M.M. Dauplat

Multicentric neoadjuvant phase II study of panitumumab combined with an anthracycline/taxane based chemotherapy in operable triple negative breast cancer: Identification of biologically-defined signatures predicting treatment impact

BACKGROUND Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors for some of which the epithelial growth factor receptor (EGFR) pathway may play an important role. We investigated the efficacy and toxicity of an anti-EGFR antibody (panitumumab) combined with a standard neoadjuvant anthracycline-taxane-based chemotherapy in patients with operable, stage II-III, TNBC. PATIENTS AND METHODS Treatment in this multicentric neoadjuvant pilot study consisted of panitumumab (9 mg/kg) for eight cycles q.3 weeks combined with four cycles of 5-fluorouracil, epidoxorubicin and cyclophosphamide (FEC100: 500/100/500 mg/m(2)) q.3 weeks, followed by four cycles of docetaxel (T: 100 mg/m(2)) q.3 weeks. Following therapy, all patients underwent surgical resection. Pathologic complete response (pCR) in assessable patients was the main end point while clinical response, toxicity and ancillary studies were secondary end points. Paraffin-embedded and frozen tumor samples were systematically collected with the aim to identify predictive biomarkers of efficacy and resistance in order to select biologically defined subpopulations for potential further clinical development of the anti-EGFR antibody. RESULTS Sixty patients were included with 47 assessable for pathologic response. The pCR rates were 46.8% [95% confidence interval (CI): 32.5% to 61.1%] and 55.3% [95% CI: 41.1% to 69.5%] according, respectively, to Chevallier and Sataloff classifications. The complete clinical response (cCR) rate was 37.5%. Conservative surgery was carried out in 87% of cases. Toxicity was manageable. The association of high EGFR and low cytokeratin 8/18 expression in tumor cells on one hand and high density of CD8+ tumor-infiltrating lymphocytes on the other hand were significantly predictive of pCR. CONCLUSIONS Panitumumab in combination with FEC100 followed by docetaxel appears efficacious, with acceptable toxicity, as neoadjuvant therapy of operable TNBC. Several biomarkers could help define large subsets of patients with a high probability of pCR, suggesting a potential interest to further develop this combination in biologically defined subgroups of patients with TNBC. CLINICAL TRIAL NUMBER NCT00933517.

Comparison of dysplasia profiles in stimulated ovaries and in those with a genetic risk for ovarian cancer.

AIM Ovarian epithelial dysplasia (OED) was first described after prophylactic oophorectomy for genetic risk of ovarian cancer. In light of Fathallas incessant ovulation theory, this study was set up to describe the presence of ovarian abnormalities (dysplasia) after ovulation induction and to compare dysplasia profiles in stimulated and genetic risk ovaries. METHODS One-hundred and twenty-four patients who had undergone salpingo-oophorectomies or ovarian cystectomies between 1990 and 2005 were reviewed. They were divided into three groups: (1) previous in vitro fertilisation (n=35); (2) prophylactic oophorectomies for genetic risk (n=27) and (3) fertile non-cancerous controls (n=62). Eleven cytological and architectural epithelial features were defined and a dysplasia score was calculated to quantify ovarian epithelial abnormalities. RESULTS Mean dysplasia score was significantly higher in the genetic risk and stimulated ovary groups than in controls (9.55 versus 3.62, p<0.0001; 7.51 versus 3.62, p<0.0002, respectively). Cytological and architectural abnormalities were more frequent in the genetic risk group, while the profile of abnormalities was different in the genetic risk and stimulated groups. CONCLUSIONS These findings support a possible relationship between OED and the use of ovulation-stimulating drugs. The increased dysplasia score in stimulated and genetic risk ovaries might be consistent with progression towards neoplastic transformation, and may justify the use of the term dysplasia or intraepithelial ovarian neoplasia. The observation of dysplasia in the stimulated group may differentiate women at risk. Conversely, the fact that the dysplasia profile after stimulation differs from that in genetic risk ovaries suggests that ovarian stimulation may predispose to a different evolution.

Multicentric neoadjuvant pilot Phase II study of cetuximab combined with docetaxel in operable triple negative breast cancer.

