Cosimo, Serena Di

Background: The potential gonadotoxicity of anti-HER2 agents remains largely unknown, and limited, conflicting evidence exists for taxanes. Antimüllerian hormone (AMH) is an established biomarker of ovarian reserve that may aid in quantifying anticancer treatment–induced gonadotoxicity. Patients and Methods: The present biomarker analysis of the randomized phase III neoadjuvant NeoALTTO trial included premenopausal women aged ≤45 years at diagnosis of HER2-positive early breast cancer with available frozen serum samples at baseline (ie, before anticancer treatments), at week 2 (ie, the “biological window” of anti-HER2 therapy alone), and/or at the time of surgery (ie, after completing paclitaxel + anti-HER2 therapy, before starting adjuvant chemotherapy). Results: The present analysis included 130 patients with a median age of 38 years (interquartile ratio [IQR], age 33–42 years). AMH values at the 3 time points differed significantly (P<.001). At baseline, median AMH levels were 1.29 ng/mL (IQR, 0.56–2.62 ng/mL). At week 2, a small but significant reduction in AMH levels was observed (median, 1.10 ng/mL; IQR, 0.45–2.09 ng/mL; P<.001). At surgery, a larger significant decline in AMH levels was observed (median, 0.01 ng/mL; IQR, 0.01–0.03 ng/mL; P<.001). Although the type of anti-HER2 treatment (trastuzumab and/or lapatinib) did not seem to impact the results, age and pretreatment ovarian reserve had a major influence on treatment-induced gonadotoxicity risk. Conclusions: This NeoALTTO biomarker analysis showed that anti-HER2 therapies alone had limited gonadotoxicity but that the addition of weekly paclitaxel resulted in marked AMH decline with possible negative implications for subsequent ovarian function and fertility.

Abstract Background: Knowledge regarding risk of treatment-induced premature ovarian insufficiency (POI) is crucial for appropriate oncofertility counseling. While the gonadotoxicity of anthracycline- and cyclophosphamide-based chemotherapy (CT) in young breast cancer (BC) patients (pts) is well established, the impact of anti-HER2 agents remains largely unknown and limited conflicting evidence exists for taxanes. Anti-Mullerian hormone (AMH) is an established biomarker of ovarian reserve; its measurements during systemic therapies may aid in diagnosis and prediction of POI. The NeoALTTO trial represented a unique setting to explore the acute gonadotoxicity of anti-HER2 therapy alone and in combination with weekly paclitaxel in pts not previously exposed to any other potentially gonadotoxic anticancer treatment. Methods: NeoALTTO (NCT00553358) is a multicenter, open-label, randomized phase III neoadjuvant trial in HER2-positive early BC pts randomized to receive lapatinib (L), trastuzumab (T), or their combination (L+T) alone for 6 weeks followed by the addition of paclitaxel for 12 weeks before surgery.The present analysis included only premenopausal women aged ≤45 years with available frozen serum samples at baseline (i.e. before administering any anticancer treatment), at week 2 (i.e. “biological window” of anti-HER2 therapy alone) and/or at surgery (i.e. after completion of paclitaxel plus anti-HER2 therapy). Central AMH testing was performed at Erasme Hospital (Brussels, Belgium) with the Roche Elecsys® AMH Plus assay (LoD=0.010 ng/ml). AMH levels during anti-HER2 therapy alone and then combined with paclitaxel were assessed as a measure of treatment impact on ovarian reserve. The impact of different anti-HER2 agents (T and/or L), pts’ age and baseline AMH levels on treatment gonadotoxicity were also investigated. Results: The present analysis included 130 pts with a median age of 38 years (IQR: 33-42 years), of whom 86 (66.2%) were ≤40 years. At baseline, median AMH levels were 1.29 ng/mL (IQR 0.56 - 2.62 ng/mL). At week 2, a small but significant reduction in AMH levels was observed (median value: 1.10 ng/mL, IQR 0.45 - 2.09 ng/mL, p&lt;0.001; median cumulative decrease: -12.5%, IQR -37.6% - 12.4%). At surgery, there was a large significant decline in AMH levels (median value: 0.01 ng/mL, IQR 0.01 - 0.03 ng/mL, p&lt;0.001; median cumulative decrease: -99.9%, IQR -100.0% - -98.7%). Type of anti-HER2 treatment (L vs. L vs. T+L) did not impact the results: there was no significant difference between treatment arms in degree of reduction in AMH levels at week 2 (p=0.763) or at surgery (p=0.700). Age and pre-treatment ovarian reserve had a major impact on treatment-induced gonadotoxicity risk, with a more profound decrease in AMH levels observed for older pts (p&lt;0.001) and those with lower baseline AMH levels (p&lt;0.001). Among pts aged &lt;35 years, there was no significant difference in AMH levels between those with (n=7) or without (n=35) a subsequent pregnancy at baseline (p=0.316), week 2 (p=0.463) or at surgery (p=0.610). Conclusions: This biomarker analysis of the NeoALTTO trial showed for the first time the potential acute gonadal damage induced by anti-HER2 therapies alone and with the addition of weekly paclitaxel. We observed a small but significant reduction in AMH levels during T and/or L alone and then a profound reduction to almost undetectable levels following the addition of weekly paclitaxel, without differences by specific anti-HER2 regimen. Age and pre-treatment AMH levels strongly impacted gonadotoxicity risk. While this analysis is limited by lack of AMH measurement in longer term follow-up, findings have important implications for future research and oncofertility counseling of premenopausal women with HER2-positive BC. Citation Format: Matteo Lambertini, Marcello Ceppi, Richard A. Anderson, David A. Cameron, Marco Bruzzone, Maria Alice Franzoi, Claudia Massarotti, Sarra El-Abed, Yingbo Wang, Christophe Lecocq, Paolo Nuciforo, Rebecca Rolyance, Lajos Pusztai, Joohyuk Sohn, Jacopo Ligato, Maria Maddalena Latocca, Luca Arecco, Barbara Pistilli, Kathryn J. Ruddy, Alberto Ballestrero, Lucia Del Mastro, Fedro A. Peccatori, Ann H. Partridge, Cristina Saura, Michael Untch, Martine Piccart, Serena Di Cosimo, Evandro de Azambuja, Isabelle Demeestere. Impact of anti-HER2 therapy alone and in association with weekly paclitaxel on the ovarian reserve of young women with HER2-positive early breast cancer: Biomarker analysis of the NeoALTTO trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD5-05.

