Clinical validation of the EndoPredict test in node-positive, chemotherapy-treated ER+/HER2- breast cancer patients: results from the GEICAM 9906 trial.

INTRODUCTION: EndoPredict (EP) is an RNA-based multigene test that predicts the likelihood of distant recurrence in patients with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer (BC) who are being treated with adjuvant endocrine therapy. Herein we report the prospective-retrospective clinical validation of EP in the node-positive, chemotherapy-treated, ER+/HER2- BC patients in the GEICAM 9906 trial. METHODS: The patients (N = 1,246) were treated either with six cycles of fluorouracil, epirubicin and cyclophosphamide (FEC) or with four cycles of FEC followed by eight weekly courses of paclitaxel (FEC-P), as well as with endocrine therapy if they had hormone receptor-positive disease. The patients were assigned to EP risk categories (low or high) according to prespecified cutoff levels. The primary endpoint in the clinical validation of EP was distant metastasis-free survival (MFS). Metastasis rates were estimated using the Kaplan-Meier method, and multivariate analysis was performed using Cox regression. RESULTS: The molecular EP score and the combined molecular and clinical EPclin score were successfully determined in 555 ER+/HER2- tumors from the 800 available samples in the GEICAM 9906 trial. On the basis of the EP, 25% of patients (n = 141) were classified as low risk. MFS was 93% in the low-risk group and 70% in the high-risk group (absolute risk reduction = 23%, hazard ratio (HR) = 4.8, 95% confidence interval (CI) = 2.5 to 9.5; P < 0.0001). Multivariate analysis showed that, in this ER+/HER2- cohort, EP results are an independent prognostic parameter after adjustment for age, grade, lymph node status, tumor size, treatment arm, ER and progesterone receptor (PR) status and proliferation index (Ki67). Using the predefined EPclin score, 13% of patients (n = 74) were assigned to the low-risk group, who had excellent outcomes and no distant recurrence events (absolute risk reduction vs high-risk group = 28%; P < 0.0001). Furthermore, EP was prognostic in premenopausal patients (HR = 6.7, 95% CI = 2.4 to 18.3; P = 0.0002) and postmenopausal patients (HR = 3.3, 95% CI = 1.3 to 8.5; P = 0.0109). There were no statistically significant differences in MFS between treatment arms (FEC vs FEC-P) in either the high- or low-risk groups. The interaction test results between the chemotherapy arm and the EP score were not significant. CONCLUSIONS: EP is an independent prognostic parameter in node-positive, ER+/HER2- BC patients treated with adjuvant chemotherapy followed by hormone therapy. EP did not predict a greater efficacy of FEC-P compared to FEC alone.

PAM50 proliferation score as a predictor of weekly paclitaxel benefit in breast cancer.

To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p < 0.001; and interaction test, p = 0.006). No significant interactions between treatment and the PAM50 subtypes or the various clinical-pathological variables, including Ki-67 and histologic grade, were identified. Finally, similar OS results were obtained in the CALGB data set, although the interaction test did not reach statistical significance (p = 0.109). The PAM50 proliferation score identifies a subset of patients with a low proliferation status that may derive a larger benefit from weekly paclitaxel.

PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers.

BACKGROUND: Many methodologies have been used in research to identify the "intrinsic" subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. METHODS: We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and "intrinsic" subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. RESULTS: ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis. CONCLUSIONS: The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.

Synchronous and metachronous bilateral breast cancer: a long-term single-institution experience.

Bilateral breast cancer (bBC) is the most common 2nd tumor in primary BC patients. However, its natural history is poorly understood as is the effect of previous adjuvant therapies. Between 1980 and 2005, we identified 3757 BC patients treated in our Institution, with 120 (3.2%) cases of bBC, 91 (2.4%) were metachronous BC (mBC), 29 (0.8%) synchronous BC (sBC). sBC defined as found before 3 months of the initial diagnosis. We performed a descriptive and an overall survival (OS) analysis. mBC appeared in young patients with a strong family history of BC. Most were diagnosed mammographically. The risk did not disappear after 15 years of follow-up. In most estrogen receptor (ER)-positive cases, the 2nd tumor was also ER-positive (concordance rate of 91%). No differences were seen according to the previous use of tamoxifen. In ER-negative cases, 43% of mBC were ER-positive. Synchronous BC (sBC) appeared in an elderly population with a strong family history. About 76% were ER-positive. ER status concordance was seen in 62%. There were no statistically significant differences in OS between patients with sBC or those with the 2nd mBC. A shorter time to appearance of the 2nd tumor predicted a worse OS. ER negativity and grade 3 tumors were negative prognostic factors. The risk of mBC does not abate with the pass of time. Contralateral mammographies should form part of follow-up. ER status concordance is high, especially in ER-positive cases. No differences were seen according to previous use of tamoxifen. Shorter disease-free intervals were linked with worse OS.

Adjuvant docetaxel for high-risk, node-negative breast cancer.

