close interactions in regulating the G1/S transition in the cell cycle. Low levels of p27 were seen in high-grade, rapidly proliferating, oestrogen receptor-negative tumours. In univariate analysis, low p27 expression was associated with a reduced relapse-free and overall survival. In multivariate analysis, p27 was not an independent predictor of survival when either histological grade or proliferative activity (S-phase fraction) was included in the model. When the combined expression of p27 and cyclin D1 was related to survival, patients with high levels of p27, regardless of their cyclin D1 status, did well, whilst those with low p27 had a poor outcome. The only exception, in the latter group, was patients with tumours expressing high levels of cyclin D1, who did as well as the high p27 group. We have shown that in clinical material p27 expression is associated with proliferative activity and while univariate analysis shows it to be a significant indicator of prognosis, this significance is lost in multivariate analysis when traditional prognostic factors are included in the model. The interest in p27 expression in mammary carcinoma lies in its behaviour when examined in combination with other G1 cell cycle regulators.

Problems with p53 immunohistochemical staining: the effect of fixation and variation in the methods of evaluation.

The availability of antibodies which recognise p53 protein in paraffin-embedded tissue has created the opportunity to use immunohistochemistry to study the expression of p53 in a wide variety of clinical material. In this paper we have investigated the relationship between the type of fixative and the pattern of p53 staining in mammary carcinoma. Optimal results were obtained from breast tissue fixed in phenol formol saline, methacarn or cold formol saline with positive staining for stabilised p53 protein occurring in 69/95 (73%) cases studied. Care must be taken in the interpretation of these results since positive staining for p53 protein is not always indicative of mutation of the p53 gene. Furthermore, a range of staining patterns is seen in mammary carcinomas, making interpretation difficult. Assessment of staining needs to be standardised in order that different studies can be compared. However, in breast carcinoma, p53 immunohistochemistry appears to give information relating to tumour grade and, independently, to prognosis.

Beta 1 and beta 4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement of cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both beta 1 and beta 4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with beta 1 and beta 4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the beta 1 chain and a variable pattern of reactivity was seen in the remaining cases. The beta 4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the beta 1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (beta 1 only). This study demonstrates that two of the main beta chains, beta 1 and beta 4, can be effectively demonstrated on methacarn and cold (4 degrees C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.

Multiple tissue core arrays in histopathology research: a validation study.

The use of multiple tissue arrays allows the examination of large cohorts of tumour tissue with economies of material and technical resources. It also permits the direct comparison of tissues on the same slide. In the present study, a series of 157 breast cancers was labelled with antibodies which recognize oestrogen (ER) and progesterone (PR) receptors and the staining obtained on whole tissue sections was compared with that from a series of multicore arrays. A highly significant association was found between the staining scores (0-7) obtained from the individual tissue sections and from the multicore arrays, although there was some discordance between the receptor status (positive/negative) of the whole section and the tissue core in 5% of cases for ER and in 6.5% of cases for PR. Multiple tissue cores represent an attractive way of dealing with large cohorts of tumours for research studies, because of the significant reduction in reagents and technical time required and the overall speed with which a study can be completed. A proportion of individual tissue cores were not representative of the diagnostic section, which limits the value of multicore arrays as a tool for patient management. However, the technique provides an efficient way of assessing the potential predictive value of novel proteins in different tumour types and in large cohorts.

Cyclin D1 and associated proteins in mammary ductal carcinoma in situ and atypical ductal hyperplasia.

Experimental studies suggest that cyclin D1 is a potential oncogene but in clinical studies of invasive breast cancer, overexpression of cyclin D1 is found to be associated with oestrogen receptor (ER) expression and low histological grade, both markers of good prognosis. Immunohistochemistry has been used to examine the relationship between cyclin D1 expression and differentiation in 36 cases of ductal carcinoma in situ (DCIS) and the interrelationship between expression of cyclin D1, its associated protein product of the retinoblastoma gene (pRb), and ER, in this group of cases. The expression of these markers has also been examined in nine cases of atypical ductal hyperplasia (ADH) and these results have been compared with the levels of expression seen in DCIS. Cyclin D1 overexpression was found in 23/36 (64 per cent) cases of DCIS and, in contrast to invasive carcinoma, there was no relationship with either differentiation or ER expression. The level of pRb expression was significantly associated with cyclin D1 expression (rS = 0.49, P = 0.001) and only two cases (6 per cent) were pRb-negative. There was no association between pRb and differentiation of DCIS or ER status. In contrast to DCIS, only one case of ADH showed overexpression of cyclin D1 (Mann-Whitney U-test, P = 0.02). All cases of ADH were ER-positive and showed moderate pRb staining, similar to that seen in well-differentiated DCIS. These results provide further evidence that overexpression of cyclin D1 plays a role early in carcinogenesis.

Cell proliferation measured by MIB1 and timing of surgery for breast cancer.

We have investigated the use of the antibody MIB1 as a proliferative and prognostic marker in breast cancer and whether changes in proliferative activity could account for differences in prognosis of premenopausal women operated on during different phases of the menstrual cycle. MIB1 expression was strongly correlated with S-phase fraction and histological grade. There was no difference in MIB1 scores between different phases of the menstrual cycle. Both MIB1 score and timing of surgery correlated significantly with duration of survival, while the two together were even stronger predictors of overall survival. Women with slowly proliferating tumours surgically removed in the luteal phase had a very good prognosis, whereas women with rapidly proliferating tumours excised at other times of the cycle had a worse prognosis.