A. de Roquancourt

Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches

Background:Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT–PCR), but are not routinely used. We evaluated the relevance of Q-RT–PCR for HER2 status determination.Methods:We compared IHC and Q-RT–PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection.Results:We observed 97.3% concordance between Q-RT–PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT–PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones.Conclusion:Q-RT–PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT–PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

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.

Complete pathological response according to hormonal status, c-erbB2 and P53 in two neoadjuvant treatments in locally advanced breast cancers.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5107 Background : We previously showed that the p53 mutation is the major factor for response to dose-dense epirubicin-cyclophosphamide regimen (PLoS Med. 2007; Lancet 2002). Some years ago Taxanes became a standart treatment in neoadjuvant setting. We study two regimens of neoadjuvant anthracycline-based chemotherapy combined with high dose cyclophosphamide (C), or with Taxotere in locally advanced breast cancers (LABC) with respect to pathologic complete response (pCR). Materials and methods : Between 1990 and 2003, 219 patients (pts) with LABC and/or inflammatory breast cancers (IBC) from one institution received 6 cycles (c) of dose dense C (1.2g/m2 d1)-epirubicin (E) (75mg/m2d1) q2w (SIM regimen). From 2003 to 2008, 132 pts with LABC and/or IBC from the same institution received 4 c of E (75mg/m2 d1) C(750mg/m2 d1) q3w followed by 4c of docetaxel 100mg/m2 d1q3w (ECT regimen). Surgery was done after chemotherapy and pCR was defined as no residual invasive tumour in breast and lymph nodes. Results: 139/219 SIM pts (63.4 %) and 96/132 ECT (72.7 %) pts had frozen biopsies, surgery after chemotherapy, ER, c-erbB2 determination (IHC and /or RT-PCR) and p53 done by yeast functional complementation assay (FASAY) and were thus included in our study. ![][1] ![][2] Conclusion: The high performance of the FASAY assay allows the demonstration of p53 role in the prediction of histological response in the ECT, as previously for the SIM regimen. In p53 mutant tumors, especially in ER- and ER- c-erbB2 negative tumors, high dose cyclophosphamide combined with E seems significantly better than Taxane regimen and needs to be studied prospectively. Future trials should determine the best regimen for p53 WT tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5107. [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif

Abstract P1-14-08: Prediction of pathological response (pCR) to neoadjuvant dose dense and intense cyclophosphamide and anthracycline in a prospective series of triple negative locally advanced breast cancers (TNLABC)

Background : Stage II-III TNBC retains a poor outcome despite high chemosensitivity. Patients (pts) with pCR after neoadjuvant chemotherapy have a good prognosis whereas non-responding pts have a 25-40% risk of distant relapse at 5 years. pCR is thus a major goal in TNBC. We previously reported that TNLABC benefit the most of dose dense dose intense cyclophosphamide (C)-epirubicin (E) ( S.Giacchetti; BJC, 2014 ) Aim : To confirm these results prospectively and analyze the predictive factors of response to high dose chemotherapy in TNBC. Patients and methods : From january 2009 to april 2015 non inflammatory TNLABC received high dose C (1200 mg/m2 d1 qw 2) with E (75 mg/m2/ d1 qw2) for 6 cycles. The pts had a breast biopsy with frozen tissue. We performed molecular studies: qRT-PCR for AR, FOXA1, PI3K and FASAY technic for p53 mutation.The percentage of stromal Tumor-infiltrating lymphocytes (TILs) was also evaluated by two independent pathologists and assessed as a continuous variable. A18F-FDG PET/CT was performed initially and after 2 courses of chemotherapy and the metabolic answer assessed as a variation of the tumor uptake (ΔSUVmax). We report here the pathological complete response (pCR) (absence of infiltrative carcinomas in the breast and in the lymph nodes) and the factors associated with pCR. Results : The characteristics of the 74 pts are listed in table 1. The median age is 48 years old, 48 pts (65.8%) are premenopausal and 79% did not have any family history of breast cancers. TIL was divided in 3 groups 50% (9 pts, 14 %). Pathological response was assessed in 66 pts, one pt progressed during chemotherapy and 6 pts did not undergo surgery yet. 28 pts were in pCR (42.4 %). With a median follow up of 25 months, 13 pts (17.8 %) progressed and 8 (11%) died. Tumor size, tumor grade, percentage of TILs, the change in 18F-fluorodeoxyglucose tumor uptake (ΔSUVmax) were significantly associated with pCR at univariate analysis. Only one factor remained significant at multivariate analysis, the ΔSUVmax, OR: 0.04 [0.007- 0.27], p = 0.0008. Conclusion : In this prospective phase III trial we confirm the efficacy of a dose dense EC in TNBC. The metabolic response evaluated with 18 F-FDG PET/CT is a strong and reliable predictor of pCR and could allow an early change of treatment for the non responders. A clinical trial is planned to test this strategy. Citation Format: Giacchetti S, De Roquancourt A, Groheux D, Piron P, Lehmann-che J, Cuvier C, Resche-rigon M, Albiter M, Roche B, Frank S, Hamy A-S, Teixeira L, Marty M, Lalloum M, Espie M. Prediction of pathological response (pCR) to neoadjuvant dose dense and intense cyclophosphamide and anthracycline in a prospective series of triple negative locally advanced breast cancers (TNLABC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-14-08.