Systemic therapy for triple negative breast cancer (TNBC) is mostly based upon chemotherapy. Epithelial Growth Factor Receptor (EGFR) is overexpressed in around 50% of TNBC and may play a role in its pathogenesis. Consequently, we performed a multicentric pilot Phase II neoadjuvant trial of cetuximab (anti‐EGFR antibody) combined with docetaxel for patients with operable, Stage II–III TNBC. Therapy consisted of weekly cetuximab (first infusion: 400 mg/m2, then 250 mg/m2) combined with six cycles of docetaxel (T: 100 mg/m2) q.3 weeks. Subsequently, all patients underwent surgery. The primary endpoint was pathological complete response (pCR) while clinical response, toxicity and ancillary studies were secondary endpoints. Paraffin‐embedded and frozen tumor samples were systematically collected in order to identify predictive biomarkers of efficacy and resistance. From a total of 35 accrued patients, 25 were assessable for pathologic response. The pCR rate was 24% [95% CI: 7.3–40.7]. Complete clinical response rate (cCR) was observed in 22% of cases. Conservative surgery was performed in 75% of patients. Toxicity, mostly cutaneous and hematologic, was manageable. The pre‐therapy ratio between CD8+ and FOXP3+ tumor‐infiltrating lymphocytes equal or higher than 2.75 was predictive of pCR: 43% versus 0%, p = 0.047. Cetuximab in combination with docetaxel displays a modest activity, but acceptable toxicity as neoadjuvant therapy of operable TNBC. Similarly to previous observations using panitumumab, another anti‐EGFR antibody, the immune component of the tumor microenvironment may play an important role in predicting TNBC response to the neoadjuvant therapy.

Analyse peropératoire et définitive du ganglion sentinelle (GS), classification de l’atteinte ganglionnaire

The sentinel lymph node (SN) status is a crucial parameter in the therapeutic approach to breast cancer. However, there is no consensus established yet with regards to the method of SN assessment. The SN assessment ex tempore determines its status during a surgical procedure, thus allowing complete axillary dissection in case of SN positivity. That analysis is not performed systematically and can be done by cytological, histological (frozen sections) or molecular methods (qRT-PCR). The methods differ in sensitivity; qRT-PCR seems to be the most sensitive. The definitive SN status is also assessable by various approaches, immunohistochemistry (IHC) being the preferred one. However, the primacy of IHC is challenged by molecular biology methods.RésuméLe statut du ganglion sentinelle (GS) est une information primordiale dans la prise en charge du cancer du sein. L’analyse de ce ganglion ne fait pas l’objet d’un consensus. L’examen extemporané permet de connaître ce statut au cours de l’intervention et de permettre une totalisation du curage axillaire en cas de positivité. Cet examen n’est pas systématique et peut se faire par le biais d’une analyse cytologique, histologique (coupe congelée) ou par QRT-PCR. La sensibilité des différentes méthodes est variable, mais la technique moléculaire semble meilleure. L’analyse définitive du ganglion varie elle aussi, le recours à l’immunohistochimie (IHC) reste actuellement préférable. Mais cette dernière pourrait être remise en question par l’utilisation de l’analyse moléculaire.

Abstract P1-08-34: Is it possible to predict the efficacy of a combination of cetuximab plus docetaxel in patients with operable, triple negative breast cancer (TNBC)? Final biomarker results from a phase II neoadjuvant trial

Background: TNBC is a heterogenous group of tumors for some of which the Epithelial Growth Factor Receptor pathway (EGFR) may play an important role. We evaluated the efficacy and toxicity of an anti-EGFR antibody (cetuximab) combined with docetaxel, which were given to the TNBC patients (pts) in the neoadjuvant setting. A biomarker analysis accompanied this trial, aiming to identify predictors of response. Methods: 35 patients with stage II-IIIA TN breast disease were prospectively included in this multicentre pilot study. Systemic therapy (ST) consisted of 6 cycles of docetaxel (100 mg/m 2 ) each 3 weeks, in combination with weekly cetuximab (first dose 400mg/m 2 then 250 mg/m 2 /week) for 6 cycles. All patients underwent surgery at completion of ST. Patient characteristics : mean age 48 [28-67]; TNM: T1: 3%, T2: 73%, T3: 24%; N0: 61%, N1-N2: 39%; mean tumor size 40 mm [15-100]; SBR: grade III: 73%, grade II: 27%. The median number of cycles was for docetaxel 6 [1-6] and for cetuximab 15 [1-18]. Pathological complete response (pCR) rate was 24% according to the Chevallier and Sataloff classifications; 28% if we consider breast pCR only. Overall clinical response rate was 57% (22% CR). Conservative surgery was performed in 75% of cases. The main side effect was skin toxicity: grade II: 39%, grade III: 36%, grade IV: 3%. Other side effects were: neutropenia grade IV: 12.7%, febrile neutropenia: 1.3%, hand-foot syndrome grade III: 3%, grade II: 3%, ungueal toxicity grade III: 3%, grade II: 33%. Paraffin-embedded and frozen samples were systematically collected before and after ST for biomarker studies. Germinal BRCA1 mutations and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki67, p53 and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry. Results: The biomarker analysis was interpretable on the samples from 21 pts (3 pCR and 18 non-pCR). We applied the ROC curve to identify the best cut-off value for Ki67, EGFR, MET, cytokeratin 5/6 and 8/18, p53, ALDH1, PTEN, P-cadherin and the FOXP3+ or CD8+ TIL counts. None of these biomarkers was predictive of pCR except for the CD8+/FOXP3+ TIL count ratio. pCR rate was higher in the pts with the ratio equal or higher than 2.75 than in the others (43% versus 0%, p = 0.047). BRCA1 mutations were detected in 16% of pts. PI3K and EGFR somatic mutations were observed in 1 and 3 patients, respectively. The presence of the mutations was not predictive of pCR. Conclusion: Similarly to the previously reported trial by our group (SABCS 2012, abstract 1081), the immune component of the tumor microenvironment plays a very important role in the TNBC response to cytotoxic therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-34.

