J.G.M. Klijn

Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer

BACKGROUND Genome-wide measures of gene expression can identify patterns of gene activity that subclassify tumours and might provide a better means than is currently available for individual risk assessment in patients with lymph-node-negative breast cancer. METHODS We analysed, with Affymetrix Human U133a GeneChips, the expression of 22000 transcripts from total RNA of frozen tumour samples from 286 lymph-node-negative patients who had not received adjuvant systemic treatment. FINDINGS In a training set of 115 tumours, we identified a 76-gene signature consisting of 60 genes for patients positive for oestrogen receptors (ER) and 16 genes for ER-negative patients. This signature showed 93% sensitivity and 48% specificity in a subsequent independent testing set of 171 lymph-node-negative patients. The gene profile was highly informative in identifying patients who developed distant metastases within 5 years (hazard ratio 5.67 [95% CI 2.59-12.4]), even when corrected for traditional prognostic factors in multivariate analysis (5.55 [2.46-12.5]). The 76-gene profile also represented a strong prognostic factor for the development of metastasis in the subgroups of 84 premenopausal patients (9.60 [2.28-40.5]), 87 postmenopausal patients (4.04 [1.57-10.4]), and 79 patients with tumours of 10-20 mm (14.1 [3.34-59.2]), a group of patients for whom prediction of prognosis is especially difficult. INTERPRETATION The identified signature provides a powerful tool for identification of patients at high risk of distant recurrence. The ability to identify patients who have a favourable prognosis could, after independent confirmation, allow clinicians to avoid adjuvant systemic therapy or to choose less aggressive therapeutic options.

Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations

Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2*1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2*1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer1, but account for only a small fraction of breast cancer susceptibility1,2. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2*1100delC, a truncating variant that abrogates the kinase activity6, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2*1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.

Breast cancer after prophylactic bilateral mastectomy in women with a BRCA1 or BRCA2 mutation

BACKGROUND Women with a BRCA1 or BRCA2 mutation have a high risk of breast cancer and may choose to undergo prophylactic bilateral total mastectomy. We investigated the efficacy of this procedure in such women. METHODS We conducted a prospective study of 139 women with a pathogenic BRCA1 or BRCA2 mutation who were enrolled in a breast-cancer surveillance program at the Rotterdam Family Cancer Clinic. At the time of enrollment, none of the women had a history of breast cancer. Seventy-six of these women eventually underwent prophylactic mastectomy, and the other 63 remained under regular surveillance. The effect of mastectomy on the incidence of breast cancer was analyzed by the Cox proportional-hazards method in which mastectomy was modeled as a time-dependent covariate. RESULTS No cases of breast cancer were observed after prophylactic mastectomy after a mean (+/-SE) follow-up of 2.9+/-1.4 years, whereas eight breast cancers developed in women under regular surveillance after a mean follow-up of 3.0+/-1.5 years (P=0.003; hazard ratio, 0; 95 percent confidence interval, 0 to 0.36). The actuarial mean five-year incidence of breast cancer among all women in the surveillance group was 17+/-7 percent. On the basis of an exponential model, the yearly incidence of breast cancer in this group was 2.5 percent. The observed number of breast cancers in the surveillance group was consistent with the expected number (ratio of observed to expected cases, 1.2; 95 percent confidence interval, 0.4 to 3.7; P=0.80). CONCLUSIONS In women with a BRCA1 or BRCA2 mutation, prophylactic bilateral total mastectomy reduces the incidence of breast cancer at three years of follow-up.

Strong Time Dependence of the 76-Gene Prognostic Signature for Node-Negative Breast Cancer Patients in the TRANSBIG Multicenter Independent Validation Series

