Rothé, Françoise

Abstract Background: Disease relapse is observed in a significant proportion of HER2-positive breast cancer (BC) patients with residual disease (RD) after neoadjuvant treatment, as well as in a subgroup of those achieving pathological complete response (pCR). As the host immune response plays a key role in modulating the activity of anti-HER2 agents, we investigated the association of T- and B-cell receptor (TCR and BCR) repertoires with pCR and event-free survival (EFS) in the NeoALTTO phase 3 trial. Methods: RNA sequencing (RNAseq) data from baseline tumor biopsies were available for 254 patients out of the 455 enrolled in the study. Among those, 166 did not achieve a pCR defined as ypT0/is. Matched RNAseq data from RD samples were available for 43 cases. TCR/BCR repertoires were extracted from RNAseq data using the MiXCR software. TCR and BCR read counts, number of clones, evenness, Shannon entropy, Gini index, length of the complementarity determining region 3, top and second top clone proportion were evaluated. Survival analysis was performed using univariate and multivariate (adjusted for tumor size, nodal status, grade, estrogen receptor [ER] status, age and treatment arm) Cox proportional hazard models, while logistic regressions were used for pCR. False discovery rate (FDR) was obtained using Benjamini & Hochberg method. Results: Baseline TCR top (odds ratio [OR]=0.63 [95% CI 0.46-0.87], FDR=0.021) and second top (OR=0.57 [0.41-0.79], FDR=0.004) clone proportion were significantly associated with a lower probability of achieving pCR in the multivariate analysis. BCR evenness (hazard ratio [HR]=1.5 [1.2-2], FDR=0.015) and Gini index (HR=0.66 [0.52-0.85], FDR=0.015) were significantly associated with EFS in the multivariate analysis. In residual disease, BCR evenness and Gini index showed a similar trend in the EFS univariate analysis, while TCR read counts, number of clones, and entropy were associated with lower HR (P<0.05), although FDR were borderline significant (0.05<FDR<0.1). A model to predict EFS including baseline BCR evenness, ER status, stromal tumor-infiltrating lymphocyte level, and pCR was able to identify 3 groups with good, intermediate and poor prognosis (Kaplan-Meier 5-year EFS rates of 95%, 80% and 58%, respectively). Of note, about 63%, 45% and 12% of the patients achieved a pCR in the three prognostic groups, respectively. Conclusions: In the NeoALTTO trial, the presence of an evenly distributed BCR repertoire was associated with worse EFS. A model integrating baseline immune-related and clinical features was able to identify patients with excellent prognosis despite RD or, conversely, with poor prognosis after pCR. We envision that our model has the potential to allow the personalization of post-operative treatment strategies after both pCR and RD in HER2-positive BC. Further validation of our findings is warranted. Citation Format: Mattia Rediti, David Venet, Françoise Rothé, Tao Qing, Marion Maetens, Ian Bradbury, Miguel A. Izquierdo, Serena Di Cosimo, Florentine Hilbers, Mohammed Bajji, Nadia Harbeck, Michael Untch, David L. Rimm, Stephen Chia, Minetta C. Liu, Cristina Saura, Jens Huober, Paolo Nuciforo, Roberto Salgado, Sherene Loi, Lajos Pusztai, Christos Sotiriou. Predictive and prognostic role of T- and B-cell receptor repertoire in HER2-positive breast cancer: An analysis of the NeoALTTO clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1998.

