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Resume Introduction La mise en place d’un controle de qualite interne est indispensable a la fiabilite des resultats du statut HER2 dans les cancers du sein. Objectifs Evaluer l’impact de notre assurance qualite interne sur les resultats HER2 dans les carcinomes mammaires infiltrants entre 2008 et 2014. Methodes Le statut HER2 etait determine par immunohistochimie en premiere intention et hybridation in situ en fluorescence (FISH) pour controle des scores 2+. Le controle de qualite interne du statut HER2 reposait sur une standardisation des phases pre-analytiques, l’utilisation de temoins externes au nombre de copies du gene HER2 connues par FISH et la surveillance reguliere de la concordance entre l’immunohistochimie et la FISH. Resultats La proportion de cas HER2-positifs correspondant a la somme des scores 3+ en immunohistochimie et 2+ amplifies en FISH variait entre 10,6 % et 13,8 % (mediane de 11,3 %). La proportion de scores 2+ amplifies en FISH variait de 13,3 % a 32,7 % sur la periode de l’etude. Le taux de concordance entre la FISH et l’immunohistochimie pour les scores 0/1+ et 3+ etait ≥ 97 %. Huit sur 12 cas discordants etaient des faux positifs resultant d’erreurs d’interpretation de l’immunohistochimie (score 2+ surcotes en 3+). Discussion La calibration de l’immunohistochimie sur la FISH pour le statut HER2 a permis de limiter la variabilite des resultats d’immunohistochimie liee a la technique ou l’interpretation. La mise en place sur chaque lame d’un temoin score 3+ est garant de l’interpretation adequate des scores 2+ et 3+ en immunohistochimie.

Background: RSPO ligands, activators of the Wnt/β-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). Methods: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT–PCR. The effect of RSPO on the Wnt/β-catenin pathway activity was determined by luciferase assay, western blotting, and qRT–PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/β-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. Results: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10−4). RSPO2 and RSPO4 stimulate Wnt/β-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. Conclusions: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Exome sequencing has recently identified highly recurrent MED12 somatic mutations in fibroadenomas (FAs) and phyllodes tumors (PTs). In the present study, based on a large series, we confirmed the presence of MED12 exon 1 and 2 mutations in 49% (41/83) of PTs, 70% (7/10) of FAs and 9.1% (1/11) of fibromatoses. We show that MED12 mutations are associated with benign behavior of phyllodes tumors, as they are detected less frequently in malignant PTs (27.6%) compared to benign (58.3%) and borderline (63.3%) PTs, respectively (p = 0.0036). Phyllodes tumors presented marked temporal heterogeneity of MED12 mutation status, as 50% (3/6) of primary and recurrent phyllodes tumor pairs with MED12 mutation presented different MED12 mutations between the primary and recurrent tumors. There was no correlation between MED12 status and genomic profiles obtained by array-CGH. MED12 mutations are associated with altered expressions of the genes involved in the WNT (PAX3, WNT3A, AXIN2), TGFB (TAGLN, TGFBR2, CTGF) and THRA (RXRA, THRA) signaling pathways. In conclusion, this study confirmed that MED12 plays a central oncogenic role in breast fibroepithelial tumorigenesis and identified a limited number of altered signaling pathways that maybe associated with MED12 mutations. MED12 exon 1 and 2 mutation status and some of the altered genes identified in this study could constitute useful diagnostic or prognostic markers, and form the basis for novel therapeutic strategies for PTs.

Reciprocal interactions between tumor cells and their microenvironment vitally impact tumor progression. In this study, we show that GM-CSF produced by primary breast tumor cells induced the activation of plasmacytoid predendritic cells (pDC), a cell type critical to anti-viral immunity. pDC that expressed the GM-CSF receptor were increased in breast tumors compared with noninvolved adjacent breast tissue. Tumor-activated pDC acquired naïve CD4+ T-cell stimulatory capacity and promoted a regulatory Th2 response. Finally, the concomitant increase of GM-CSF and pDC was significantly associated with relatively more aggressive breast cancer subtypes. Our results characterize the first tumor-derived factor that can activate pDC to promote a regulatory Th2 response, with implications for therapeutic targeting of a tumor-immune axis of growing recognition in its significance to cancer. Cancer Res; 75(14); 2775–87. ©2015 AACR.

