Tamoxifen resistance in early breast cancer: statistical modelling of tissue markers to improve risk prediction.

BACKGROUND: For over two decades, the Nottingham Prognostic Index (NPI) has been used in the United Kingdom to calculate risk scores and inform management about breast cancer patients. It is derived using just three clinical variables - nodal involvement, tumour size and grade. New scientific methods now make cost-effective measurement of many biological characteristics of tumour tissue from breast cancer biopsy samples possible. However, the number of potential explanatory variables to be considered presents a statistical challenge. The aim of this study was to investigate whether in ER+ tamoxifen-treated breast cancer patients, biological variables can add value to NPI predictors, to provide improved prognostic stratification in terms of overall recurrence-free survival (RFS) and also in terms of remaining recurrence free while on tamoxifen treatment (RFoT). A particular goal was to enable the discrimination of patients with a very low risk of recurrence. METHODS: Tissue samples of 401 cases were analysed by microarray technology, providing biomarker data for 72 variables in total, from AKT, BAD, HER, MTOR, PgR, MAPK and RAS families. Only biomarkers screened as potentially informative (i.e., exhibiting univariate association with recurrence) were offered to the multivariate model. The multiple imputation method was used to deal with missing values, and bootstrap sampling was used to assess internal validity and refine the model. RESULTS: Neither the RFS nor RFoT models derived included Grade, but both had better predictive and discrimination ability than NPI. A slight difference was observed between models in terms of biomarkers included, and, in particular, the RFoT model alone included HER2. The estimated 7-year RFS rates in the lowest-risk groups by RFS and RFoT models were 95 and 97%, respectively, whereas the corresponding rate for the lowest-risk group of NPI was 89%. CONCLUSION: The findings demonstrate considerable potential for improved prognostic modelling by incorporation of biological variables into risk prediction. In particular, the ability to identify a low-risk group with minimal risk of recurrence is likely to have clinical appeal. With larger data sets and longer follow-up, this modelling approach has the potential to enhance an understanding of the interplay of biological characteristics, treatment and cancer recurrence.

Poor survival outcomes in HER2-positive breast cancer patients with low-grade, node-negative tumours.

We present a retrospective analysis on a cohort of low-grade, node-negative patients showing that human epidermal growth factor receptor 2 (HER2) status significantly affects the survival in this otherwise very good prognostic group. Our results provide support for the use of adjuvant trastuzumab in patients who are typically classified as having very good prognosis, not routinely offered standard chemotherapy, and who as such do not fit current UK prescribing guidelines for trastuzumab.

Is the biology of breast cancer changing? A study of hormone receptor status 1984-1986 and 1996-1997.

Using archived tumours, those from 1984-1986 and 1996-1997 underwent immunohistochemistry for hormone receptors and grade analysis. A significant shift towards more ER-positive and low-grade disease was found; this appears to reflect screening practices, but could still influence survival.

Frequent amplifications and deletions of G1/S-phase transition genes, CCND1 and MYC in early breast cancers: a potential role in G1/S escape.

Uncontrolled growth of cancer cells can be related to dysfunctional cell cycle control, including entry into S-phase, initiating cell division. Cyclin CCND3 and CCNE1 along with CDK2 and CDK6 regulate this checkpoint, and genetic changes, detectable by fluorescence in situ hybridization, are hypothesized to increase the aggressiveness of breast cancer, thereby influencing patient survival. Genomic change was investigated in 106 primary breast cancer samples, where the combined gene copy number changes in one of these four cell cycle regulatory factors was observed in 22% of the 98 tumors of successful analysis, distributed with 15 deletions and 7 amplifications. A trend towards decreased survival was observed with the aberrations, suggesting a prognostic potential of this set of markers, which was supported by an association with tumor grade. For validation of the new set of FISH probes for the G1/S-phase cell cycle factors, two additional markers, frequently amplified in breast cancers, were included in this study: The G1/S phase control gene CCND1 and the proliferation marker MYC. Both markers were amplified in 14% and deleted in 5% of the cases. This is the first report of genomic deletions of MYC in breast cancer.