Abstract P4-13-03: Hormone replacement therapy, is there an increased risk of in situ breast cancer ? data from a french cohort

Background: Use of hormone replacement therapy (HRT) has been associated with an increased risk of breast cancer. Few studies focus on correlation between HRT and in situ breast cancer A large cohort study, the Million Women Study ( Reeves GK et al. Lancet Oncol. 2006 ) have shown that the use of HRT has been associated with an increased incidence of in situ breast cancer (RR = 1, 55, IC=1, 4–1,72, p = 0,03). Purpose: We investigated the association between HRT use and duration and in situ breast cancer incidence from a data base of infraclinic breast lesions. Materials & Methods: From 2007 to 2011, 2708 patients (pts) with a non palpable breast lesion were referred to our breast disease unit for exploration (Saint Louis Hospital, Paris). Radiological abnormalities were screened by mammography and/or breast ultrasound, and/or MRI. Out of 2708 pts, 1668 had a biopsy. All biopsies were seen by an anatomopathologist dedicated to breast. Here, we focus on the 1017 postmenopausal women (61%). Results: Biopsies revaled invasive breast cancers in 222 pts (22%), in situ breast cancers in 164 pts (16%) [10 lobular in situ carcinoma and 154 ductal in situ carcinoma], high risk breast lesions in 103 pts (10%) and benign breast lesions in 528 patients (52%). Characteristics of the 164 patients with in situ breast cancer: median age 62 [IQR: 57 to 68], at least one pregnancy (mean number of pregnancies per woman, 1.6) 75,6%, family history of breast cancer:30,2%. Among these 164 patients, 42.6 % had used an oral contraceptive and 53% of them a HRT, with a mean duration of use of 7 years. The HRT use was not significantly associated with in situ breast cancer (p = 0.66) as well as the duration of HRT. Discussion: In this study of postmenopausal women, HRT use, whatever is the duration, was not associated with in situ breast cancer. Although a lack of statistical power may be invoked to explain these results, these findings suggest that the type of the HRT could be important to explain the difference between our study and the Million Women Study. In our study, the HRT used is in the majority of the pts, percutaneous oestrogens and natural progesterone as it is the most commun HRT used in France ( Fournier A, Int J Cancer. 2005 ) during this period of time. Use and type of HRT are different in USA where conjugated estrogens and medroxy progestogerone acetate were commonly used. Conclusion: We therefore encourage further studies to better appreciate the links between type of HRTs and in situ breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-13-03.