Purpose: Recently, a 76-gene prognostic signature able to predict distant metastases in lymph node–negative (N−) breast cancer patients was reported. The aims of this study conducted by TRANSBIG were to independently validate these results and to compare the outcome with clinical risk assessment. Experimental Design: Gene expression profiling of frozen samples from 198 N− systemically untreated patients was done at the Bordet Institute, blinded to clinical data and independent of Veridex. Genomic risk was defined by Veridex, blinded to clinical data. Survival analyses, done by an independent statistician, were done with the genomic risk and adjusted for the clinical risk, defined by Adjuvant! Online. Results: The actual 5- and 10-year time to distant metastasis were 98% (88-100%) and 94% (83-98%), respectively, for the good profile group and 76% (68-82%) and 73% (65-79%), respectively, for the poor profile group. The actual 5- and 10-year overall survival were 98% (88-100%) and 87% (73-94%), respectively, for the good profile group and 84% (77-89%) and 72% (63-78%), respectively, for the poor profile group. We observed a strong time dependence of this signature, leading to an adjusted hazard ratio of 13.58 (1.85-99.63) and 8.20 (1.10-60.90) at 5 years and 5.11 (1.57-16.67) and 2.55 (1.07-6.10) at 10 years for time to distant metastasis and overall survival, respectively. Conclusion: This independent validation confirmed the performance of the 76-gene signature and adds to the growing evidence that gene expression signatures are of clinical relevance, especially for identifying patients at high risk of early distant metastases.

Definition of Clinically Distinct Molecular Subtypes in Estrogen Receptor–Positive Breast Carcinomas Through Genomic Grade

PURPOSE A number of microarray studies have reported distinct molecular profiles of breast cancers (BC), such as basal-like, ErbB2-like, and two to three luminal-like subtypes. These were associated with different clinical outcomes. However, although the basal and the ErbB2 subtypes are repeatedly recognized, identification of estrogen receptor (ER) -positive subtypes has been inconsistent. Therefore, refinement of their molecular definition is needed. MATERIALS AND METHODS We have previously reported a gene expression grade index (GGI), which defines histologic grade based on gene expression profiles. Using this algorithm, we assigned ER-positive BC to either high-or low-genomic grade subgroups and compared these with previously reported ER-positive molecular classifications. As further validation, we classified 666 ER-positive samples into subtypes and assessed their clinical outcome. RESULTS Two ER-positive molecular subgroups (high and low genomic grade) could be defined using the GGI. Despite tracking a single biologic pathway, these were highly comparable to the previously described luminal A and B classification and significantly correlated to the risk groups produced using the 21-gene recurrence score. The two subtypes were associated with statistically distinct clinical outcome in both systemically untreated and tamoxifen-treated populations. CONCLUSION The use of genomic grade can identify two clinically distinct ER-positive molecular subtypes in a simple and highly reproducible manner across multiple data sets. This study emphasizes the important role of proliferation-related genes in predicting prognosis in ER-positive BC.

Oral Contraceptives and the Risk of Hereditary Ovarian Cancer

Background Women with mutations in either the BRCA1 or the BRCA2 gene have a high lifetime risk of ovarian cancer. Oral contraceptives protect against ovarian cancer in general, but it is not known whether they also protect against hereditary forms of ovarian cancer. Methods We enrolled 207 women with hereditary ovarian cancer and 161 of their sisters as controls in a case–control study. All the patients carried a pathogenic mutation in either BRCA1 (179 women) or BRCA2 (28 women). The control women were enrolled regardless of whether or not they had either mutation. Lifetime histories of oral-contraceptive use were obtained by interview or by written questionnaire and were compared between patients and control women, after adjustment for year of birth and parity. Results The adjusted odds ratio for ovarian cancer associated with any past use of oral contraceptives was 0.5 (95 percent confidence interval, 0.3 to 0.8). The risk decreased with increasing duration of use (P for trend, <0.001); use for six or...

Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype.

