John M.S. Bartlett

Assessment of Ki67 in Breast Cancer: Recommendations from the International Ki67 in Breast Cancer Working Group

Uncontrolled proliferation is a hallmark of cancer. In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Increasingly, Ki67 is measured in these scenarios for clinical research, including as a primary efficacy endpoint for clinical trials, and sometimes for clinical management. At present, the enormous variation in analytical practice markedly limits the value of Ki67 in each of these contexts. On March 12, 2010, an international panel of investigators with substantial expertise in the assessment of Ki67 and in the development of biomarker guidelines was convened in London by the co-chairs of the Breast International Group and North American Breast Cancer Group Biomarker Working Party to consider evidence for potential applications. Comprehensive recommendations on preanalytical and analytical assessment, and interpretation and scoring of Ki67 were formulated based on current evidence. These recommendations are geared toward achieving a harmonized methodology, create greater between-laboratory and between-study comparability, and allow earlier valid applications of this marker in clinical practice.

Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations.

The goal of this review is to systematically address a number of issues raised in the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) guidelines on testing for the human epidermal growth factor receptor 2 (HER-2) alteration. A group of investigators who are experienced in the conduct and interpretation of HER-2 assay methods reviewed the ASCO-CAP guidelines and address several areas of the HER-2 testing guidelines with a particular emphasis on biologic and methodologic considerations. Although HER-2 status determined by immunohistochemistry (IHC) and the status determined by fluorescent in situ hybridization (FISH) are significantly correlated, we feel that standard considerations of laboratory testing, including test accuracy, reproducibility, and precision, as well as the current data favor FISH over IHC assay methods for determining HER-2 status. These considerations are clearly important in clinical practice because HER2 amplification is directly linked to protein expression levels in breast cancer. However, this protein is not consistently analyzed in formalin-fixed tissues as a result of variability in fixation methods and times and the impact of fixation on HER-2 protein antigenicity. Conversely, gene amplification and FISH are significantly less dependent on tissue fixation methods, making this assay more reproducible between central and peripheral laboratories than IHC. Moreover, review of the existing data demonstrate that FISH is more strongly correlated with responsiveness to either trastuzumab or lapatinib treatment. Until other methods achieve similar test accuracy, reproducibility, and predictive value, we suggest FISH as the primary HER-2 testing modality for women with breast cancer who are candidates for HER-2-targeted therapies.

Adjuvant tamoxifen and exemestane in early breast cancer (TEAM): a randomised phase 3 trial

BACKGROUND Aromatase inhibitors improved disease-free survival compared with tamoxifen when given as an initial adjuvant treatment or after 2-3 years of tamoxifen to postmenopausal women with hormone-receptor-positive breast cancer. We therefore compared the long-term effects of exemestane monotherapy with sequential treatment (tamoxifen followed by exemestane). METHODS The Tamoxifen Exemestane Adjuvant Multinational (TEAM) phase 3 trial was conducted in hospitals in nine countries. Postmenopausal women (median age 64 years, range 35-96) with hormone-receptor-positive breast cancer were randomly assigned in a 1:1 ratio to open-label exemestane (25 mg once a day, orally) alone or following tamoxifen (20 mg once a day, orally) for 5 years. Randomisation was by use of a computer-generated random permuted block method. The primary endpoint was disease-free survival (DFS) at 5 years. Main analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, NCT00279448, NCT00032136, and NCT00036270; NTR 267; Ethics Commission Trial27/2001; and UMIN, C000000057. FINDINGS 9779 patients were assigned to sequential treatment (n=4875) or exemestane alone (n=4904), and 4868 and 4898 were analysed by intention to treat, respectively. 4154 (85%) patients in the sequential group and 4186 (86%) in the exemestane alone group were disease free at 5 years (hazard ratio 0·97, 95% CI 0·88-1·08; p=0·60). In the safety analysis, sequential treatment was associated with a higher incidence of gynaecological symptoms (942 [20%] of 4814 vs 523 [11%] of 4852), venous thrombosis (99 [2%] vs 47 [1%]), and endometrial abnormalities (191 [4%] vs 19 [<1%]) than was exemestane alone. Musculoskeletal adverse events (2448 [50%] vs 2133 [44%]), hypertension (303 [6%] vs 219 [5%]), and hyperlipidaemia (230 [5%] vs 136 [3%]) were reported more frequently with exemestane alone. INTERPRETATION Treatment regimens of exemestane alone or after tamoxifen might be judged to be appropriate options for postmenopausal women with hormone-receptor-positive early breast cancer. FUNDING Pfizer.

