P. Cottu

Long-term survival of advanced triple-negative breast cancers with a dose-intense cyclophosphamide/anthracycline neoadjuvant regimen.

Background:Triple-negative (TN) breast cancers exhibit major initial responses to neoadjuvant chemotherapy, but generally have a poor outcome. Because of the lack of validated drug targets, chemotherapy remains an important therapeutic tool in these cancers.Methods:We report the survival of two consecutive series of 267 locally advanced breast cancers (LABC) treated with two different neoadjuvant regimens, either a dose-dense and dose-intense cyclophosphamide–anthracycline (AC) association (historically called SIM) or a conventional sequential association of cyclophosphamide and anthracycline, followed by taxanes (EC-T). We compared pathological responses and survival rates of these two groups and studied their association with tumours features.Results:Although the two regimens showed equivalent pathological complete response (pCR) in the whole population (16 and 12%), the SIM regimen yielded a non-statistically higher pCR rate than EC-T (48% vs 24%, P=0.087) in TN tumours. In the SIM protocol, DFS was statistically higher for TN than for non-TN patients (P=0.019), although we showed that the TN status was associated with an increased initial risk of recurrence in both regimens. This effect gradually decreased and after 2 years, TN was associated with a significantly decreased likelihood of relapse in SIM-treated LABC (hazard ratio (HR)=0.25 (95% CI: 0.07–0.86), P=0.028).Conclusions:AC dose intensification treatment is associated with a very favourable long-term survival rate in TN breast cancers. These observations call for a prospective assessment of such dose-intense AC-based regimens in locally advanced TN tumours.

High-dose sequential epirubicin and cyclophosphamide with peripheral blood stem cell support for advanced breast cancer: results of a phase II study

The aim of this study was to evaluate the feasibility of a high-dose intensity and high-dose density multicycle epirubicin and cyclophosphamide regimen with peripheral blood stem cells (PBSC) and haematopoietic growth factor (G-CSF) support in advanced breast cancer patients. From August 1994 to September 1999, 56 breast cancer patients (8 stage IIIB and 48 stage IV) received 205 courses of cyclophosphamide 3 g m–2and epirubicin 100 mg m–2every 14 days. G-CSF 5 μg kg–1day–1was administered from day 3 to neutrophil recovery. 4 courses were planned. PBSC were collected after course 1, and reinfused after courses 3 and 4, with ≥ 2 × 106CD34+ PBSC kg–1required for each reinfusion. 48 patients (86%) received all 4 planned courses. Early withdrawal was consecutive to infectious complications (n= 4), severe asthenia (n= 3), haemorrhagic cystitis (n= 1). A median number of 10.8 × 106CD34+ PBSC kg–1(range, 3–80) was harvested with 1 or 2 apheresis in 48 patients (94%). Median relative dose intensity was 91.3% (range, 72–102%). Grade 4 neutrophil toxicity was observed in 100% of patients. Febrile neutropenia was observed in 40% of courses (median duration 2 days). Red blood cells and platelets had to be transfused in 54% and 27% of courses, respectively. There were no toxic deaths. Objective response rate was 69% in stage IV patients (31/45 evaluable pts), with a 16% complete response rate. Their median progression-free and overall survivals were 22.5 and 37 months, respectively. This epirubicine-containing high-dose regimen appeared feasible, albeit with high toxicity. Time-related progression parameters exceed commonly reported ones. Controlled studies of upfront sequential high-dose chemotherapy are still needed to evaluate its real benefit.

Abstract P6-04-14: Targeting the PI3K/mTOR pathway in patient-derived xenograft models of endocrine resistant luminal breast cancer