Phosphorylated c-Src in the nucleus is associated with improved patient outcome in ER-positive breast cancer.

Elevated c-Src protein expression has been shown in breast cancer and in vitro evidence suggests a role in endocrine resistance. To investigate whether c-Src is involved in endocrine resistance, we examined the expression of both total and activated c-Src in human breast cancer specimens from a cohort of oestrogen receptor (ER)-positive tamoxifen-treated breast cancer patients. Tissue microarray technology was employed to analyse 262 tumour specimens taken before tamoxifen treatment. Immunohistochemistry using total c-Src and activated c-Src antibodies was performed. Kaplan-Meier survival curves were constructed and log-rank test were performed. High level of nuclear activated Src was significantly associated with improved overall survival (P=0.047) and lower recurrence rates on tamoxifen (P=0.02). Improved patient outcome was only seen with activated Src in the nucleus. Nuclear activated Src expression was significantly associated with node-negative disease and a lower NPI (P<0.05). On subgroup analysis, only ER-positive/progesterone receptor (PgR)-positive tumours were associated with improved survival (P=0.004). This shows that c-Src activity is increased in breast cancer and that activated Src within the nucleus of ER-positive tumours predicts an improved outcome. In ER/PgR-positive disease, activated Src kinase does not appear to be involved in de novo endocrine resistance. Further study is required in ER-negative breast cancer as this may represent a cohort in which it is associated with poor outcome.

Automated telephone follow-up after breast cancer: an acceptability and feasibility pilot study.

Traditional clinical follow-up after breast cancer is inefficient at detecting relapse and is poorly suited to detecting and ameliorating psychological problems. There is interest in developing more effective and efficient methods of follow-up. We report a prospective cohort study of the acceptability and feasibility of remote, automated telephone follow-up after breast cancer. Women with a history of breast cancer were approached at their annual follow-up visit. For participants, the follow-up questionnaire was administered on paper at baseline. In place of a clinic visit following year, the women completed the same questionnaire using an automated telephone system. All patients were given mammograms. A semi-structured interview was then conducted to assess the acceptability. The potential impact on clinic usage was assessed. In all, 110 of 121 women (91%) agreed to participate. Seventy-five patients (71%) completed follow-up using the new automated system 1 year later. Seventy-one of the 75 patients found the system easy to use. Forty-nine of the 75 (65.33%) liked the system and were happy to use it as their sole method of follow-up. A further 12% were happy to use it as part of their follow-up. In only 10.66% of participants were concerns raised which led to clinic attendance. Automated questionnaire-based telephone follow-up is acceptable to women and has the potential to reduce attendance at clinic. Further studies to validate this method further are planned.

Patients' expectations for follow-up in breast cancer--a preliminary, questionnaire-based study.

The current National Institute for Clinical Excellence (NICE) guidelines recommends that only 2-3 years of follow-up after breast cancer be provided. Clinicians are unwilling to implement these guidelines. However, little has been done to establish patients expectations before they embark on their regular follow-up programme. We have sought the opinions of patients in a questionnaire-based prospective cohort study. Expectations for length and frequency of follow-up were established in women prior to attending their first scheduled follow-up review 1 year after treatment. In addition, patients were asked their opinions on what clinics were designed to achieve. An attempt was made to establish whether patients would be happy with less follow-up when informed of the inefficiency of routine clinic visits. Most women expect some follow-up, but expectations for length and frequency vary dramatically. Most believe follow-up is for the detection of relapse, but very few see psychological support or side effect detection as being central to clinicians' aims. One third of women would be happy to not come back to clinic at all when told how infrequently routine clinical examination detects metastatic disease.

Genetic alterations of CCND1 and EMSY in breast cancers.

