Loi, Sherene

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 .

AbstractImmune checkpoint inhibitor therapies targeting PD‐1/PD‐L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD‐L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab‐paclitaxel. However, concerns regarding variability between immunohistochemical PD‐L1 assay performance and inter‐reader reproducibility have been raised. High tumor‐infiltrating lymphocytes (TILs) have also been associated with response to PD‐1/PD‐L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin–stained slides and have shown reliable inter‐reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD‐L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD‐L1 and TIL analyses as a more comprehensive immuno‐oncological biomarker for patient selection for PD‐1/PD‐L1 inhibition‐based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk‐management framework that may help mitigate the risks of suboptimal patient selection for immuno‐therapeutic approaches in clinical trials and daily practice based on combined TILs/PD‐L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Background We investigated the clinical outcomes and behavior of HER2+ EBC according to centrally tested HR status in pts treated with modern chemotherapy (CT) and trastuzumab (T)-based regimens. Patients and methods ALTTO is an international phase 3 trial in HER2+ EBC pts randomized to 4 adjuvant anti-HER2 treatment arms: T alone, lapatinib (L) alone, their sequence (T-&gt;L) or their combination (T+L). Pts in the L alone arm were excluded from the present analysis. HER2 and HR status were centrally tested for all patients. HER2 status was defined based on the 2007 ASCO/CAP guidelines. HR-positive (HR+) was defined as ≥1% tumor cells with expression of estrogen (ER) and/or progesterone receptors.We investigated the prognosis of pts with HR+ or negative (HR-) HER2+ EBC, the risk of developing early (years 0-5) and late (years 6-8) recurrences, the factors impacting this risk, and the patterns of relapse according to HR status. Univariate and multivariate Cox proportional hazard models were used to compare hazard rates within the HR subgroups; a conditional landmark analysis was conducted to investigate late recurrences. For the hazard ratios for HR status, an interaction between log(time) and HR status was included as proportional hazard assumption was not satisfied. Results This analysis included 6,271 HER2+ pts of whom 3,601 (57%) had HR+ and 2,670(43%) HR- EBC. Median follow-up was 6.93 years (range 6.81-6.96). 5-year disease-free survival (DFS) was 86% (85-87) in HR+ and 83% (82-85) in HR- pts; 8-year DFS was 80% (78-81) in HR+ and 79% (77-81) in HR- pts. The mean annual hazards of recurrence in years 0-5 were 3% in HR+ and 4% in HR- pts, while in years 6-8 they were 3% in HR+ and 2% in HR- pts. The hazard ratios for HR status (ref=HR-) were 0.60 (0.51-0.72) overall, 0.74 (0.65-0.85) for years 0-5 and 1.61 (1.17-2.24) for years 6-8. In HR+ pts, risk factors for recurrence overall were age ≥65 (p=0.01), tumor size pT3-4 (p&lt;0.001), nodal status pN1-3 (p&lt;0.001), tumor grade 2 or 3 (p&lt;0.001), anthracycline-only CT (p&lt;0.001), no use of aromatase inhibitors (p&lt;0.001), and ER&lt;10% (p=0.01). Main risk factors for recurrence in both years 0-5 and years 6-8 were pN2-3 (p=0.04 for both) and high (≥5) HER2 FISH ratio (p=0.02 for both). In HR- pts, risk factors for recurrence overall were BMI (obesity or underweight; p=0.01), pT3-4 (p&lt;0.001), pN1-3 (p&lt;0.001) and anthracycline-only CT (p&lt;0.001). No significant risk factors for recurrence in years 0-5 were identified. Main risk factor for recurrence in years 6-8 was BMI (obesity; p=0.002). HR+ pts had lower incidence of visceral disease (58.8% vs. 69.6%; p=0.005) and lung metastasis (18.5% vs. 27.8%; p=0.006); bone was the most common site of first recurrence (30%). Conclusions This large analysis of centrally tested HR+ and HR- HER2+ EBC provides strong evidence on the existence of two different diseases within the HER2+ population based on HR expression. These up-to-date estimates of pts outcomes after modern CT and T-based regimens may be used as “historical control” for the design of future de-escalation or escalation trials separately in HR+ and HR- HER2+ EBC pts. Citation Format: Lambertini M, Campbell C, Gelber RD, Viale G, McCullough A, Tonghua L, Hilbers F, Korde LA, Werner O, Chumsri S, Jackisch C, Wolff AC, Vaz-Luis I, Ferreira AR, Prat A, Moreno Aspitia A, Piccart M, Loi S, de Azambuja E. Dissecting the effect of hormone receptor (HR) status expression in patients (pts) with HER2-positive (HER2+) early breast cancer (EBC): Exploratory analysis from the ALTTO (BIG 2-06) trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-14.

