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HER2-enriched subtype and pathological complete response in HER2-positive breast cancer: A systematic review and meta-analysis

  • Author Footnotes
    1 Co-first authors for equal contribution.
    Francesco Schettini
    Footnotes
    1 Co-first authors for equal contribution.
    Affiliations
    Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy

    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain
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  • Author Footnotes
    1 Co-first authors for equal contribution.
    Tomás Pascual
    Footnotes
    1 Co-first authors for equal contribution.
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Benedetta Conte
    Affiliations
    Department of Medical Oncology, Ospedale Policlinico San Martino, University of Genova, Genova, Italy
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  • Nuria Chic
    Affiliations
    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Fara Brasó-Maristany
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Patricia Galván
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain
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  • Olga Martínez
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Barbara Adamo
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Maria Vidal
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Montserrat Muñoz
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
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  • Aranzazu Fernández-Martinez
    Affiliations
    Department of Genetics, University of North Carolina, Chapel Hill, USA
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  • Carla Rognoni
    Affiliations
    Centre for Research on Health and Social Care Management (CERGAS), SDA Bocconi School of Management, Milan, Italy
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  • Gaia Griguolo
    Affiliations
    Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy

    Division of Medical Oncology 2, Istituto Oncologico Veneto – IRCCSS, Padova, Italy
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  • Valentina Guarneri
    Affiliations
    Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy

    Division of Medical Oncology 2, Istituto Oncologico Veneto – IRCCSS, Padova, Italy
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  • Pier Franco Conte
    Affiliations
    Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy

    Division of Medical Oncology 2, Istituto Oncologico Veneto – IRCCSS, Padova, Italy
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  • Mariavittoria Locci
    Affiliations
    Department of Neuroscience, Reproductive Medicine, Odontostomatology, University of Naples Federico II, Naples, Italy
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  • Jan C. Brase
    Affiliations
    Novartis Pharma AG, Basel, Switzerland
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  • Blanca Gonzalez-Farre
    Affiliations
    Department of Pathology, Hospital Clinic, Barcelona, Spain
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  • Patricia Villagrasa
    Affiliations
    SOLTI Breast Cancer Research Group, Barcelona, Spain
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  • Sabino De Placido
    Affiliations
    Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy
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  • Rachel Schiff
    Affiliations
    Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    Department of Medicine, Baylor College of Medicine, Houston, TX, USA

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
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  • Jamunarani Veeraraghavan
    Affiliations
    Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA
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  • Mothaffar F. Rimawi
    Affiliations
    Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    Department of Medicine, Baylor College of Medicine, Houston, TX, USA
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  • C. Kent Osborne
    Affiliations
    Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    Department of Medicine, Baylor College of Medicine, Houston, TX, USA

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
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  • Sonia Pernas
    Affiliations
    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Department of Medical Oncology, Institut Català d'Oncologia-H. U. Bellvitge-IDIBELL, Barcelona, Spain
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  • Charles M. Perou
    Affiliations
    Department of Genetics, University of North Carolina, Chapel Hill, USA
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  • Lisa A. Carey
    Affiliations
    Department of Medicine, University of North Carolina, Chapel Hill, USA
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  • Aleix Prat
    Correspondence
    Corresponding author at: Translational Genomic and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute (IDIBAPS) and Department of Medical Oncology, Hospital Clinic, Carrer de Villarroel, 170, 08036 Barcelona, Spain.
    Affiliations
    Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain

    SOLTI Breast Cancer Research Group, Barcelona, Spain

    Department of Medical Oncology, Hospital Clínic, Barcelona, Spain
    Search for articles by this author
  • Author Footnotes
    1 Co-first authors for equal contribution.
Published:January 17, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101965

      Highlights

      • We correlated the breast cancer intrinsic subtypes with pCR in HER2+ disease.
      • The HER2-E subtype was significantly and consistently associated with pCR after anti-HER2-based therapy.
      • The HER2-E subtype was associated with pCR irrespective of hormone receptor status.
      • The HER2-E subtype was associated with pCR also with chemo-free neoadjuvant schemes.

      Abstract

      Background

      HER2-positive (HER2+) breast cancer (BC) comprises all the four PAM50 molecular subtypes. Among these, the HER2-Enriched (HER2-E) appear to be associated with higher pathological complete response (pCR) rates following anti-HER2-based regimens. Here, we present a meta-analysis to validate the association of the HER2-E subtype with pCR following anti-HER2-based neoadjuvant treatments with or without chemotherapy (CT).

      Methods

      A systematic literature search was performed in February 2019. The primary objective was to compare the association between HER2-E subtype (versus others) and pCR. Selected secondary objectives were to compare the association between 1) HER2-E subtype and pCR in CT-free studies, 2) HER2-E subtype within hormone receptor (HR)-negative and HR+ disease and 3) HR-negative disease (versus HR+) and pCR in all patients and within HER2-E subtype. A random-effect model was applied. The Higgins’ I2 was used to quantify heterogeneity.

      Results

      Sixteen studies were included, 5 of which tested CT-free regimens. HER2-E subtype was significantly associated with pCR in all patients (odds ratio [OR] = 3.50, p < 0.001, I2 = 33%), in HR+ (OR = 3.61, p < 0.001, I2 = 1%) and HR-negative tumors (OR = 2.28, p = 0.01, I2 = 47%). In CT-free studies, HER2-E subtype was associated with pCR in all patients (OR = 5.52, p < 0.001, I2 = 0%) and in HR + disease (OR = 4.08, p = 0.001, I2 = 0%). HR-negative status was significantly associated with pCR compared to HR + status in all patients (OR = 2.41, p < 0.001, I2 = 30%) and within the HER2-E subtype (OR = 1.76, p < 0.001, I2 = 0%).

