Caroline J Witton

Can Molecular Markers Predict When to Implement Treatment with Aromatase Inhibitors in Invasive Breast Cancer

Purpose: Resistance to tamoxifen is linked to overexpression of HER2, and aromatase inhibitors show particular benefit in progesterone receptor (PR)–negative patients. We previously reported reduced survival in patients overexpressing HER1, HER2, and HER3. We now show that both HER1-3 and PR status predicts for early relapse in estrogen receptor (ER)–positive tamoxifen-treated breast cancer patients. Experimental Design: Tissue microarray technology was used to analyze 402 ER-positive tamoxifen-treated patients. Immunohistochemistry using epidermal growth factor receptor, HER2, HER3, HER4, and PR antibodies was done. Kaplan-Meier life table and Cox Regression analysis (log-rank testing of differences in breast cancer–related relapse on tamoxifen) was done. Results: HER1-3 (but not HER4) overexpression predicted for early relapse on tamoxifen (P = 0.0060). PR-negative cases were also significantly more likely to relapse while on tamoxifen (P= 0.017). HER1-3-positive and/or PR-negative patients combined as a “high-risk” group were significantly more likely to relapse on tamoxifen in univariate (P < 0.0001) and Coxs multivariate analysis (P = 0.0069). However, this applied to early relapse on tamoxifen only, as any disease relapse after 3 years of tamoxifen was unrelated to PR/HER status. Conclusions: We show that HER1-3 and PR status can identify time-dependent de novo tamoxifen resistance with risk declining markedly after 3 years of tamoxifen treatment. These results parallel data from the ATAC and Intergroup Exemastane Study trials which suggest that whereas PR-negative patients derive greater benefit from initial aromatase inhibitor treatment, PR status has no effect on response when given as delayed treatment to those disease free on tamoxifen after 3 years.

Outcome and Human Epithelial Growth Factor Receptor (HER) 1-4 status in invasive breast carcinomas with proliferation indices evaluated using bromodeoxyuridine (BrdU) labelling

BackgroundWe have shown previously that whilst overexpression of HER1, 2 and 3 is associated with poor prognosis in breast cancer, HER4 is associated with a good prognosis. Cell proliferation is a key component of aggressive cancers and is driven by growth factors. In this study bromodeoxyuridine-derived proliferation indices are correlated with clinical outcome and HER1-4 status to further clarify the differing roles for the HER family at a biological level.Patients and Methods78 invasive breast cancers had BrdU in vivo labelling to determine the labelling index (BLI) and the potential tumour doubling time (Tpot). Long term clinical follow up was available for these patients. Using immunohistochemistry we established the HER1-4 status in 55 patients from the BrdU cohort.ResultsWe demonstrate a significant correlation between high BLI values and breast cancer specific death (p = 0.0174). Low Tpottimes were also significantly correlated with breast cancer specific death (p = 0.0258). However BLI did not independently predict survival in Coxs multiple regression analysis when combined with other prognostic factors such as size, grade and nodal status.Tumours found to be positive for HER 1, 2 or 3 had significantly (p = 0.041) higher labelling indices, with HER1 also showing significantly higher indices when considered independently (p = 0.024). Conversely HER4 positivity significantly correlated (p = 0.013) with low BLI values in line with previous data associating this receptor with good prognosis tumours.ConclusionsThese results support the hypothesis that HER1-3 are associated with driving tumour proliferation whilst HER4 is involved in a non-proliferative or even protective role.

Is PTEN loss associated with clinical outcome measures in human prostate cancer

Inactivating PTEN mutations are commonly found in prostate cancer, resulting in an increased activation of Akt. In this study, we investigate the role of PTEN deletion and protein expression in the development of hormone-refractory prostate cancer using matched hormone-sensitive and hormone-refractory tumours. Fluorescent in situ hybridisation and immunohistochemistry was carried out to investigate PTEN gene deletion and PTEN protein expression in the transition from hormone-sensitive to hormone-refractory prostate cancer utilising 68 matched hormone sensitive and hormone-refractory tumour pairs (one before and one after hormone relapse). Heterogeneous PTEN gene deletion was observed in 23% of hormone sensitive tumours. This increased significantly to 52% in hormone-refractory tumours (P=0.044). PTEN protein expression was observed in the membrane, cytoplasm and the nucleus. In hormone sensitive tumours, low levels of cytoplasmic PTEN was independently associated with shorter time to relapse compared to high levels of PTEN (P=0.028, hazard ratio 0.51 (95%CI 0.27–0.93). Loss of PTEN expression in the nucleus of hormone sensitive tumours was independently associated with disease-specific survival (P=0.031, hazard ratio 0.52, 95%CI 0.29–0.95). The results from this study demonstrate a role for both cytoplasmic and nuclear PTEN in progression of prostate cancer to the hormone-refractory state.

A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ERbeta in archival formalin-fixed paraffin embedded human breast tissue

SummaryEstrogen receptor (ER) α is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor, ERb has been described, and a number of studies have examined its expression in breast tissue. However elucidation of the role played by ERb has been hampered by published immunohistochemical studies employing a variety of protocols and scoring systems such that inter-laboratory comparisons are difficult. Here we present a multi-centre study designed to critically evaluate inter-laboratory differences in methodology. Six UK and Irish centres participated in this study. A small series of breast cancers were stained using centre-specific laboratory protocols and scored using both centrespecific and standard scoring protocols. There was generally poor agreement as to what constituted a positive or negative case when centre-specific scoring systems were used with less than half of all cases in agreement. Concordance was improved when a standard scoring system was used but varied according to threshold for positivity employed and primary antibody. Our results emphasise the need for further studies addressing the role of ERb to be based on a wider consensus on criteria for positivity. Ideally this should be based on calibration against clinical outcome.

Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH

Kirkegaard T, Witton C J, Edwards J, Nielsen K V, Jensen L B, Campbell F M, Cooke T G & Bartlett J M S
(2010) Histopathology56, 203–211

HER4 in breast cancer: comparison of antibodies against intra- and extra-cellular domains of HER4

IntroductionWe have previously linked HER4 expression with increased survival in breast cancer. However, other reports have associated HER4 with adverse prognostic significance. One possible explanation for the conflicting reports may be that these results are antibody dependent. The HER4 protein is enzymatically cleaved, which may alter the function of its intracellular domain (ICD). We have therefore compared the staining patterns of antibodies against its intracellular and extracellular domains using tissue microarray technology.MethodsImmunohistochemistry was performed and evaluated on tumours from 402 tamoxifen treated oestrogen receptor positive patients. The HFR1 antibody recognises the ICD of HER4 and thus recognises both the intact receptor and the cleaved ICD. The H4.77.16 clone recognises an extracellular domain of HER4 and thus detects the full length receptor only.ResultsBoth antibodies demonstrated nuclear, cytoplasmic and membranous staining. Concordance between the membrane staining patterns was high (88.44%, kappa 0.426). The HFR1 antibody, however, demonstrated generally higher levels of cytoplasmic staining (concordance 74.77%, kappa 0.351). The antibodies demonstrated very different patterns of nuclear staining. Over 60% of patients stained with the H4.77.16 had no nuclear staining whereas the vast majority showed staining with the HFR1 antibody (concordance 40.12%, kappa 0.051). Neither antibody demonstrated relationships between membranous or cytoplasmic HER4 staining and survival, although associations were seen with known poor prognostic markers. Cases with H4.77.16-determined nuclear staining had significantly poorer survival outcomes.ConclusionThe difference in antigen site may explain the different staining patterns we have seen with respect to location; with each antibody appearing to select for distinct compartments. Thus, HFR1 may select for cytoplasmic and nuclear HER4 ICD, whilst H4.77.16 selects for membranous HER4 and/or HER4 being recycled in cytoplasm or nucleus. This ability to distinguish between site and function of HER4 and its fragments is particularly important, with recent evidence highlighting the different functions of nuclear and mitochondrial HER4.

Structure of HER receptors and intracellular localisation of downstream effector elements gives insight into mechanism of tumour growth promotion

Since the discovery that the receptor HER2 (erbB2/neu) was overexpressed in 20–30% of breast cancers, and its subsequent association with poor prognosis, there has been much interest in this protein. The production of the humanised antibody to HER2 (Herceptin [trastuzumab]) and the use of Herceptin to treat patients with HER2 positive tumours has emphasised the potential of targeted treatments for breast cancer. The differences between HER2 and the other members of the type I receptor family (epidermal growth factor receptor [EGFR], HER3, HER4) have also been researched. HER2 differs in that it has no known ligand and when overexpressed HER2 can form dimers. In contrast the other receptors have several known ligands that promote dimer formation on binding. HER receptors can form both homo and heterodimers with HER2 acting as the preferred dimer partner. The activation of these receptors either by overexpression or ligand binding leads to the activation of intracellular growth promoting pathways and thus promotes tumour growth.

Determining sensitivity to rapamycin and its analogues in breast cancer patients

Mammalian target of rapamycin (mTOR) is a kinase with sequence homology to phosphoinositol-3 kinase (PI3K). It is a downstream mediator in the PI3K/AKT pathway, which regulates proliferation, survival, mobility and angiogenesis. The targets of mTOR include p70s6 kinase and 4E-BP1 (for review, see Bjornsti and Houghton [1]). Rapamycin is an antibiotic and fungicide isolated from Streptomyces hygroscopicus that inhibits mTOR activity and has been approved as an immunosuppressive drug in organ transplant patients. Interest in rapamycin and analogues as cancer treatments is growing [2] because of the observation that the PI3K/AKT pathway is often altered in cancers. This can occur via mutation in the tumour suppressor gene PTEN, which downregulates the pathway, or over-expression of receptors (such as HER2) that stimulate the pathway. It can also occur via over-expression of other proteins in the pathway (such as AKT/protein kinase B) due to gene amplification or failure to break down the proteins. Rapamycin itself has poor aqueous solubility and is not stable, and so several analogues (CCI-779, RAD001 and AP23573) have been developed that are being tested in clinical trials for cancer treatment. These new drugs can potentially be used for the treatment of breast cancer once those patients who will respond to the drug can be identified. In this review I summarize two recent papers that provide insight into the determinants of sensitivity to rapamycin and the potential synergism with conventional chemotherapies.

8th Nottingham International Breast Cancer Conference, Nottingham, UK, 16–19 September 2003

The four-day biennial 8th Nottingham Breast Cancer Conference held at the East Midlands Conference Centre, University of Nottingham, UK (16–19 September 2003) once again proved to be a successful event. Recent advances in clinical and scientific research were presented to an international audience, covering a broad spectrum of breast cancer issues including prediction, diagnosis and treatment. Delegates were encouraged to participate in workshop sessions, which allowed the comprehensive discussion of existing and promising future advances in breast cancer care.