BACKGROUND: A regimen of docetaxel, doxorubicin, and cyclophosphamide (TAC) is superior to a regimen of fluorouracil, doxorubicin, and cyclophosphamide (FAC) when used as adjuvant therapy in women with node-positive breast cancer. The value of taxanes in the treatment of node-negative disease has not been determined. METHODS: We randomly assigned 1060 women with axillary-node-negative breast cancer and at least one high-risk factor for recurrence (according to the 1998 St. Gallen criteria) to treatment with TAC or FAC every 3 weeks for six cycles after surgery. The primary end point was disease-free survival after at least 5 years of follow-up. Secondary end points included overall survival and toxicity. RESULTS: At a median follow-up of 77 months, the proportion of patients alive and disease-free was higher among the 539 women in the TAC group (87.8%) than among the 521 women in the FAC group (81.8%), representing a 32% reduction in the risk of recurrence with TAC (hazard ratio, 0.68; 95% confidence interval [CI], 0.49 to 0.93; P=0.01 by the log-rank test). This benefit was consistent, regardless of hormone-receptor status, menopausal status, or number of high-risk factors. The difference in survival rates (TAC, 95.2%; FAC, 93.5%) was not significant (hazard ratio, 0.76; 95% CI, 0.45 to 1.26); however, the number of events was small (TAC, 26; FAC, 34). Rates of grade 3 or 4 adverse events were 28.2% with TAC and 17.0% with FAC (P<0.001). Toxicity associated with TAC was diminished when primary prophylaxis with granulocyte colony-stimulating factor was provided. CONCLUSIONS: As compared with adjuvant FAC, adjuvant TAC improved the rate of disease-free survival among women with high-risk, node-negative breast cancer. (Funded by GEICAM and Sanofi-Aventis; ClinicalTrials.gov number, NCT00121992.).

Randomized phase 3 trial of fluorouracil, epirubicin, and cyclophosphamide alone or followed by Paclitaxel for early breast cancer.

BACKGROUND: Taxanes are among the most active drugs for the treatment of metastatic breast cancer, and, as a consequence, they have also been studied in the adjuvant setting. METHODS: After breast cancer surgery, women with lymph node-positive disease were randomly assigned to treatment with fluorouracil, epirubicin, and cyclophosphamide (FEC) or with FEC followed by weekly paclitaxel (FEC-P). The primary endpoint of study-5-year disease-free survival (DFS)-was assessed by Kaplan-Meier analysis. Secondary endpoints included overall survival and analysis of the prognostic and predictive value of clinical and molecular (hormone receptors by immunohistochemistry and HER2 by fluorescence in situ hybridization) markers. Associations and interactions were assessed with a multivariable Cox proportional hazards model for DFS for the following covariates: age, menopausal status, tumor size, lymph node status, type of chemotherapy, tumor size, positive lymph nodes, HER2 status, and hormone receptor status. All statistical tests were two-sided. RESULTS: Among the 1246 eligible patients, estimated rates of DFS at 5 years were 78.5% in the FEC-P arm and 72.1% in the FEC arm (difference = 6.4%, 95% confidence interval [CI] = 1.6% to 11.2%; P = .006). FEC-P treatment was associated with a 23% reduction in the risk of relapse compared with FEC treatment (146 relapses in the 614 patients in the FEC-P arm vs 193 relapses in the 632 patients in the FEC arm, hazard ratio [HR] = 0.77, 95% CI = 0.62 to 0.95; P = .022) and a 22% reduction in the risk of death (73 and 95 deaths, respectively, HR = 0.78, 95% CI = 0.57 to 1.06; P = .110). Among the 928 patients for whom tumor samples were centrally analyzed, type of chemotherapy (FEC vs FEC-P) (P = .017), number of involved axillary lymph nodes (P < .001), tumor size (P = .020), hormone receptor status (P = .004), and HER2 status (P = .006) were all associated with DFS. We found no statistically significant interaction between HER2 status and paclitaxel treatment or between hormone receptor status and paclitaxel treatment. CONCLUSIONS: Among patients with operable breast cancer, FEC-P treatment statistically significantly reduced the risk of relapse compared with FEC as adjuvant therapy.

Time-to-progression in breast cancer: a stratification model for clinical trials.

The development of new anti-tumour drugs without clear cytoreductive activity has necessitated changes in the design of clinical trials. Defining the "time" parameter has become the essential objective of the majority of these trials. However, in breast cancer, this parameter is highly variable and, as such, difficult to quantify. We developed a useful tool that takes into account the inter-relatedness of all the variables known to have the capacity to predict the time-to-progression (TTP) in advanced breast cancer. From the Alamo database (GEICAM), we selected 1798 patients diagnosed as having metastatic breast cancer. Univariate analysis was performed using the method of Kaplan-Meier. Multivariate analysis was with the Cox regression method. The variables that were shown to have independent predictive value for the TTP were: non-visceral metastatic disease, single metastases, hormonal receptor positive N/T ratio<2 and disease-free interval (DFI) > or = 24 months. Taking into account the variables that had reached an independent predictive value, we constructed a model of scoring in which the patients were grouped according to the TTP. Using our new scoring model, it is possible to group patients with metastatic breast cancer according to the predicted TTP. This can be a useful tool at the time of selecting and stratifying patients on entry into new randomised clinical trials.