AIMS: CCND1 and EMSY, on 11q13, are frequently amplified in breast cancer. CCND1 is implicated in cell cycle progression and EMSY is a BRCA2-associated repressor protein. The aim was to investigate gene copy numbers of CCND1 and EMSY and to determine if CCND1 amplification is associated with reduced survival of tamoxifen-treated breast cancer patients. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) was performed on 111 consecutive and 354 oestrogen receptor (ER)+ tamoxifen-treated breast cancers. In the consecutive set, CCND1 and EMSY were amplified in 14.8% and 7.2%, respectively, and deleted in 8.7% and 13.5%, respectively. In the ER+ set, CCND1 and EMSY were amplified in 20.6% and 9.6%, respectively, and deleted in 1.7% and 4.2%, respectively. CCND1 and EMSY gene amplifications were associated with decreased overall survival (OS) (P = 0.03 and P = 0.04, respectively) of patients in the ER+ set. CONCLUSION: As hypothesized, CCND1 amplifications are associated with poor OS in ER+ patients. EMSY amplification is also associated with poor OS. However, as >70% of EMSY amplifications were CCND1 amplified, EMSY may not have any additional effect on survival of ER+ breast cancer.

Follow-up in breast cancer: does routine clinical examination improve outcome? A systematic review of the literature.

Multiple guidelines exist for the follow-up of breast cancer, with no agreement on frequency or duration. The contribution of routine clinical examination for the detection of potentially treatable relapse, and the impact this has on survival, is unknown. In this study, we systematically review the literature to establish the proportion of potentially treatable locoregional relapses and new contralateral breast cancers detected by clinical examination, mammography and patient self-examination. We analyse whether method of detection of relapse influences outcome. The methods used were systematic review of the literature. MEDLINE, EMBASE, CancerLit, Web of Sciences and EBM reviews were the data sources for the systematic review. All studies with information on proportion of relapses detected by clinical examination, mammography and self-examination were included. A total of 30-40% of potentially treatable relapses are detected by patient self-examination. In studies published before 2000, 15% of such relapse is mammographically detected with 46% detected by routine clinical examination. In those published after 2000, 40% are mammographically detected with 15% detected on routine clinical examination. Patients with ipsilateral breast relapse detected clinically appear to do less well than those with relapse detected by self-examination or mammography. Routine clinical surveillance is responsible for detection of fewer potentially treatable relapses in more modern cohorts as experience with mammography increases. There is no evidence to suggest that clinical examination confers a survival advantage compared with other methods of detection. The data in this analysis suggest that a review of the guidelines on follow-up after breast cancer should be undertaken.

Alternative methods of follow up in breast cancer: a systematic review of the literature.

Regular clinical follow up after breast cancer is a common practice. Evidence from retrospective reviews casts doubt on the efficacy of this practice and the various guidelines for follow up show little concordance. Our aim was to investigate what alternative follow-up methods (including reduced frequency of visits) have been subjected to controlled trial and to establish what evidence exists from controlled trials to advise the guidelines. The study involved systematic review of the literature using MEDLINE, Embase, CancerLit, Web of Sciences and EBM reviews as data sources. Methods included reviewing all randomised controlled trials comparing different follow-up frequencies or comparing an alternative method with clinical follow up after breast cancer. All outcome measures addressed in the trials were analysed. Two trials compared frequency of traditional follow up. Five trials assessed alternative methods. All were of inadequate power or duration to establish ideal frequency of clinic visits or safety of alternative follow-up methods. Alternative follow up had no detrimental effect on satisfaction or outcome. Few trials have been conducted, all of which are underpowered to establish safety of reducing or replacing clinic visits. Alternative methods of follow up are acceptable to patients and may be associated with other benefits. Larger trials are required.

Bad expression predicts outcome in patients treated with tamoxifen.

AIMS: Activation of the PI3K/Akt signal transduction pathway has been linked to endocrine resistance in tamoxifen treated breast cancer patients. Activation of the PI3K/Akt pathway causes phosphorylation of Bad leading to modulation of cellular apoptosis. The present study was carried out to test the hypothesis that disruption of apoptosis in breast cancer, via Akt activation, is linked with hormone resistance. METHODS: Immunohistochemistry (IHC) was performed on 402 oestrogen receptor (ER) positive breast cancers using antibodies against Bad, pBad (ser 112), Bcl-2, Bcl-xl and Bax. RESULTS: Bad, pBad (ser 112), Bcl-2 and Bax expression was observed in the cellular cytoplasmic compartment only. Patients, whose tumours had high levels of Bad expression, had a significantly improved disease-free survival when compared to patients whose tumours had low levels of Bad expression (P = 0.049). Activation of the PI3K/Akt pathway by either heregulin or oestrogen had no effect on expression of Bad, Bcl-2, Bax or Bcl-xl. However, heregulin increased pBad (ser 112) expression. DISCUSSION: Data presented here shows that Bad expression is associated with relapse in tamoxifen-treated breast cancer patients, supporting our hypothesis that the apoptosis pathway is involved in tamoxifen resistance.