Purpose: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

Trastuzumab-Associated Cardiac Adverse Effects in the Herceptin Adjuvant Trial

PURPOSE The purpose of this analysis was to investigate trastuzumab-associated cardiac adverse effects in breast cancer patients after completion of (neo)adjuvant chemotherapy with or without radiotherapy. PATIENTS AND METHODS The Herceptin Adjuvant (HERA) trial is a three-group, multicenter, open-label randomized trial that compared 1 or 2 years of trastuzumab given once every 3 weeks with observation in patients with HER-2-positive breast cancer. Only patients who after completion of (neo)adjuvant chemotherapy with or without radiotherapy had normal left ventricular ejection fraction (LVEF > or = 55%) were eligible. A repeat LVEF assessment was performed in case of cardiac dysfunction. RESULTS Data were available for 1,693 patients randomly assigned to 1 year trastuzumab and 1,693 patients randomly assigned to observation. The incidence of trastuzumab discontinuation due to cardiac disorders was low (4.3%). The incidence of cardiac end points was higher in the trastuzumab group compared with observation (severe congestive heart failure [CHF], 0.60% v 0.00%; symptomatic CHF, 2.15% v 0.12%; confirmed significant LVEF drops, 3.04% v 0.53%). Most patients with cardiac dysfunction recovered in fewer than 6 months. Patients with trastuzumab-associated cardiac dysfunction were treated with higher cumulative doses of doxorubicin (287 mg/m(2) v 257 mg/m(2)) or epirubicin (480 mg/m(2) v 422 mg/m(2)) and had a lower screening LVEF and a higher body mass index. CONCLUSION Given the clear benefit in disease-free survival, the low incidence of cardiac adverse events, and the suggestion that cardiac dysfunction might be reversible, adjuvant trastuzumab should be considered for treatment of breast cancer patients who fulfill the HERA trial eligibility criteria.

Breast Cancer Risk Following Bilateral Oophorectomy in BRCA1 and BRCA2 Mutation Carriers: An International Case-Control Study

PURPOSE The purpose of this study was to estimate the extent of protection offered against breast cancer by prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations and to determine to what extent risk reduction varies with age at oophorectomy, age at diagnosis, and time elapsed since surgery. PATIENTS AND METHODS We analyzed 1,439 patients with breast cancer and 1,866 matched controls derived from a registry of BRCA1 and BRCA2 carriers. We estimated odds ratios (ORs) of breast cancer for having had a bilateral oophorectomy, using conditional logistic regression, matched for parity and for oral contraceptive use. RESULTS A previous history of oophorectomy was associated with a significant reduction in breast cancer risk of 56% for BRCA1 carriers (OR = 0.44; 95% CI, 0.29 to 0.66) and of 46% for BRCA2 carriers (OR = 0.57; 95% CI, 0.28 to 1.15). The risk reduction was greater if the oophorectomy was performed before age 40 (OR = 0.36; 95% CI, 0.20 to 0.64 for BRCA1 carriers) than after age 40 (OR = 0.53; 95% CI, 0.30 to 0.91). The protective effect was evident for 15 years post-oophorectomy (OR = 0.39; 95% CI, 0.26 to 0.57). CONCLUSION Oophorectomy is an effective means of reducing the risk of breast cancer in carriers of BRCA1 mutations. The data suggest oophorectomy is protective in BRCA2 carriers as well, but needs to be confirmed in other studies.

Multicenter Validation of a Gene Expression–Based Prognostic Signature in Lymph Node–Negative Primary Breast Cancer

PURPOSE We previously identified in a single-center study a 76-gene prognostic signature for lymph node-negative (LNN) breast cancer patients. The aim of this study was to validate this gene signature in an independent more diverse population of LNN patients from multiple institutions. PATIENTS AND METHODS Using custom-designed DNA chips we analyzed the expression of the 76 genes in RNA of frozen tumor samples from 180 LNN patients who did not receive adjuvant systemic treatment. RESULTS In this independent validation, the 76-gene signature was highly informative in identifying patients with distant metastasis within 5 years (hazard ratio, [HR], 7.41; 95% CI, 2.63 to 20.9), even when corrected for traditional prognostic factors in multivariate analysis (HR, 11.36; 95% CI, 2.67 to 48.4). The actuarial 5- and 10-year distant metastasis-free survival were 96% (95% CI, 89% to 99%) and 94% (95% CI, 83% to 98%), respectively, for the good profile group and 74% (95% CI, 64% to 81%) and 65% (53% to 74%), respectively for the poor profile group. The sensitivity for 5-yr distant metastasis-free survival was 90%, and the specificity was 50%. The positive and negative predictive values were 38% (95% CI, 29% to 47%) and 94% (95% CI, 86% to 97%), respectively. The 76-gene signature was confirmed as a strong prognostic factor in subgroups of estrogen receptor-positive patients, pre- and postmenopausal patients, and patients with tumor sizes 20 mm or smaller. The subgroup of patients with estrogen receptor-negative tumors was considered too small to perform a separate analysis. CONCLUSION Our data provide a strong methodologic and clinical multicenter validation of the predefined prognostic 76-gene signature in LNN breast cancer patients.