Multidisciplinary Meeting on Male Breast Cancer: Summary and Research Recommendations

Male breast cancer is a rare disease, accounting for less than 1% of all breast cancer diagnoses worldwide. Most data on male breast cancer comes from small single-institution studies, and because of the paucity of data, the optimal treatment for male breast cancer is not known. This article summarizes a multidisciplinary international meeting on male breast cancer, sponsored by the National Institutes of Health Office of Rare Diseases and the National Cancer Institute Divisions of Cancer Epidemiology and Genetics and Cancer Treatment and Diagnosis. The meeting included representatives from the fields of epidemiology, genetics, pathology and molecular biology, health services research, and clinical oncology and the advocacy community, with a comprehensive review of the data. Presentations focused on highlighting differences and similarities between breast cancer in males and females. To enhance our understanding of male breast cancer, international consortia are necessary. Therefore, the Breast International Group and North American Breast Cancer Group have joined efforts to develop an International Male Breast Cancer Program and to pool epidemiologic data, clinical information, and tumor specimens. This international collaboration will also facilitate the future planning of clinical trials that can address essential questions in the treatment of male breast cancer.

Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization.

Purpose: Chromogenic in situ hybridization (CISH) is a new modification of the fluorescence in situ hybridization (FISH) technique for detection of oncogene amplification in archival tumor samples. In CISH, the oncogene probe is detected using a peroxidase reaction, allowing use of transmitted light microscopy. We compared detection of HER-2/neu amplification by CISH with a Food and Drug Administration-approved two-color FISH test in an interlaboratory setting. Experimental Design: Formalin-fixed paraffin-embedded tumor samples from 197 breast cancers were analyzed for HER-2 amplification by CISH. Two-color FISH (PathVysion) CISH of 17 centromere was done if the observer considered it necessary to ascertain amplification status in tumors with borderline HER-2 CISH copy numbers. Results: Paired CISH/FISH results were available from 192 (97%) of 197 cases, no clear difference in success rates of either method was observed. Centromere 17 CISH was considered necessary in seven tumors. CISH and two-color FISH results were concordant in 180 cases (93.8%). There were 92 and 88 tumors found HER-2 amplified and nonamplified, respectively, by both methods. Eight tumors were amplified by CISH but not by FISH, and four tumors exhibited the opposite condition (kappa coefficient 0.875). In 7 of 12 cases differences between the two methods could have related to a lack of CISH chromosome 17 information. The remaining cases were explained by difficult histology (ductal carcinoma in situ, poor representativity, dense lymphocytic infiltration, or intratumoral heterogeneity). Conclusions: These results indicate that CISH could provide an accurate and practical alternative to FISH for clinical diagnosis of HER-2/neu oncogene amplification in archival formalin-fixed breast cancer samples.

Sequential docetaxel as adjuvant chemotherapy for early breast cancer (TACT): an open-label, phase III, randomised controlled trial

Summary Background Incorporation of a taxane as adjuvant treatment for early breast cancer offers potential for further improvement of anthracycline-based treatment. The UK TACT study (CRUK01/001) investigated whether sequential docetaxel after anthracycline chemotherapy would improve patient outcome compared with standard chemotherapy of similar duration. Methods In this multicentre, open-label, phase III, randomised controlled trial, 4162 women (aged >18 years) with node-positive or high-risk node-negative operable early breast cancer were randomly assigned by computer-generated permuted block randomisation to receive FEC (fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2 at 3-weekly intervals) for four cycles followed by docetaxel (100 mg/m2 at 3-weekly intervals) for four cycles (n=2073) or control (n=2089). For the control regimen, centres chose either FEC for eight cycles (n=1265) or epirubicin (100 mg/m2 at 3-weekly intervals) for four cycles followed by CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 at 4-weekly intervals) for four cycles (n=824). The primary endpoint was disease-free survival. Analysis was by intention to treat (ITT). This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN79718493. Findings All randomised patients were included in the ITT population. With a median follow-up of 62 months, disease-free survival events were seen in 517 of 2073 patients in the experimental group compared with 539 of 2089 controls (hazard ratio [HR] 0·95, 95% CI 0·85–1·08; p=0·44). 75·6% (95% CI 73·7–77·5) of patients in the experimental group and 74·3% (72·3–76·2) of controls were alive and disease-free at 5 years. The proportion of patients who reported any acute grade 3 or 4 adverse event was significantly greater in the experimental group than in the control group (p<0·0001); the most frequent events were neutropenia (937 events vs 797 events), leucopenia (507 vs 362), and lethargy (456 vs 272). Interpretation This study did not show any overall gain from the addition of docetaxel to standard anthracycline chemotherapy. Exploration of predictive biomarker-defined subgroups might have the potential to better target the use of taxane-based therapy. Funding Cancer Research UK (CRUK 01/001), Sanofi-Aventis, Pfizer, and Roche.