Background. Most ER+ tumors will eventually escape endocrine treatments. PI3K/mTOR pathway targeting has improved clinical results in endocrine treatments resistant (ETR) patients, however with unsustained responses. Our laboratory has developed several models of ETR luminal breast cancer (LBC) patient derived xenografts (PDX) suitable for biological studies (Cottu et al, AACR 2012 & IMPAKT 2012). The aims of the present study were 1) to analyze the biological factors associated with acquired ETR in relevant models of LBC; 2) to determine the therapeutic interest of combining everolimus with different ET models resistant to different endocrine treatments (ET). Methods. Implementation of LBC models has been reported (Cottu et al, BCRT 2012). ETR models were derived by selecting tumors growing under continuous ET, subsequently re-engrafted and rechallenged with endocrine therapies (tamoxifen, letrozole, fulvestrant), and with everolimus alone and combined with ETs. When possible, initial sensitive tumors were simultaneously treated with the same protocols. At progression, or after at least 120 days of continuous therapies, tumor tissue was retrieved. Tissue microarrays (TMA) were performed to analyze the biological modifications associated to ET and mTOR targeting. Triplicate immunohistochemistry of P-AKT/AKT, P-mTOR/mTOR and P-S6 were performed in addition to standard markers. Transcriptomic and RTPCR analyses are also being conducted. Results. Three models resistant to tamoxifen and 2 models resistant to surgical ovariectomy used a surrogate to estrogen deprivation, were developed from 3 initial PDX, and were maintained as independent hormone-resistant variants. All these models retained a luminal phenotype, based on IHC and transcriptomic analyses and had a wt PI3KCA. ETR models had an expression profile very similar to the initial PDX, suggesting that minor changes may be associated with the acquisition of an ETR phenotype. Three of these models have been tested. One tamoxifen resistant model confirmed a resistance specific to tamoxifen only and a high level of sensitivity to a fulvestrant everolimus combination. A second PDX yielded 2 other models resistant to tamoxifen and ovariectomy. These 2 models developed a resistance to all ET modalities. Therapeutic testing with combinations of everolimus and ETs were highly efficient, but everolimus alone was as efficient in these 2 models with complete and durable responses. Activation of the PI3K pathway was demonstrated in all cases by IHC analyses (expression of pAKT and pS6), and complementary ongoing RTPCR analyses. Everolimus based effects were associated with tumor fibrosis, a strong inhibition of Ki67 and pS6, but not always pAKT. The expression of ER was not modified by treatments, except in the fulvestrant-treated mice where it was strongly inhibited. Conclusion. We have developed and validated a platform of ETR models derived from our LBC original PDX. These models retained a luminal phenotype although highly resistant to ET. Activation of the PI3K pathway has been confirmed in ETR models, and everolimus alone, or combined with ET confirms high and durable efficacy. Updated mechanistic analyses performed at different time points will be presented at the meeting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-14.

Abstract S6-7: Establishment and Characterization of Human Luminal Breast Cancer Xenografts

Background: Despite an initial response to hormone therapy, luminal breast cancers (BC) are defined by a persisting long term risk of relapse. Preclinical models of estrogen-dependent human BC are therefore requested for a better understanding of estrogen receptor (ER) and hormone resistance biology. Because of a very low tumor take in immunodeficient mice, most in vivo models of estrogen-dependent human breast tumors are derived from human cancer cell lines. We report here the establishment and the characterization of new primary human luminal breast cancer xenografts directly obtained from fresh human tumor samples. Methods: As of December 2009, 453 fresh human BC samples have been engrafted in the interscapular fatpad of nude mice, of which 405 were retained for further studies (32 were non infiltrating or non-breast carcinoma, and 16 were axillary metastatic lymph node from a simultaneously engrafted primay tumor). ER was expressed in 313 tumors (77.3%), progesterone receptor in 175/291 informative tumors (60.1%), Her2 in 39/315 tumors (12.4%), and 60 tumors were triple negative. After transplantation, mice were kept in a dedicated unit and fed with water containing estrogen. Validation of the xenografts was obtained by a large phenotypic and genotypic profiling including: pathological and immunohistochemical (IHC) examination, dedicated gene expression (RT-qPCR), genomic (BAC CGH arrays) and transcriptomic (Affymetrix u133 RNA chips) analyses, and therapeutic assessment (estrogen deprivation, ovariectomy, LHRH agonists, letrozole, tamoxifen, fulvestrant). Results: Among the 405 human xenografted tumors, 8 luminal models have been established (2%), 7 from ER+/PR+ tumors and 1 from an axillary relapse of an ER-/HER2+ tumor. In all tumor/xenograft pairs, histopathological analyses showed an impressive morphological concordance. One had a strong mucinous component, and all of them were grade II/III tumors. Out of the 7 ER+/PR+ models, 3 were also HER2 positive. RNA expression by RT-qPCR confirmed ER, PR and HER2 status for the 7 ER+/ER+ tumors, and confirmed the ER+ status of the ER-/HER2+ derived tumorgraft. CGH arrays analyses demonstrated striking similarities of the genomic profile between the original tumors and their corresponding xenografts. Array CGH analyses were also performed at several passages, showing stable profile of the tumors during successive in vivo passages. Transcriptomic profile are still ongoing and will define each xenograft as either luminal A or B tumor subtypes. The tumor growth of all luminal BC models was estrogen dependent as demonstrated by estrogen supplementation and ovariectomy. Finally, therapeutic characterization of the xenografts showed that tamoxifen had a delayed but significant anti-tumor effect, whereas fulvestrant was the most efficient hormone therapy with durable complete responses observed in 3/3 evaluable models. Updated and extended results will be presented during the meeting. Conclusions: We have durably established and characterized 8 primary human luminal BC xenografts. In order to identify new therapeutic approaches of hormone resistant BC, we have now planed to decipher in these well-defined preclinical models the molecular variations associated with emergence of resistance to hormone therapies Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-7.