Abstract P1-11-14: Neoadjuvant Chemotherapy in Lobular and Ductal Carcinoma: Comparison of Clinical, Pathological Response Rates and Survival

Introduction: Neoadjuvant chemotherapy is a standard of care in locally advanced breast carcinomas. The major purpose of neoadjuvant chemotherapy is breast conserving surgery. Advantages in survival have only been shown in patients undergoing achieving pathological complete response (pCR). Lobular carcinomas seem less chemosensitive than ductal carcinomas to neoadjuvant chemotherapy (NCT). Purpose: To compare the clinical and pathological response rate and the outcome of lobular (ILC) versus ductal (IDC) invasive breast carcinomas after NCT. Patients and methods: Between 1985 and 2010, 385 patients with locally advanced and/or inflammatory breast carcinomas from Saint Louis hospital received neoadjuvant chemotherapy. Forty-four (11, 4%) ILC and 341 (88, 6%) IDC were diagnosed by surgical or core needle biopsy before CT. All patients received anthracyclines based CT,181 (47%) additionally received a taxane (four cycles of epirubicin 75 mg/m 2 and cyclophosphamide 750 mg/m 2 then four cycles of taxotere 100mg/m2 or six cycles of a dose dense regimen of 75 mg/m2 epirubicin and 1200mg/m2 cyclophosphamide, every 14 days),and underwent breast surgical excision (lumpectomy or mastectomy) and axillary node dissection.Clinical response was defined by the lack of palpable tumor in the breast before surgery.Pathological complete response was defined by no residual invasive tumor in breast. Radiotherapy and hormonotherapy were delivered to patients, when appliable. Results: Clinical response to NCT was higher for ILC (27, 3%) than IDC (13,4%). ILC with clinical response tended to have higher histological grade 2 (91,6 % versus 30,4% for IDC),more estrogens receptors (RE) positivity (91,6% vs 45,6%), p53 wild type (66,6% vs 30,4%) and HER2- negative tumors (91,6% vs 76%) Pathologic response (pCR) rate was lower for ILC than for IDC (2, 7% vs 9, 3%). Only one ILC (grade 2, RE+, HER2 negative, p53 wild type) underwent pCR. Thirty two (9, 3%) IDC haved pCR: 15,6% haved histological grade 2, 15,6% RE positivity,9,3% p53 wild type, 62,5% HER2 negative. Thirteen percent of ILD and 9 % of ILC haved breast conservating surgery .At a median follow up of 60 months, ILC patients tended to have longer overall survival (55% vs 48%) and recurrence free survival (44% vs 36,8%) than IDC. Conclusions: ILC was characterized by better clinical response rates but lower pathologic response and breast conservating surgery rates .Despite the low pCR rate, patients with ILC tended to have better outcomes than did patients with IDC. Pathological complete response to NCT in ILC did not seem to have prognostic significance. Further data are warranted to help clarify the characteristics genomics and proteomics of ILC which explains this better outcome. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-14.

Abstract P6-05-06: Clinical Presentation of Molecular Apocrine Subgroup of Breast Cancer: A Rather Aggressive Group of Tumor