Abstract Background: Pre-clinical data and results from early phase clinical trials point to synergistic antitumor activity and potential efficacy of palbociclib when given in combination with anti-HER2 therapy (tx). The aim of PATINA is to determine the effect of adding palbociclib to anti-HER2 and ET maintenance after induction tx in the 1st line setting for HR+/HER2+ MBC. Trial Design: The PATINA trial (AFT-38/NCT02947685) is a pivotal, open-label, international, phase III study. Following 6-8 cycles of chemotherapy (taxane or vinorelbine) with anti-HER2 tx for MBC participants will be randomized 1:1 to standard anti-HER2 tx (trastuzumab +/- pertuzumab) in combination with ET with or without palbociclib until disease progression. ET options are either an aromatase inhibitor or fulvestrant. Premenopausal patients (pts) must receive ovarian suppression. The trial is open to men or women with histologically confirmed HR+/HER2+ MBC provided they are without evidence of disease progression by local assessment after induction tx. Total planned accrual is 496 pts. Primary objective is to demonstrate that the combination of palbociclib with anti-HER2 tx plus ET is superior to anti-HER2 tx plus ET alone in prolonging progression-free survival (PFS). Key secondary objectives are measures of tumor control, overall survival, safety and Quality of Life. The main translational science objective is to compare PFS estimates according to PIK3CA mutation status. The study has a 90% power to detect a hazard ratio of 0.667 in favor of the palbociclib arm. All pts approached to participate in PATINA will be asked to share remaining biospecimens and clinical data with the Mastering Breast Cancer Initiative. This initiative was created in order to understand the natural history of MBC and how it envolves over time with the aim to develop new treatments for this patient population. Recruitment has started in 07/2017 and is planned for approximately 22 months in 140 sites in Australia, Germany, Italy, France, New Zealand, Spain, and the US. 69 pts have been recruited so far. The study is sponsored by Alliance Foundation Trials (AFT) and financially supported by Pfizer Citation Format: Metzger O, Mandrekar S, Loibl S, Mundhenke C, Seiler S, Valagussa P, Lim E, Tripathy D, Winer EP, Huang C, Carey L, Francis P, Miller K, Goetz MP, Prat A, Loi S, Krop I, Gianni L, Delalogue S, Vaz-Luis I, Dockter T, Lanzillotti J, Ciruelos E, DeMichele AM. PATINA: A randomized, open label, phase III trial to evaluate the efficacy and safety of palbociclib + anti-HER2 therapy + endocrine therapy (ET) vs. anti-HER2 therapy + ET after induction treatment for hormone receptor positive (HR+)/HER2-positive metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-02-07.

1088 Background: Standard first-line treatment for mTNBC is chemotherapy. However, outcomes are poor, and new treatment options are needed. Cohort B of KEYNOTE-086 (NCT02447003) assessed the safety and antitumor activity of pembrolizumab as first-line therapy for patients (pts) with PD-L1–positive mTNBC. Methods: Men and women with centrally confirmed mTNBC, no prior systemic anticancer therapy for metastatic disease, ECOG PS 0-1, and a tumor PD-L1 combined positive score (CPS) ≥1% received pembrolizumab 200 mg Q3W for 24 mo or until disease progression, intolerable toxicity, or investigator or pt decision. Tumor imaging was performed Q9W for 12 mo and Q12W thereafter. Clinically stable pts with PD could remain on pembrolizumab until PD was confirmed on subsequent assessment. Primary end point was safety. Secondary end points included ORR, DOR, and PFS (RECIST v1.1, central review). Planned enrollment was 80 pts. This analysis included all pts who had ≥18 wk of follow-up as of Nov 10, 2016. Results: 79 of the first 137 pts with PD-L1–evaluable tumors (58%) had PD-L1 CPS ≥1%. Of the first 52 pts enrolled, 100% were women, median age was 53 y, 40% had elevated LDH, 69% had visceral metastases, and 87% received prior (neo)adjuvant therapy. After a median follow-up of 7.0 mo (range 4.4-12.5), 15 (29%) pts remained on pembrolizumab. Treatment-related AEs occurred in 37 (71%) pts, most commonly fatigue (31%), nausea (15%), and diarrhea (13%). 4 (8%) pts experienced 5 grade 3-4 treatment-related AEs: back pain, fatigue, hyponatremia, hypotension, and migraine (n = 1 each). No pts died or discontinued pembrolizumab due to an AE. ORR was 23% (95% CI 14%-36%). Best overall response was CR in 4%, PR in 19%, SD in 17%, PD in 58%, and not assessed in 2%. Median time to response was 8.7 wk (range 8.1-17.7). Median DOR was 8.4 mo (range, 2.1+ to 8.4), with 8 (67%) responses ongoing at cutoff. Median PFS was 2.1 mo (95% CI, 2.0-3.9); estimated 6-mo PFS rate was 29%. Conclusions: Data from the first 52 pts enrolled in KEYNOTE-086 cohort B suggest that pembrolizumab monotherapy has a manageable safety profile and promising antitumor activity as first-line therapy for PD-L1–positive mTNBC. Clinical trial information: NCT02447003.