Background: The association between obesity and prognosis in HER2-positive early breast cancer remains unclear, with limited data available. This study aimed to determine the impact of body mass index (BMI) at baseline and weight change after 2 years on outcomes of patients with HER2-positive early breast cancer. Methods: ALTTO was a randomized phase III trial in patients with HER2-positive early breast cancer. BMI was collected at randomization and 2 years after. WHO BMI categories were used: underweight, <18.5 kg/m2; normal weight, 18.5 to <25 kg/m2; overweight, ≥25 to <30 kg/m2; and obese ≥30 kg/m2. A weight change from baseline of ≥5.0% and ≤5.0% was categorized as weight gain and weight loss. The impact of BMI at randomization and of weight change on disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS) were investigated with multivariate analyses, adjusting for baseline patients and tumor characteristics. Results: A total of 8,381 patients were included: 187 (2.2%), 3,797 (45.3%), 2,690 (32.1%), and 1,707 (20.4%) were underweight, normal weight, overweight, and obese at baseline, respectively. Compared with normal weight, being obese at randomization was associated with a significantly worse DDFS (adjusted hazard ratio [aHR], 1.25; 95% CI, 1.04–1.50) and OS (aHR, 1.27; 95% CI, 1.01–1.60), but no significant difference in DFS (aHR, 1.14; 95% CI, 0.97–1.32). Weight loss ≥5.0% at 2 years after randomization was associated with significantly poorer DFS (aHR, 1.34; 95% CI, 1.05–1.71), DDFS (aHR, 1.46; 95% CI, 1.07–1.98), and OS (aHR, 1.83; 95% CI, 1.18–2.84). Hormone receptor and menopausal status but not anti-HER2 treatment type influenced outcomes. Toxicities were more frequent in obese patients. Conclusions: In patients with HER2-positive early breast cancer, obesity at baseline is a poor prognostic factor. Weight loss during treatment and follow-up negatively impacts clinical outcomes. Dietary counseling should be part of survivorship care programs.