Abstract P3-14-19: Panitumumab in combination with FEC 100 (5-fluorouracil, epirubicin, cyclophosphamide) followed by docetaxel for operable, triple negative breast cancer (TNBC): Patient outcome

Background: Panitumumab is an antibody targeting the epidermal growth factor receptor (EGFR) for which an important role has been suggested in TNBC. Consequently, we evaluated a combination of the standard chemotherapy (FEC 100 followed by docetaxel) with panitumumab as neoadjuvant therapy of operable TNBC. Complete pathologic response (pCR) was the primary endpoint, with clinical response, toxicity, and outcome as secondary endpoints. An investigation of biomarkers possibly predictive of pCR accompanied this trial. Here we focus on tumor recurrence, after a median follow up of 33 months [25-40] as on April, 1, 2013. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic neoadjuvant treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm [20-120]; SBR grade III: 71.7%; pCR rate: 55.3% and 46.8% (the Sataloff and the Chevallier classifications, respectively). Paraffin-embedded and frozen tumor samples were collected before and after ST for biomarker analysis. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry. Results : .We have observed 9 recurrences: 1 local and 8 distant recidives, including 1 both local and distant. The distant recidives (metastases) were as follows: brain (4 pts); brain and lungs (1 pt); lungs only (1 pt), pleura (1 pt); liver (1 pt). 6 out of the 8 metastatic pts died and all were non-pCR post-ST. The 2 alive pts had brain metastases, but reached a pCR after the ST. Among the 9 relapsed pts 6 were 55 years old or less at the diagnosis. Seven out of those 9 pts had tumors with the clinical size equal or higher than 4 cm. As previously reported (SABCS 2012, abstract 1081), the pCR-predictive biomarkers in this study were high CD8+ TIL count (p = 3.4.10 −6 ) and high ratio between the CD8+ and FOXP3+ TIL counts (CD8+/FOXP3+ > 1.23, p = 8.5.10 −5 ). With this in mind, we have evaluated whether those parameters, assessed before or after the ST, could predict the recurrences. No difference was found in the preoperative CD8+ and the FOXP3+ TIL counts, as well as in the CD8+/FOXP3+ ratio, between the patiens who have recurred and the others. Conclusion : As it has been reported in previous studies, in our cohort of TNBC pts, the relapses occurred early after the administration of the last systemic treatment. The patients who relapsed died rapidly and most of them have not reached pCR after the ST. In addition, half of the metastatic pts got brain deposits. This implies that research on the resistance factors in TNBC should focus on those important for seeding of the “sanctuaries”, like brain. This research is ongoing in our group. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-14-19.

Abstract P5-08-07: HER3, MET and immune pathways play important roles in the resistance of triple negative breast cancer (TNBC) after neoadjuvant anti-EGFR (panitumumab) combined with FEC 100