511 Background: Clinicopathological and molecular features, including estrogen receptor (ER) status and PAM50 subtypes, have shown an association with immunogenicity and tumor-infiltrating lymphocyte (TIL) levels in breast cancer (BC). To investigate the complexity of the immune response in HER2+ BC, we explored the association of T- and B-cell receptor (TCR and BCR) repertoires with clinicopathological characteristics, PAM50 subtypes and outcome in the NeoALTTO phase III trial. Methods: RNA sequencing (RNAseq) data from baseline tumor biopsies were available for 254 out of the 455 patients enrolled. TCR and BCR repertoires were extracted from RNAseq data using the MiXCR software. Repertoire and diversity measures (read counts, number of clones, evenness, Gini index, Shannon entropy, length of the complementarity-determining region 3 [CDR3], top and second top clone proportions) were estimated. PAM50 subtypes were computed from RNAseq data. Univariate and multivariate (adjusted for clinicopathological characteristics, TIL levels dichotomized using the median value of 12.5% and treatment arm) Cox proportional hazard models were used for survival analysis, while logistic regressions were used for pathological complete response (pCR), defined as ypT0/is. All results reported had a false discovery rate (FDR) <0.05. Results: Higher BCR read counts, number of clones and Gini index were significantly associated with ER-negative as well as grade 3 tumors. Among the PAM50 subtypes, HER2-enriched (HER2-E) showed significantly higher BCR read counts, number of clones and Gini index along with lower evenness compared to luminal A and B, as well as higher length of CDR3 than luminal A. Of note, basal-like showed similar BCR diversity measures to HER2-E. No significant differences were noted for TCR diversity measures. In multivariate analyses, neither TCR nor BCR features were associated with pCR, while BCR evenness (HR 1.5; 95%CI 1.1-2.1) and Gini index (HR 0.66; 95%CI 0.5-0.88) were associated with event-free survival. Conclusions: BCR repertoire measures suggest a clonal expansion in HER2-E and basal-like PAM50 subtypes. Furthermore, the implementation of BCR-derived biomarkers can help to identify patients with an improved clinical outcome after neoadjuvant anti-HER2 treatment. Our findings highlight the heterogeneity of the immune response within HER2+ BC and provide support for biomarker-driven treatment strategies including immunotherapy in this BC subtype. Further validation is required. Clinical trial information: NCT00553358 .

Abstract Background: CTCs are associated with clinical outcomes in metastatic breast cancer irrespective of ER/PR/HER2 status. Some data support the prognostic relevance of serial CTC enumeration relative to adjuvant chemotherapy in early stage breast cancer. However, data from a large scale study focused on HER2 directed therapy for HER2+ disease have been lacking. We therefore sought to prospectively evaluate the effect of trastuzumab +/- lapatinib on CTCs and assess the prognostic/predictive value of CTC monitoring in HER2+ early stage breast cancer patients (pts). Methods: The Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO; NCT00490139) Trial is an international, randomized, open-label phase III study of two targeted agents for HER2+ breast cancer. From June 2007 to July 2011, 8381 pts were randomised from 946 sites in 44 countries to 1 of 4 arms with sequential or concurrent chemotherapy: (i) 52 wks of trastuzumab (T); (ii) 52 wks of oral lapatinib (L); (iii) 12 or 18 wks of T followed by a washout and then 34 or 38 wks of L; or (iv) 52 wks of L+T. 540 (6%) pts provided optional informed consent and up to 30 mL peripheral blood suitable for CTC analyses at baseline with additional collections at 13 or 19 wks, 52 wks, 18 mos, 24 mos, and recurrence. CTC analyses are being conducted in three laboratories (Mayo Clinic Rochester, n=431; Institut Jules Bordet and University of Munich, n=109). 2-3 x 10 mL CellSave™ samples are pooled and processed at each time point for CTC enumeration and HER2 expression using the immunomagnetic/immunofluorescence assay (CellSearch™). A round-robin concordance project was done between Mayo Clinic Rochester and Institut Jules Bordet before embarking on the primary correlative work. Results: At baseline, 20% pts had detectable (i.e., ≥1) EpCAM+/CK+/DAPI+/CD45- CTCs, and 16% pts had detectable EpCAM+/CK+/DAPI+/CD45-/HER2+ CTCs. Correlative analyses with clinical outcome are ongoing with plans for completion by Fall 2014. Conclusions: CTCs were detected in 20% of pts with HER2+ early stage breast cancer. This is similar to the frequency of detection in mixed early stage breast cancer populations relative to ER/PR and HER2 status. Concordance of enumeration and HER2 assessments between the two experienced laboratories, and correlation between disease free survival and CTC findings (from serial samples collected at baseline, during the course of HER2 directed therapy, and at set intervals of follow-up) will be reported. Citation Format: Minetta C Liu, Brigitte Rack, Amylou C Dueck, David W Hillman, Michael B Campion, Monica M Reinholz, Kevin C Halling, Christos Sotiriou, Françoise Rothé, Marion Maetens, Ghizlane Rouas, Wolfgang Janni, Antonio C Wolff, Lyndsay N Harris, Julie R Gralow, Kathleen I Pritchard, Susan Ellard, Nguyet A Le-Lindqwister, Frances Boyle, Evandro De Azambuja, Martine J Piccart-Gebhart, Michail Ignatiadis, Edith A Perez. Circulating tumor cell (CTC) enumeration and HER2 assessment as predictors of breast cancer outcomes in the ALTTO (BIG 2-06, Alliance N063D) Trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-01.