// Marie-Paule Sablin 1, * , Coraline Dubot 1, 2, * , Jerzy Klijanienko 3 , Sophie Vacher 2 , Lamia Ouafi 3 , Walid Chemlali 2 , Martial Caly 3 , Xavier Sastre-Garau 3 , Emmanuelle Lappartient 3 , Odette Mariani 3 , Jose Rodriguez 4 , Thomas Jouffroy 4 , Angelique Girod 4 , Valentin Calugaru 5 , Caroline Hoffmann 4 , Rosette Lidereau 2 , Frederique Berger 4, 6 , Maud Kamal 1 , Ivan Bieche 2, 7 , Christophe Le Tourneau 1, 8 1 Department of Medical Oncology, Institut Curie, Paris and Saint-Cloud, France 2 Unit of Pharmacogenomics, Department of Genetics, Institut Curie, Paris, France 3 Department of Biopathology, Institut Curie, Paris, France 4 Department of Surgery, Institut Curie, Paris, France 5 Department of Radiotherapy, Institut Curie, Paris, France 6 Department of Biostatistics, Institut Curie, Paris, France 7 EA7331, Paris Descartes University, Sorbonne Paris Cite, Faculty of Pharmaceutical and Biological Sciences, Paris, France 8 EA7285, Versailles-Saint-Quentin-en-Yvelines University, Versailles, France * These authors equally contributed to this work Correspondence to: Coraline Dubot, email: coraline.dubot@curie.fr Keywords: head and neck squamous cell carcinoma, gene expression, clinical prognostic and theranognostic biomarkers Received: January 21, 2016 Accepted: June 01, 2016 Published: June 18, 2016 ABSTRACT Background: We aimed at identifying druggable molecular alterations at the RNA level from untreated HNSCC patients, and assessing their prognostic significance. Methods: We retrieved 96 HNSCC patients who underwent primary surgery. Real-time quantitative RT-PCR was used to analyze a panel of 42 genes coding for major druggable proteins. Univariate and multivariate analyses were performed to assess the prognostic significance of overexpressed genes. Results: Median age was 56 years [35–78]. Most of patients were men (80%) with a history of alcohol (70.4%) and/or tobacco consumption (72.5%). Twelve patients (12%) were HPV-positive. Most significantly overexpressed genes involved cell cycle regulation (CCND1 [27%], CDK6 [21%]), tyrosine kinase receptors ( MET [18%], EGFR [14%]), angiogenesis ( PGF [301%], VEGFA [14%]), and immune system ( PDL1/CD274 [28%]). PIK3CA expression was an independent prognostic marker, associated with shorter disease-free survival. Conclusions: We identified druggable overexpressed genes associated with a poor outcome that might be of interest for personalizing treatment of HNSCC patients.

ANRIL, a long noncoding RNA (lncRNA), has recently been reported to have a direct role in recruiting polycomb repressive complexes PRC2 and PRC1 to regulate the expression of the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. Expression analysis of ANRIL, EZH2, SUZ12, EED, JARID2, CBX7, BMI1, p16, p15 , and p14/ARF genes was evaluated in a large cohort of invasive breast carcinomas (IBC, n = 456) by qRT-PCR and immunohistochemistry (IHC) was performed on CBX7, EZH2, p14, p15, p16, H3K27me3, and H3K27ac. We observed significant overexpression in IBCs of ANRIL (19.7%) and EZH2 (77.0%) and an underexpression of CBX7 (39.7%). Correlations were identified between these genes, their expression patterns, and several classical clinical and pathologic parameters, molecular subtypes, and patient outcomes, as well as with proliferation, epithelial–mesenchymal transition, and breast cancer stem cell markers. Multivariate analysis revealed that combined EZH2/CBX7 status is an independent prognostic factor ( P = 0.001). In addition, several miRNAs negatively associated with CBX7 underexpression and EZH2 overexpression. These data demonstrate a complex pattern of interactions between lncRNA ANRIL , several miRNAs, PRC2/PRC1 subunits, and p15/CDKN2B-p16/CDKN2A-p14/ARF locus and suggest that their expression should be considered together to evaluate antitumoral drugs, in particular the BET bromodomain inhibitors. Implications: This study suggests that the global pattern of expression rather than expression of individual family members should be taken into account when defining functionality of repressive Polycomb complexes and therapeutic targeting potential. Mol Cancer Res; 14(7); 623–33. ©2016 AACR. This article is featured in Highlights of This Issue, [p. 587][1] [1]: /lookup/volpage/14/587?iss=7