Background: Our group has previously reported (SABCS 2012, abstract 1081) a 2-fold higher pathologic complete response (pCR) rate in TNBC treated with panitumumab+FEC100 (47%) than with the standard therapy alone. However, in a half of the patients (pts) a residual tumor (RT) was detected. Having in mind the detrimental impact of resistant TNBC on patient outcome, we have investigated the tumor tissue factors that might have been important in development of resistance to this treatment. Methods: 40 TNBC pts, treated with 8 cycles the anti-EGFR antibody panitumumab combined with FEC 100 for the first 4 and with docetaxel only for the last 4 cycles, were eligible. All pts underwent breast surgery after the neoadjuvant chemotherapy (NACT) completion. Formalin-fixed, paraffin-embedded tumor samples were collected before and after NACT for biomarker analysis. EGFR, HER3, IGF-1R, MET and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry (IHC). Expression of the kinases was graded semi-quantitatively (histoscores). Results: Out of 40 treated pts, 21 demonstrated a RT (53%, according to the Chevallier classification, PMID 8338056). The IHC data were available for 17 pts. Since high EGFR expression was important for pCR achievement in this study (SABC 2012), we first investigated the expressions of EGFR, HER3 and MET in the RT. The results are presented in Table 1. Overall, among the 9 cases (cs) with increased post-NACT HER3 expression (HER3 up), only 1 had increased MET (MET up), while among the 7 cs with no change in HER3 post-CTNA (HER3 flat), 4 had MET expression increased. Interestingly, although the pre-NACT IGF-1R expression was a strong predictor of pCR (p = 0.028, SABCS 2012), no significant changes in this kinase expression were observed in the RT, compared to the pre-NACT levels. Most RT had low IGF-1R scores ( The density of TIL post-NACT correlated principally with the% of tumor mass (TM) reduction. Among the 7 pts with average reduction of 20%, there was only 1 cs with a good InSitu Immune Response (ISIR: dense TIL, with CD8+/FOXP3+ > 1.2). On the contrary, among the 9 pts with average TM reduction of 90%, 8 demonstrated a good ISIR. Conclusion: The results indicate that HER3 and MET play significant roles in development of TNBC resistance to the anti-EGFR agents. Those molecules seem to drive 2 different resistance pathways in TNBC, as it has been shown in other malignancies. As several anti-HER3 or anti-MET drugs are nowadays available, we find very important investigating the expression of HER3 and MET in each TNBC pt pre- and post-NACT, in order to rapidly detect and combat the resistance. The ISIR-stimulating agents may bring an additional therapeutic benefit to those patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-07.

Abstract P3-06-20: Is it possible to predict the efficacy of a combination of Panitumumab plus FEC 100 followed by docetaxel (T) for patients with triple negative breast cancer (TNBC)? Final biomarker results from a phase II neoadjuvant trial.

Background: TNBC is an heterogeneous group of tumors for some of which the Epithelial Growth Factor Receptor pathway (EGFR) may play an important role. We evaluated the efficacy and toxicity of an anti-EGFR antibody (panitumumab) combined with a standard neoadjuvant chemotherapy in order to identify predictive biomarkers of efficacy and target biologically defined subpopulations for potential further development. Methods: Sixty patients with stage II-IIIA disease were prospectively included in this multicentre neoadjuvant study. Systemic therapy (ST) consisted of panitumumab (9 mg/kg q.3 weeks x8) combined with FEC 100 (500/100/500 mg/m 2 q.3 weeks x4) followed by 4 cycles of T (100 mg/m 2 q.3weeks x4). All patients underwent surgery at completion of ST. Paraffin-embedded and frozen samples were systematically collected before and after ST for biologic studies. Patients characteristics are as follows: mean age 50 [27–72]; median tumor size: 40 mm [20–120]; invasive ductal carcinoma: 96.7%; Scarff-Bloom-Richardson Grade III: 71.7%, grade II: 28.3%. Complete pathological response (pCR) rate was 52.3% [95% IC: 37.3–67.5] (Sataloff9s classification) and 46.7% [95% IC: 31.6–61.4](Chevallier9s classification). Conservative surgery was performed in 88% of cases. Skin toxicity was the main side-effect: Cutaneous toxicity grade IV: 5%, grade III: 30%, grade II: 20%. Neutropenia grade IV: 27%; febrile neutropenia: 5%. Infection: 0%. Hand-foot syndrome grade III: 3.3%. Ungueal toxicity grade III: 1.6%, grade II: 20%. Results: We performed a ROC curve to identify the best cut-off value for KI-67, EGFR, cytokeratin 5–6 and p53 in order to predict a pCR. Tumors with more than 40% of positive cells for KI-67 and tumors with a score for EGFR greater than 70 tend to be associated with pCR according to Chevallier9s classification (p = 0.06). No predictive value was identified for Cytokeratin 5–6 and p53 (p = 0.61 and p = 0.27, respectively). Immunohistochemistry results show that two thirds of tumors have more than 40% of positive cells for KI-67 and that two thirds of tumors present a score for EGFR greater than 70. About half of the tumors express cytokeratin 5–6 and p53 (cut off: 1%). Chi-squared tests were performed to assess relations between cutaneous toxicities and pCR. The cutaneous toxicities were not predictive of pCR (p = 0.94) and no correlations were found with KI-67, EGFR, Cytokeratin 5–6 and p53. In terms of BRCA1 and BRCA2 status, 35 tumors were analysed so far: BRCA1: 6 mutations (17%); BRCA2 (30 patients): 1 mutation (3.3%). Conclusions: These results suggest the possibility to identify a subpopulation with high probability of pCR (KI-67 > 40%, EGFR score > 70). Further biological studies are ongoing and will be presented at the meeting, including EGFR polymorphisms, C-met, ALDH1, pCadherine and PTEN. This will help us further define subpopulations of TNBC patients, potential targets for antiEGFR development. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-20.