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&lt;0.05), although FDR were borderline significant (0.05&lt;FDR&lt;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: The level of immune suppression in an individual breast cancer (BC) patient is relevant for having a major benefit from treatments that act via or are directed to the immune response, such as anti-HER2 agents and immune checkpoint molecules, respectively. The link(s) between the tumor immune microenvironment and treatment responses has been investigated in HER2-positive BC. This study analyzed the association between baseline expression of genes regulating T cell activities (PD1, CTLA4, LAG3, TIM3, PDL1, PDL2, FOXP3, IL10, TGFβ (1-3), FOXP1 and other related genes) and pathologic complete response (pCR) rates in the NeoALTTO trial. Methods: NeoALTTO randomized early-stage HER2-positive BC patients to neoadjuvant trastuzumab (T), lapatinib (L) or T+L for 6 weeks followed by 12 weeks of concurrent paclitaxel. Anthracyclines were given after surgery (FEC). Patients with RNA sequencing data from their baseline tumor samples (N=254 patients out of 455 from the original cohort) were included in the current study. The primary endpoint, pCR, was defined as ypT0/is ypN0. Logistic regression was used for analysis of pCR. Cox regression univariate and multivariate (adjusted for clinicopathological parameters and treatment arms) analyses were performed. Results: In the total cohort analyzed, high PD1 (odds ratio, OR: 1.4, P=0.03), PDL2 (OR: 1.4, P=0.04), CTLA4 (OR: 1.4, P=0.03) and TGFβ3 (OR: 1.4, P=0.02) expression was associated with an increased probability of achieving pCR at the multivariate analysis. ERBB2 (HER2) expression was associated with pCR in the three arms (T: OR: 2.7, P=0.02; L: OR: 2.3, P=0.01; T+L: OR: 5, P&lt;0.001). Additionally in the T+L arm, PAM50 HER2-enriched subtype (OR: 2.9, P=0.03), as well as immune genes PD1 (OR: 2.1, P=0.01), PDL1 (OR: 1.8, P=0.03) and CTLA4 (OR: 2; P=0.01), were also associated with pCR. Conclusions: In the NeoALTTO population examined, PD1, PDL2, CTLA-4 and TGFβ3 expression at diagnosis were all associated with improved pCR rates after anti-HER2 treatment. The effect of these treatments seemed to be dependent on HER2 expression levels, which was particularly relevant in the T+L arm. These observations confirm previous findings that link immune infiltrates to higher pCR rates, demonstrate the clinical significance of the PD-1 pathway and additionally show that CTLA-4 and TGFβ3 could also be important in early stage HER2-positive BC. The association of tumors linked with immune regulatory gene expression with positive responses to neoadjuvant anti-HER2 agents suggests that they act in a way that reinvigorates the antitumor immunity in the face of tumor-mediated suppression. Patients whose tumors highly express these genes may be good candidates for immunotherapy before the start or after the neoadjuvant treatment when residual disease is detected at surgery, although these hypotheses require further confirmation. Citation Format: Cinzia Solinas, Pushpamali De Silva, David Venet, Soizic Garaud, Chunyan Gu-Trantien, Florentine Hilbers, Evandro de Azambuja, Olena Werner, Lorena De la Peña, Amylou Dueck, Serena Di Cosimo, Istvan Lang, Jens Huober, Sherko Küemmel, Carsten Denkert, Roberto Salgado, Christos Sotiriou, Martine Piccart-Gebhart, Debora Fumagalli, Karen Willard-Gallo. Immune regulatory gene expression and clinical outcome in the NeoALTTO trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3132.

Background Limited data exist on the safety of using anti–human epidermal growth factor receptor 2 (HER2) targeted agents during pregnancy. To date, only retrospective studies have assessed the prognosis of patients with a pregnancy after prior early breast cancer, with no data in HER2‐positive patients. Methods The Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimization (NeoALTTO) trial and the Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization (ALTTO) trial were randomized phase 3 trials for patients with HER2‐positive early breast cancer. In both trials, pregnancy information was prospectively collected. Pregnancy outcomes were compared between patients unintentionally exposed to trastuzumab and/or lapatinib during gestation (the exposed group) and those who became pregnant after trastuzumab and/or lapatinib completion (the unexposed group). In the ALTTO trial, disease‐free survival (DFS) was compared between pregnant patients and those aged 40 years or younger without a subsequent pregnancy via an extended Cox model with time‐varying covariates to account for a guarantee‐time bias. Results Ninety‐two patients (12 in the exposed group and 80 in the unexposed group) had a pregnancy: 7 in the NeoALTTO trial and 85 in the ALTTO trial. Seven patients (58.3%) in the exposed group and 10 patients (12.5%) in the unexposed group opted for an induced abortion; in the unexposed group, 10 patients (12.5%) had a spontaneous abortion. No pregnancy/delivery complications were reported for the remaining cases, who successfully completed their pregnancy, with the exception of 1 fetus with trisomy 21 (Down syndrome). No significant difference in DFS (adjusted hazard ratio, 1.12; 95% confidence interval, 0.52‐2.42) was observed between young patients with a pregnancy (n = 85) and young patients without a pregnancy (n = 1307). Conclusions For patients with HER2‐positive early breast cancer, having a pregnancy after treatment completion appears to be safe without compromising fetal outcome or maternal prognosis.