Low expression of HER2 protein in breast cancer is biologically significant.

HER2 status is routinely tested using immunohistochemistry or FISH following the licensing of a therapeutic agent targeting HER2. However, neither of these methods provides quantitative information relating to the 70-80% of patients with levels of expression lower than the assay detection thresholds. In this study, radioimmunohistochemistry was used to detect quantitative HER2 protein expression in 178 breast cancers. Survival analysis was performed, as were correlations with known prognostic variables and with overexpression of other HER family members. It is demonstrated that the populations expressing very high and very low levels of HER2 are each associated with increased risk of cancer-specific death on survival analysis (p = 0.0043). The group with low levels of HER2 was more likely to be of higher grade, EGFR-positive and ER/HER3/HER4-negative. HER2-positive cases were frequently ER-negative/HER3-positive, whilst cases with normal HER2 expression were often ER-positive/HER4-positive. The aggressive nature of the tumour group with low HER2 expression may be explained by actions of other HER family members, particularly EGFR, but whether these or other factors have a negative regulatory effect on HER2 expression remains to be determined.

Does deprivation affect breast cancer management?

We evaluated whether social deprivation affected decision-making for breast cancer surgery. Of 3419 patients, 53.6% had mastectomy and this was predicted by deprivation, age, tumour size and hospital, all of which retained significance on multivariate analysis, except deprivation. Pathological characteristics and surgical decision-making determined choice of operation not deprivation.

Cyclooxygenase 2 (COX2) expression is associated with poor outcome in ER-negative, but not ER-positive, breast cancer.

AIMS: Inflammation and hormonal signalling induce cyclooxygenase 2 (COX2) expression in solid tumours. COX2 expression is linked to neovascularization and tumour growth. HER2 modulates colorectal cancer COX2 expression. We investigated interactions between COX2 and HER1-4 in breast cancer. METHODS AND RESULTS: COX2 expression was localized to epithelial cells with 21.2% of cases expressing higher levels than normal epithelium. Elevated COX2 expression was not associated with size, grade, high Nottingham prognostic index (NPI) or oestrogen receptor (ER) negativity. No association was observed between COX2 and HER1-4 expression. High COX2 expression was associated with reduced disease-free survival (P = 0.03) and disease-related survival in ER-negative (P = 0.046) but not ER-positive disease (P = 0.835). CONCLUSION: HER1, 2, 3 and 4 are not associated with high breast tumour COX2 expression. COX2 is frequently expressed in breast carcinoma cells and adjacent epithelium. COX2 may be an important factor in promoting tumour progression in ER-negative tumours and a potential drug target in breast tumours.

A role for BRCA1 in sporadic breast cancer.

To test the hypothesis that altered expression of BRCA1 protein may play an important role in sporadic breast cancer development, 50 randomly selected primary breast cancers (frozen sections, 5 years' median follow-up) were immunolabelled with two monoclonal BRCA1 antibodies (MS110 and MS13). MS110 labelling was exclusively nuclear showing no relation to outcome or tumour pathology. Western blotting demonstrated crossreactivity, suggesting antibody nonspecificity. MS13 labelling was predominantly cytoplasmic. Intense labelling predicted decreased overall survival (P=0.012), disease-free survival (P=0.029), oestrogen receptor negativity (P=0.0004) and c-erbB-2 overexpression (P=0.006). Western blotting detected a 110 kDa molecule consistent with BRCA1 delta11b splice variant. BRCA1 protein is postulated to function as a tumour suppressor. We demonstrate cytoplasmic localisation in sporadic breast cancer suggesting excess delta11b splice variant production, reduced production of full-length BRCA1 and thus postulate reduced tumour suppressor activity. BRCA1 protein appears to have a significant role in both sporadic and hereditary breast cancers.