Abstract Background: Patients with previously untreated mTNBC typically receive cytotoxic chemotherapy as first-line therapy for metastatic disease. However, efficacy and safety are suboptimal. KEYNOTE-086 (NCT02447003) is a multicohort, single-arm, phase 2 study of pembrolizumab monotherapy for mTNBC. In cohort B, we assessed the safety and antitumor activity of pembrolizumab as first-line therapy for patients with PD-L1-positive mTNBC. Methods: Key eligibility criteria for cohort B were age ≥18 y, centrally confirmed TNBC, no prior systemic anticancer therapy for metastatic disease, ECOG performance status 0-1, measurable disease per RECIST v1.1 by central review, no radiographic evidence of brain metastases, and a tumor PD-L1 combined positive score (CPS) ≥1. Pembrolizumab 200 mg was given once every 3 wk for 24 mo or until disease progression, intolerable toxicity, or investigator or patient decision. Tumor imaging was performed every 9 wk for 12 mo and every 12 wk thereafter. Clinically stable patients with radiologic progression could remain on pembrolizumab until progression was confirmed on subsequent assessment. Primary end point was safety. Secondary end points included ORR, duration of response, and PFS (all RECIST v1.1 by central review) and OS. Results: Of the 206 patients with tumors evaluable for PD-L1 expression, 128 (62%) had PD-L1 CPS ≥1. Of these, 84 met all eligibility criteria and enrolled. All patients were women, median age was 52.5 y, 29 (35%) had ECOG PS 1, 40 (48%) had elevated LDH, 55 (65%) had visceral ± nonvisceral metastases, and 73 (87%) received prior (neo)adjuvant therapy. All patients received ≥1 pembrolizumab dose, and after median follow-up of 10.6 mo, 18 (21%) remained on pembrolizumab. Treatment-related AEs occurred in 53 (63%) patients and were of grade 3-4 severity in 7 (8%); no patients died or discontinued pembrolizumab because of treatment-related AEs. The most common treatment-related AEs were fatigue (26%), nausea (13%), and diarrhea (12%). No grade 3-4 treatment-related AE occurred in &gt;1 patient. The most common immune-mediated AE was hypothyroidism (10%). Three patients had complete response and 16 had partial response for an ORR of 23% (95% CI 15-33). Of the 11 patients with a best response of stable disease, 1 had stable disease for ≥24 wk, leading to a disease control rate of 24% (95% CI 16-34). 12 of 19 (63%) responses were ongoing at data cutoff, and median duration of response was 8.4 mo (range 2.1+ to 13.9+). Median PFS was 2.1 mo (95% CI 2.0-2.2), with an estimated 6-mo PFS rate of 26%. Median OS was 16.1 mo (95% CI 11.3-NR), with an estimated 6-mo OS rate of 83%. Conclusions: Pembrolizumab monotherapy continues to demonstrate a favorable safety profile and robust, durable antitumor activity in patients with PD-L1-positive, previously untreated mTNBC. Citation Format: Adams S, Loi S, Toppmeyer DL, Cescon DW, De Laurentiis M, Nanda R, Winer EP, Mukai H, Tamura K, Armstrong AC, Liu MC, Iwata H, Ryvo L, Wimberger P, Rugo HS, Tan A, D'Aquanno C, Ding Y, Karantza V, Schmid P. KEYNOTE-086 cohort B: Pembrolizumab monotherapy for PD-L1–positive, previously untreated, metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-10.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.