Abstract Background: Preliminary results from the Success and Remagus trials showed that CTC detection by CellSearch® has adverse prognostic value in non-metastatic breast cancer. Moreover, several investigators have characterized HER2 expression on CTCs in early breast cancer. Since the majority of CTC-positive women with non-metastatic breast cancer have only 1 CTC detected / blood volume analyzed, we wanted to evaluate the inter-reader variability in this setting. This is a crucial step before moving forward with multi-lab/multi-center prospective testing of the clinical utility of the CellSearch® technology in non-metastatic breast cancer. Methods: Five galleries of CellSearch® images from 3 European and one US institution (a total of 307 images) were mailed to 22 independent readers from 14 European and US academic laboratories and 8 readers from two CellSearch® Veridex laboratories in a blinded fashion. These images came mainly from studies on CTC and/or HER2−positive CTC detection in breast cancer and included healthy women (negative controls), women with metastatic (positive controls) and non-metastatic disease (Pierga et al SABCS 2010, Pierga et al ASCO 2011, Riedthorf et al CCR 2010, Ignatiadis et al PLoS ONE 2011). Each reader reported the images as either CTC-negative or CTC-positive/HER2−negative or CTC-positive/HER2−positive. Kappa statistics were used to assess inter-reader agreement. The 8 Veridex readers were summarized by a majority voting system to derive a gold standard in order to compare each independent reader using discordance rates. Results: Kappa statistics showed moderate to good agreement between independent readers depending on the gallery evaluated (gallery 1 K: 0.55, gallery 2 K:0.57 gallery 3 K:0.64, gallery 4 K:0.72, gallery 5 K:0.85). These differences were attributed to differences in scoring difficulty of each gallery. Discordances in CTC-positive vs CTC-negative events between each reader and the gold standard ranged from 2.3%-31.3% with 7 readers showing discordance rates &lt;5%, 11 readers between 5–10%, 10 readers between 10–14% and only 2 readers showing discordance rates &gt;14% as compared to the gold standard (median discordance rate: 9.3%). Image analysis showed that investigators with high discordance rates compared to the Veridex gold standard were not always taking into account “morphological characteristics” for defining an event as CTC-positive. Discordant results between readers were mainly due to discordance in CTC definition and to a lesser extent in the definition of HER2−positivity. Indeed, discordances in assigning a CTC-positive event as either HER2−negative or HER2−positive ranged from 0.3−13.7% with 19 readers showing discordance rates &lt;5%, 7 readers between 5–10% and 4 readers with discordance rates &gt;10% (median discordance rate: 3.9%). Conclusion: In non-metastatic breast cancer, we observed low discordance between most independent readers and the gold standard for defining a CellSearch® event as CTC with very few readers showing high discordance rates. Concordance can be improved through appropriate training and the use of all the tools provided by the CellSearch® system for image interpretation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-14.