BACKGROUND The objective of this study was to determine whether HER2 expression levels in breast carcinomas were modified by chemotherapy or during the metastatic process. METHODS HER2 expression was analyzed on sequential tissue specimens taken from the primary tumor before patients received preoperative chemotherapy (CT) and from post-CT residual breast tumor or at a metastatic site. The first group of patients included 59 women who presented with T2–T4,N1–N2 breast carcinoma and were treated by preoperative anthracycline-based CT and then underwent surgery. The second group included 44 patients with metastatic breast carcinoma localized to the lung (27 patients) or to the liver (17 patients). HER2 status was determined by immunohistochemistry using an anti-p185HER/neu monoclonal antibody and was classified as overexpressed or not overexpressed. RESULTS Among the patients who received preoperative CT, HER2 overexpression was observed in 15 of 59 patients (25%). A complete pathologic response was observed in 2 of these 15 patients. HER2 still was overexpressed in 11 of 13 remaining residual tumors and was no longer detectable in 2 tumors. In addition, the 29 tumors with no HER2 overexpression before CT remained negative after treatment. In patients with metastatic breast carcinoma, HER2 was overexpressed in 11 of 44 primary tumors (25%). In 9 of these 11 tumors, HER2 overexpression was maintained in the metastases (9 pulmonary metastases and 4 hepatic metastases). In two patients who had low levels of HER2 overexpression in their primary tumors, no staining was observed in the secondary tumor (one pulmonary tumor and one liver tumor). There were no tumors in which the overexpression of HER2 was found only in the metastasis. CONCLUSIONS The current study showed that, in most patients, HER2 overexpression was unchanged after CT and in metastatic sites. No HER2 negative primary tumors became HER2 positive after patients received CT or during the metastatic process. In a few patients, a diminution in the level of HER2 expression was observed after CT or in secondary tumors. This may have been due to a transitory state of altered tumor cells or to the selection of HER2 negative tumor cells clones. Cancer 2002;94:2169–73. © 2002 American Cancer Society. DOI 10.1002/cncr.10456

Transcriptomic analyses identified four major groups among invasive ductal carcinomas, associated with different clinical outcomes. Their definitions in practice is still matter of debate. Our aims were 1) to validate definitions based on immunohistochemical markers and grade: luminal A= ER and or PR+ve, grade I HER2−ve, luminal B= ER+ve and/or PR+ve, HER2+ve or grade III, HER2 enriched carcinomas= HER2+ve and ER-ve, basal-like/triple negative carcinomas= grade III, ER-ve, PR-ve, HER2−ve and expression of at least one of the basal-like markers (CK5/6, CK14, EGFR); 2) to refine this immunohistochemical definition. 142 consecutive tumors were selected in our tumour bank (42 triple-negative and grade III; 31 HER2+ve and ER-ve; 35 luminal B ER or PR+ve and grade III (7 cases) or HER2+ve (28 cases); luminal A (34 ER+ve or PR+ve, grade I)). Transcriptomic analyses were performed using Affymetrix U133+2 arrays (good quality RNAs obtained for all frozen specimens). The molecular classes determined according to proposed definitions were compared to those obtained with unsupervised clustering analyses using datas after GC-RMA normalisation (with the intrinsic gene list genes and with the highest variance genes). Marker patterns of expression (CK 8/18, 5/6, 14, EGFR, BCL2 and Ki67) were analysed within each molecular class. Based on the phenotypical definition and grade, 10 and 9% of cases were misclassified respectively using unsupervised clustering either with the intrinsic gene list or the highest variance genes. The misclassified tumors were luminal B HER2+ve case with low ER level of expression, or HER2+ve and ER-ve cases classified among the triple-negative group. 39 out of 42 (93%) triple negative expressed at least one of the basal markers. RP expression pattern differed between luminal A and B carcinomas. All luminal A showed at least > 15% of positive cells and 65% of them harboured > 50% of positive cells. In contrast, luminal B showed < 15% of positive cells in 70% of the cases. Bcl2 was negative in 40% of the luminal B cases and positive (> 20% stained cells) in more than 90% of the luminal A cases. CK14 was positive (i.e. > 1% positive cell) in 65% of the triple negative cases, compared to CK5/6 positive in 58% of the cases. Ki67 was > 20% in 90% of the triple negative and in 55% of luminal B cases compared to less than 5% of the luminal A cases. 20 and 30% of HER2+ve ER-ve carcinomas expressed CK5/6, CK14 and EGFR associated in more than 90% of the cases to CK8/18 positivity (> 20% positive cells). Identification of molecular classes of breast carcinomas was accurately determined by immunohistochemistry and grade. Low level of RP expression and BCL2 negativity were part of the luminal B phenotype. CK14 was more sensitive in this population of triple negative carcinomas to identify basal-like carcinomas. KI67 was highly expressed in the vast majority if not all breast triple negative carcinomas. HER2+ve ER-ve carcinomas can express basal markers but in contrast to basal-like carcinomas, associated in the large majority of the cases to CK8/18 expression. The accurate determination of molecular groups of breast carcinomas should be a key parameter for development of targeted therapies within each group. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-03.