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.

HER2 testing in the UK: further update to recommendations

These guidelines update the previous UK HER2 testing guidelines and have been formulated to give advice on methodology, interpretation and quality assurance to ensure that HER2 testing results are accurate, reliable and timely with the expansion of testing to all patients with breast cancer at the time of primary diagnosis. The recommendations for testing are the use of immunohistochemistry but with analysis of equivocal cases by in situ hybridisation to clarify their HER2 status or the use of frontline fluorescence in situ hybridisation (FISH) testing for those laboratories wishing to do so; the inclusion of a chromosome 17 probe is strongly recommended. Laboratories using chromogenic or silver in situ hybridisation should perform an initial validation against FISH. For immunohistochemistry and in situ hybridisation there must be participation in the appropriate National External Quality Assurance scheme.

Predictive markers of anthracycline benefit: a prospectively planned analysis of the UK National Epirubicin Adjuvant Trial (NEAT/BR9601)

BACKGROUND The NEAT/BR9601 trial showed benefit for addition of anthracyclines to cyclophosphamide, methotrexate, and fluorouracil (CMF) as adjuvant treatment for early breast cancer. We investigated prospectively predictive biomarkers of anthracycline benefit including HER2 and TOP2A. METHODS 1941 tumours from 2391 women recruited to NEAT/BR9601 were analysed on tissue microarrays for HER2 and TOP2A amplification and deletion, HER1-3 and Ki67 expression, and duplication of chromosome 17 centromere enumeration probe (Ch17CEP). Log-rank analyses identified factors affecting relapse-free and overall survival, and regression models tested independent prognostic effect of markers, with adjustment for known prognostic factors (age, nodal status, oestrogen-receptor status, grade, and tumour size). The predictive value of markers was tested by treatment interactions for relapse-free and overall survival. FINDINGS 1762 patients were analysed. 21% of tumours (n=367) were HER2 amplified, 10% were TOP2A amplified (n=169), 11% showed TOP2A deleted (n=191), 23% showed Ch17CEP duplication (n=406), and 61% had high (>13.0%) Ki67 (n=1136). In univariate analyses, only HER2 amplification and TOP2A deletion were significant prognostic factors for relapse-free (hazard ratio [HR] 1.59, 95% CI 1.32-1.92, p<0.0001; and 1.52, 1.20-1.92, p=0.0006, respectively) and overall survival (1.79, 1.47-2.19, p<0.0001; and 1.62, 1.26-2.08, p=0.0002 respectively). We detected no significant interaction with anthracycline benefit for Ki67, HER2, HER1-3, or TOP2A. By contrast, in multivariate analyses, Ch17CEP duplication was associated with significant improvements in both relapse-free (HR 0.92, 95% CI 0.72-1.18 for tumours with normal Ch17CEP vs 0.52, 0.34-0.81 for tumours with abnormal Ch17CEP; p for interaction=0.004) and overall survival (0.94, 0.72-1.24 vs 0.57, 0.36-0.92; p for interaction=0.02) with anthracycline use. INTERPRETATION In women with early breast cancer receiving adjuvant chemotherapy, the most powerful predictor of benefit from anthracyclines is Ch17CEP duplication. In view of the location of HER2/TOP2A on chromosome 17, Ch17CEP duplication might explain the inconsistencies in previous studies of factors predicting benefit from anthracyclines. FUNDING Cancer Research UK and the Scottish Breast Cancer Clinical Trials Group.