Chromosome 17 aneusomy is associated with poor prognostic factors in invasive breast carcinoma.

Aberrations of chromosome 17 are common in breast cancer. Fluorescence in situ hybridization (FISH) enables gene or chromosome copy number to be assessed in situ in archival tissues and related to morphology and clinical outcome. In this study direct labeled DNA probes for the chromosome 17 alpha satellite and the HER2/neu gene were applied simultaneously to 5 micron sections of 214 formalin-fixed paraffin-embedded invasive primary breast carcinomas. A high proportion (54%) of invasive breast carcinomas displayed aneusomy of chromosome 17. Polysomy 17 correlated with multiple copies of HER2/neu (p = < 0.001), but not with HER2/neu amplification. Eighty-six patients without HER2/neu amplification had aneusomy 17. Fifty-eight of the 86 patients that had aneusomy 17 had high HER2/neu copy number. Twelve patients with normal copy number for chromosome 17 had amplification of HER2/neu and 30 patients had amplification of HER2/neu with aneusomy 17. Aneusomy 17 was associated with grade 3 carcinoma (p = 0.008), ER negativity (p = 0.0032) and a Nottingham prognostic index of greater than 5.4 (p = 0.039) but was not associated with survival by univariate analysis. In conclusion, the determination of chromosome 17 copy number should be incorporated in assessment of HER2/neu status, as this will give an accurate measure of amplification of HER2/neu and may also be helpful in determining suitability for breast carcinoma trials.

Mitoxantrone and methotrexate chemotherapy with and without mitomycin C in the regional treatment of locally advanced breast cancer.

Fifty patients with locally advanced breast cancer received regional chemotherapy delivered angiographically via the internal mammary artery and varying vessels supplying the lateral aspect of the breast. Thirty three patients received mitomycin C, methotrexate and mitoxantrone, and 17 patients received methotrexate and mitoxantrone only. There was no significant difference in clinical response between the two groups. However, in patients who received mitomycin C, severe local skin toxicity occurred in nine patients resulting in delay of further therapy and considerable morbidity. Mitomycin C should not be administered regionally in patients with locally advanced breast cancer.

Mucinous breast carcinoma.

A case of mucinous breast carcinoma is discussed in which several unusual features in presentation and difficulties in management are addressed.

Evaluating HER2 amplification and overexpression in breast cancer.

The development of Herceptin (Trazumatab) makes testing for HER2 status important for choosing optimal therapy in breast cancer. This study addresses the precision, accuracy, and reproducibility of HER2 assays. HER2 was assessed retrospectively by immunohistochemistry (IHC) with Dako 'Herceptest', by IHC with the monoclonal antibody CB11, and by fluorescence in situ hybridization (FISH, PathVysion), in a series of 216 formalin-fixed breast carcinomas including 191 for which quantitative HER2 data from radioimmunohistochemistry (Q-IHC) were available. All tests were scored independently by two observers. Positivity rates varied between Herceptest (12.6%), FISH (19.4%), and CB11 IHC (28.5%). Kappa values showed that IHC-based tests were more susceptible to inter-observer variation (kappa=0.67 and 0.74 for Herceptest and CB11, respectively) than FISH (kappa=0.973). Overall test accuracy (see the Materials and methods section) for CB11 IHC (83.8%) was lower than Herceptest (87.4%) or FISH (93.2%). FISH predicted p185 HER2 overexpression (determined by Q-IHC) better (concordance index C.Ind. 0.90) than CB11 IHC (C.Ind.=0.85) or Herceptest (C.Ind.=0.81). Of 42 cases with gene amplification by FISH, 67% were positive in the Herceptest (2+ or 3+) vs. 83% with CB11. Of 174 cases negative by FISH, 96% were negative in the Herceptest and 68% with CB11. In conclusion, FISH is the most accurate, reproducible, and precise predictor of HER2 overexpression in routine diagnostic laboratories.