IntroductionEpigenetic modifications such as aberrant DNA methylation has long been associated with tumorogenesis. Little is known, however, about how these modifications appear in cancer progression. Comparing the methylome of breast carcinomas and locoregional evolutions could shed light on this process.MethodsThe methylome profiles of 48 primary breast carcinomas (PT) and their matched axillary metastases (PT/AM pairs, 20 cases), local recurrences (PT/LR pairs, 17 cases) or contralateral breast carcinomas (PT/CL pairs, 11 cases) were analyzed. Univariate and multivariate analyzes were performed to determine differentially methylated probes (DMPs), and a similarity score was defined to compare methylation profiles. Correlation with copy-number based score was calculated and metastatic-free survival was compared between methods.Results49 DMPs were found for the PT/AM set, but none for the others (FDR < 5%). Hierarchical clustering clustered 75% of the PT/AM, 47% of the PT/LR, and none of the PT/CL pairs together. A methylation-based score (MS) was defined as a clonality measure. The PT/AM set contained a high proportion of clonal pairs while PT/LR pairs were evenly split between high and low MS score, suggesting two groups: true recurrences (TR) and new primary tumors (NP). CL were classified as new tumors. MS score was significantly correlated with copy-number based scores. There was no significant difference between the metastatic-free survival of groups of patients based on different classifications.ConclusionEpigenomic alterations are well suited to study clonality and track cancer progression. Methylation-based classification of TR and NP performed as well as clinical and copy-number based methods suggesting that these phenomenons are tightly linked.

BackgroundGenomic grade (GG) is a 97-gene signature which improves the accuracy and prognostic value of histological grade (HG) in invasive breast carcinoma. Since most of the genes included in the GG are involved in cell proliferation, we performed a retrospective study to compare the prognostic value of GG, Mitotic Index and Ki67 score.MethodsA series of 163 consecutive breast cancers was retained (pT1-2, pN0, pM0, 10-yr follow-up). GG was computed using MapQuant Dx(R).ResultsGG was low (GG-1) in 48%, high (GG-3) in 31% and equivocal in 21% of cases. For HG-2 tumors, 50% were classified as GG-1, 18% as GG-3 whereas 31% remained equivocal. In a subgroup of 132 ER+/HER2- tumors GG was the most significant prognostic factor in multivariate Cox regression analysis adjusted for age and tumor size (HR = 5.23, p = 0.02).ConclusionsIn a reference comprehensive cancer center setting, compared to histological grade, GG added significant information on cell proliferation in breast cancers. In patients with HG-2 carcinoma, applying the GG to guide the treatment scheme could lead to a reduction in adjuvant therapy prescription. However, based on the results observed and considering (i) the relatively close prognostic values of GG and Ki67, (ii) the reclassification of about 30% of HG-2 tumors as Equivocal GG and (iii) the economical and technical requirements of the MapQuant micro-array GG test, the availability in the near future of a PCR-based Genomic Grade test with improved performances may lead to an introduction in clinical routine of this test for histological grade 2, ER positive, HER2 negative breast carcinoma.

Background Adjuvant! Online is a web-based application designed to provide 10 years survival probability of patients with breast cancer. Several predictors have not been assessed in the original Adjuvant! Online study. We provide the validation of Adjuvant! Online algorithm on two breast cancer datasets, and we determined whether the accuracy of Adjuvant! Online is improved with other well-known prognostic factors. Patients and Methods The French data set is composed of 456 women with early breast cancer. The Dutch data set is composed of 295 women less than 52 years of age. Agreement between observation and Adjuvant! Online prediction was checked, and logistic models were performed to estimate the prognostic information added by risk factors to Adjuvant! Online prediction. Results Adjuvant! Online prediction was overall well-calibrated in the French data set but failed in some subgroups of such high grade and HER2 positive patients. HER2 status, Mitotic Index and Ki67 added significant information to Adjuvant! Online prediction. In the Dutch data set, the overall 10-year survival was overestimated by Adjuvant! Online, particularly in patients less than 40 years old. Conclusion Adjuvant! Online needs to be updated to adjust overoptimistic results in young and high grade patients, and should consider new predictors such as Ki67, HER2 and Mitotic Index.

702 Background: Level of circulating Tumoral Cells (TC) have been recently shown to predict survival in MBC (Cristofanilli, M, NEJM, 2004, 351, 781). We have previously shown that the detection of ...