Background: Estrogen receptor negative (ER-) breast cancer represents 30% of breast cancers. This heterogeneous group comprises at least the basal and HER2+ subgroups. Recent data, as well as our own, has observed that the HER2+ subtype is highly heterogeneous. Several teams have identified a new “apocrine” molecular subgroup of cancer, characterized by androgen receptor (AR) expresion in an ER-context. Here, we have retrospectively identified, based on a transcriptionnal signature, these apocrine molecular tumours and described their clinical presentation and evolution. Material and Methods: We retrospectively identified 60 patients treated in St Louis Hospital (Paris) from 1995 to 2008 and presenting the signature of the molecular apocrine subgroup (ERA-, AR+, FOXA1+) by Q-RT-PCR. Results: Mean age at diagnosis was 53,5 y.o. Tumours size were T2 or more in 78% cases. Histological types were ductal invasive with intraductal component (n=22), histological apocrine (n=3), and paget disease (n=4). Tumor grade was 3 in 68%, and 2 in 21%, with lymphovascular invasion in 37%. Excluding patients receiving neo-adjuvant chemotherapy, lymph node status was negative in 41%, and positive in 52% (1 to 3, 32%, more than 4N+, 20%). By immunohistochemistry 97.4% were PR-and 58.72% overexpressed HER2. Surgery was conservative in 46%, and 48% patients underwent mastectomy. Sixteen patients received neoadjuvant chemotherapy (27%), 41 received adjuvant chemotherapy (68%), 16 received hormonal therapy (27%), and 16 received trastuzumab (27%). With a median follow up of 60 months, 34 events (local recurrence n=9, contralateral n=3, distant metastasis n=22), and 13 deaths occurred. Median disease free survival was 48 months. Discussion: In this cohort of apocrine molecular carcinomas, tumor phenotypes appears to be rather aggressive, with a high proportion of poor prognosis factors (grade SBR3, lymphovascular invasion, node involvement), and are generally well-correlated to a poor clinical outcome in this population that received heterogeneous treatments. Further data are needed to precisely characterise this particular breast cancer subtype, notably patients who are not eligible to Herceptin-based regimen. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-06.

Abstract P6-09-06: Oral Contraceptive Use and Overall Breast Disease Risk: Cross Sectional Retrospective Study

Background : Oral contraception (OC) is one of the most widely used means for birth control in the world. In several studies, OC has been associated with a slightly increased risk of breast cancer in current user, and with a decreased risk of benign disease. Little is known about atypical high risk lesions. Our goal was to investigate whether OC use was a risk factor or a protective factor for benign, high risk or malignant breast lesions. Material and methods : From 2001 to 2007, all non-palpable breast lesions referred to biopsy or cytology in Saint Louis hospital were prospectively registered. Demographic and clinical data including oral contraceptive pill use extent were reported. We defined benign lesions (fibroadenoma, blunt duct adenosis, fibrocystic changes, epithelial hyperplasia, others), high risk lesions (atypical ductal hyperplasia, atypical lobular hyperplasia, lobular carcinoma in situ and malignant lesions (ductal carcinoma in situ, invasive ductal or lobular carcinoma). The aim was to analyse the correlation between the duration of OC use and the occurrence of the breast disease classified by histology groups. Patients with previous history of benign breast disease or malignancy were excluded. Results: The analysis was performed on 1329 breast lesions. The breakdown of the lesions were as follows: 819 benign lesions (fibroadenoma n=155, blunt duct adenosis n=169, fibrocystic disease n=194, epithelial hyperplasia n=132, others n=170), 104 high risk lesions (atypical ductal hyperplasia n=54, atypical lobular hyperplasia n=29, lobular carcinoma in situ n=21), and 406 malignant lesions (ductal carcinoma in situ n=158, invasive ductal or lobular carcinoma n=248). The duration of oral contraception use was not significantly associated with the occurrence of benign, high risk or malignant breast disease. When focusing on benign lesion subtypes, no association was observed either. Older age was significantly correlated to the occurrence of atypia or carcinoma. Significant difference existed in the median age of apparition 55 y.o and 57 y.o respectively, versus 53 y.o for benign lesion (p Conclusion: In this cross sectional retrospective study, the duration of OC use was not associated with differential occurrence of benign, high risk and malignant breast lesion. Although a lack of statistical power may be invoked to explain the results, we are called to believe that the magnitude of an effect of OC use is small, if ever it exists. Results [Table 1] Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-09-06.

Correlation between 18F Fluorodeoxyglucose (FDG) Uptake and Tumor Characteristics in Locally Advanced Breast Cancers.