Influence of timing of initiation of adjuvant chemotherapy over survival in breast cancer: a negative outcome study by the Spanish Breast Cancer Research Group (GEICAM).

PURPOSE: This retrospective trial evaluates whether the timing of initiation of the adjuvant chemotherapy has any influence over survival in early-stage breast cancer. PATIENTS AND METHODS: A total of 2782 patients from El Alamo project (from 1990 to 1997; n = 15,400) were selected with stages I-III, surgery and adjuvant chemotherapy. Data were gathered about prognostic factors such as age, tumor size, vessel permeation (vascular or lymphatic), histological grade, and number of involved nodes, hormonal receptor status and administration of hormone therapy. The time interval between surgery and initiation of chemotherapy, and dates of relapse, second primary breast tumor and death were recorded. Patients were assigned in four groups according to the surgery-chemotherapy interval: <3 weeks (group A), 3-6 weeks (group B), 6-9 weeks (group C) and >9 weeks (group D). RESULTS: There were no differences in disease-free survival (DFS), nor in 5-year overall survival (OS), according to the timing of initiation of adjuvant chemotherapy. Cox proportional hazards model was used to adjust analysis for known prognostic factors but the effect of surgery-chemotherapy interval remained non-significant. The variable timing of initiation of adjuvant chemotherapy has also been assessed as a continuous variable and no differences have been detected. CONCLUSION: The optimum time of initiation of adjuvant chemotherapy in early stages of breast cancer is unknown. The delay in the initiation of adjuvant chemotherapy has no influence over survival in the analyzed time intervals. Retrospective analysis like this one with enough statistical power would be necessary to detect differences among groups.

[BRCA1 and BRCA2 mutations in patients with familial breast cancer]. / Mutaciones en BRCA1 y BRCA2 en pacientes con historia familiar de cáncer de mama.

BACKGROUND AND OBJECTIVE: Familial breast (BC) and ovarian cancer (OC) has been associated with germ-line mutations in the cancer susceptibility genes BRCA1 and BRCA2. The mutational spectra vary depending on the study populations and the criteria applied for patient selection. The present study reports the mutations found in 48 families of the Valencian Community studied in the University Hospital La Fe of Valencia (Spain). PATIENTS AND METHOD: We analysed the BRCA1 and BRCA2 genes of 48 families (55 patients) with a family history of BC an/or OC. The method consists of DNA extraction, followed by a polymerase chain reaction, mutational analysis by heteroduplex formation in conformation-sensitive-gel electrophoresis and sequencing of the heteroduplex detected. RESULTS: We report eight different deleterious mutations, four in BRCA1 and four in BRCA2 and four unclassified variants (UV). We also identified the presence of the BRCA1 3889_3890delG mutation for the first time in the Spanish population. In BRCA2, we report two mutations not described in the breast cancer information care, the 5025delT which is completely new and the 9206_9219del14, which has already been described in Spanish population. We also describe for the first time the presence of UV 8038C>T in this gene. CONCLUSIONS: The mutational spectra and the presence of new mutations in the limited number of patients give support to the relevance of the study population in the mutational spectra.

[Epidemiological study of the GEICAM group about breast cancer in Spain (1990-1993): El Alamo project]. / Estudio epidemiológico del grupo GEICAM sobre el cáncer de mama en España (1990-1993): proyecto El Alamo.

BACKGROUND AND OBJECTIVE: The aim of the El Alamo project was to define the demographic and clinic characteristics, treatment and evolution of women with invasive breast cancer diagnosed in hospitals of the GEICAM group (Spanish Breast Cancer Research Group) between 1990 and 1993. PATIENTS AND METHOD: Data from 4,532 patients were included. Forms were completed according to the medical history of patients, and collected in the GEICAM scientific office, where they were added to a data base. RESULTS: 32 hospitals from 19 provinces and 11 regional communities participated in the study. Mean age of the 4,532 patients was 56.72 years, 1,428 (31.5%) were premenopausal and 2,988 (65.9%) were postmenopausal. Stage II tumors were most frequent (55.5%). Among patients with stage I, II and III at diagnosis, surgery was the first treatment in most (90.7%), radical mastectomy being the most frequent procedure performed (79.7%). 70.4% of 1941 patients with positive axillary node and 37.4% of 1,806 patients without axillary affection received adjuvant chemotherapy with or without hormone therapy. CONCLUSIONS: El Alamo project represents the largest data base on breast cancer in Spain and the results are similar to those observed in other countries such as the USA.