Chirurgie conservatrice du cancer du sein: évaluation des berges d’exérèse lors de la chirurgie première

Conservative surgery is the actual gold standard for the management of low-stages (i.e. I and II) breast cancers. The completion of clearmargins isaprerequisite for a complete local treatment and a decrease of local recurrences. Atight collaboration between pathologists and surgeons is mandatory. There is no consensus for the definition of the cut of a clear margin. Different management processes have been reported through the literature, ranging fromgrossmanagement to cytologic or histopathologic methods for margin evaluation. Such approaches have to be correlated to the clinical and radiological setting. They have to be performed in line with tumor characteristics, and treatment plans. The perspectives are a better evaluation of surgical margins using nuclear medicine or molecular biology tools to decrease the number of second surgical procedures.RésuméLe traitement conservateur est actuellement la référence dans la prise en charge des cancers du sein de stades I et II. L’obtention de marges saines est un facteur primordial pour la diminution du taux de récidives locales. Elle ne fait l’objet d’aucun consensus tant pour la technique d’évaluation des berges chirurgicales que pour la valeur seuil limite d’une marge d’exérèse de sécurité. Diverses approches ont été évaluées dans la littérature, depuis la prise en charge macroscopique de la pièce à sa réception par le pathologiste à l’emploi de méthodes cytologiques et histologiques d’évaluation des berges. De telles pratiques sont évidemment à replacer dans un contexte radioclinique et doivent faire l’objet d’une collaboration étroite entre les différents spécialistes. Les perspectives sont une amélioration de l’évaluation des limites et/ou des marges tumorales à l’aide de techniques de médecine nucléaire, et/ou de biologie moléculaire afin de diminuer le nombre de reprises chirurgicales.

Abstract P1-01-15: Intraoperative Imprint Cytology Examination of Sentinel Lymph Nodes after Neoadjuvant Chemotherapy in Breast Cancer Patients

Background: Intraoperative imprint cytology (IC) is one of several accurate, proved methods to detect tumor cells in sentinel lymph nodes (SLN) from patients with operable breast cancer. In patients treated by neoadjuvant chemotherapy (NAC), studies have demonstrated the feasibility and accuracy of SLN biopsy procedure. The aim of this study was to evaluate the validity of tumor cell detection using IC in SLN from patients who underwent NAC. Material and methods: Patients with infiltrating breast carcinoma receiving NAC (n= 132) were studied prospectively. At surgery, SLN biopsy followed by axillary lymph node dissection was performed. Intraoperative SLN evaluation using IC was performed in 80 out of 132 patients (60%). In the same period, IC has been evaluated at random for 100 chemonaive patients in the same institution. Results: Sentinel lymph node metastases were correctly identified using IC in 58 of 80 (72%) patients. False negative results were observed in 21 patients. The overall sensitivity and specificity of IC for the identification of lymph node metastases was respectively 38.2% and 97.8%. The positive predictive value was 92.9% and the negative predictive value was 68.2%. In univariate analysis and multivariate logistic regression analysis, patients with micrometastases or ITC in SLN have 2.3 times higher risk of a false negative IC result than patients with macrometastases of the SLN (P= 2.1.10 -4 ; RR= 2.3; 95% confidence interval [1.37-3.85]). For chemonaive patient, sensitivity, specificity, PPV and NPV were respectively 47.4%, 98.8%, 90% and 88.9%. Conclusion: The accuracy of intraoperative IC observed in our study is similar to that reported previously in patients without NAC, indicating that IC is a clinically acceptable method for intraoperative pathological examination of SLN for patients after NAC. Variations in sensitivity are related to the proportion of cases with micrometastases and ITC, as it was also shown in chemonaive patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-01-15.