Background: This study analysed the correlation between [ 18 F]fluorodeoxyglucose (FDG) uptake, assessed by positron emission tomography (PET), and prognostic factors in locally advanced breast cancers Material and Methods: All locally advanced breast cancers seen at Saint Louis hospital and treated with neo-adjuvant chemotherapy (NAC) have a FDG PET after core needle biopsy and before chemotherapy. We correlated the tumor characteristics: T-stage, histological grade, estrogen and progesterone receptors, c-erbB2 over-expression (immuno-histochemistry determination) and P53 (determinate on frozen biopsies by the FASAYmethod) to FDG standardized uptake value (SUV max). The statistics tests used are student test (comparison of 2 means) and kendall correlation. Results: From June 2006 to April 2009, 91 patients with locally advanced breast tumors have both PET scan and frozen tissue before NAC. Median age at diagnosis is 48 (26-81) and 45 % are post menopausal. Conclusion: This study indicates that FDG-PET uptake is correlated with the phenotype of breast tumours. Over-expression of c-erbB2 does not influence FDG uptake. Triple negative tumours and p53 mutated tumors have a high initial SUV which can reflect their aggressiveness and their chemotherapy sensitivity. The knowledge of SUV uptake according to tumor characteristics allows a better understanding of the role of FDG-PET in the prediction of neoadjuvant chemotherapy response. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5010.

The Role of FDG PET for Early Prediction of Response after Two Cycles of Epirubicin + Cyclophosphamide Neoadjuvant Chemotherapy in Breast Cancer.

Background: Previous studies showed a possible role of Fluorodeoxyglucose Positron Emission Tomography (FDG PET) in monitoring response to neoadjuvant chemotherapy (NACT) in breast cancer patients. Most studies, however, mixed various chemotherapy protocols. We assessed the ability of FDG PET to predict response after two cycles of epirubicin + cyclophosphamide (EC) and compared it with histopathological response as determined by the Sataloff scale after completion of chemotherapy.Material and Methods: From 07/2007 to 05/2009, 54 patients seen at Saint Louis hospital underwent FDG PET at baseline and after the second cycle of NACT. We present data for the first 22 consecutive patients for whom pathology data are available. Standard chemotherapy treatment was four cycles of epirubicin + cyclophosphamide followed by four cycles of docetaxel. The study was performed according to the guidelines of the institutional ethical committee. The standardized uptake value (SUVmax) of FDG was measured with a PET-CT instrument at baseline and after the second cycle of chemotherapy. The change in SUV was expressed as Δ SUVmax (%) = 100 X (2 nd cycle SUVmax - baseline SUVmax)/baseline SUVmax. A Δ SUVmax cutoff value of -45% was used to differentiate metabolic responders and non-responders. Histopathological response was assessed on fresh surgical specimens (mastectomy or lumpectomy) by an experienced pathologist and graded according to the scale established by Sataloff: total or near-total therapeutic effect (grade A), more than 50% therapeutic effect but less than total or near-total effect (grade B), less than 50% therapeutic effect but visible effect (grade C), or no therapeutic effect (grade D). For the analysis, grades A and B were considered as histopathological responders and grades C and D were as non-responders.Results: Initial T-stage was T2 in 12 cases, T3 in 6 cases and T4 in 4 cases. There were 21 cases of invasive ductal carcinoma and 1 case of invasive lobular carcinoma. Mean SUVmax in initial PET was 7.22 (ranges from 2.7 to 18.5) and mean SUVmax after 2 cycles of chemotherapy was 4.77 (1.4-15.3). Breast-conserving surgery was performed in 12 patients and mastectomy in 10 patients. Nine (41%) of 22 patients were classified as histopathologic responders and 13 (59%) as non-responders. PET after two cycles of NACT revealed 9 patients (41%) as responders and 13 (59%) as non-responders. Among 9 metabolic responders, 7 were true positive, and 2 were false positive. Among 13 metabolic non-responders, 11 were true negative, and 2 were false negative. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of FDG PET after two cycles of NACT were 78%, 85%, 78%, 85% and 82% respectively.Discussion: NACT has proved useful in stage II and III breast cancer, to reduce tumour volume, increasing the chance of breast-conserving surgery. In order to minimize adverse effects of NACT, non-responders must be identified as early as possible. Our preliminary results on a small series of patients show that FDG PET can differentiate responders from non-responders with good accuracy after two cycles of neoadjuvant chemotherapy with EC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5009.