      Conclusions

      The HER2-E biomarker identifies patients with a higher likelihood of achieving a pCR following neoadjuvant anti-HER2-based therapy beyond HR status and CT use. Future trial designs to escalate or de-escalate systemic therapy in HER2+ disease should consider this genomic biomarker.

      Keywords

      Introduction

      Breast cancer (BC) with overexpression and/or amplification of the Human Epidermal Growth Factor Receptor 2 (HER2-positive) represents 11–30% of all breast tumors [
      • Cronin K.A.
      • Harlan L.C.
      • Dodd K.W.
      • Abrams J.S.
      • Ballard-Barbash R.
      Population-based estimate of the prevalence of HER-2 positive breast cancer tumors for early stage patients in the US.
      ]. HER2 positivity is defined today by immunohistochemistry (IHC) as complete and strong membrane staining (i.e. score of 3 + ) in ≥10% of cancer cells, and/or by in situ immunofluorescence (ISH) techniques as amplified using a HER2/CEP17 ratio cutoff of ≥2.0 and an average HER2 gene copy number ≥4.0 signals per cell [
      • Wolff A.C.
      • Hammond M.E.H.
      • Allison K.H.
      • et al.
      Human Epidermal growth factor receptor 2 testing in breast cancer: american society of clinical oncology/college of american pathologists clinical practice guideline focused update.
      ]. This consensus definition is based on the methods and cutoffs used over the years in pivotal trials that led to the approval of trastuzumab [
      • Slamon D.J.
      • Leyland-Jones B.
      • Shak S.
      • et al.
      Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
      ], pertuzumab [
      • Swain S.M.
      • Kim S.-B.
      • Cortés J.
      • et al.
      Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic breast cancer (CLEOPATRA study): overall survival results from a randomised, double-blind, placebo-controlled, phase 3 study.
      ], neratinib [
      • Martin M.
      • Holmes F.A.
      • Ejlertsen B.
      • et al.
      Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial.
      ], lapatinib [
      • Geyer C.E.
      • Forster J.
      • Lindquist D.
      • et al.
      Lapatinib plus capecitabine for HER2-positive advanced breast cancer.
      ] and T-DM1 [
      • Verma S.
      • Miles D.
      • Gianni L.
      • et al.
      Trastuzumab emtansine for HER2-positive advanced breast cancer.
      ] in HER2+ breast cancer.
      The current HER2 definition do not sufficiently consider HER2+ disease′s clinical and biological heterogeneity. On one hand, high variability in patient́s response and survival outcomes following anti-HER2-based therapy is common [
      • Mounsey L.A.
      • Deal A.M.
      • Keith K.C.
      • et al.
      Changing natural history of HER2-positive breast cancer metastatic to the brain in the era of new targeted therapies.
      ,
      • Prat A.
      • Pineda E.
      • Adamo B.
      • et al.
      Clinical implications of the intrinsic molecular subtypes of breast cancer.
      ]. On the other hand, high biological variability exists within HER2+ disease [
      • Prat A.
      • Carey L.A.
      • Adamo B.
      • et al.
      Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
      ,
      • Prat A.
      • Pascual T.
      • De Angelis C.
      • et al.
      HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
      ,
      • Cancer Genome Atlas Network
      Comprehensive molecular portraits of human breast tumors.
      ]. For example, all the BC intrinsic subtypes [i.e. Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like] can be identified through gene expression profiling [
      • Prat A.
      • Pineda E.
      • Adamo B.
      • et al.
      Clinical implications of the intrinsic molecular subtypes of breast cancer.
      ,
      • Prat A.
      • Carey L.A.
      • Adamo B.
      • et al.
      Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
      ,
      • Prat A.
      • Perou C.M.
      Deconstructing the molecular portraits of breast cancer.
      ]. Among them, the HER2-E subtype is the most frequent (31–76%), shows the highest levels of ERBB2 mRNA and protein and appears to be the subtype with the highest activation of the EGFR-HER2 signaling pathway [
      • Prat A.
      • Pascual T.
      • De Angelis C.
      • et al.
      HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
      ,
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. Importantly, these biological entities within HER2+ disease are not fully recapitulated by hormone receptor (HR) status since 40% of HER2+/HR+ tumors are HER2-E and 15% of HER2+/HR-negative tumors are Basal-like [
      • Prat A.
      • Carey L.A.
      • Adamo B.
      • et al.
      Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
      ,
      • Prat A.
      • Pascual T.
      • De Angelis C.
      • et al.
      HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
      ,
      • Cejalvo J.M.
      • Pascual T.
      • Fernández-Martínez A.
      • et al.
      Clinical implications of the non-luminal intrinsic subtypes in hormone receptor-positive breast cancer.
      ].
      To date, no biomarker has demonstrated clinical utility in HER2+ early disease beyond HER2 and HR status [
      • Curigliano G.
      • Burstein H.J.
      • Winer E.P.
      • Gnant M.
      • Dubsky P.
      • Loibl S.
      • Colleoni M.
      • Regan M.M.
      • Piccart-Gebhart M.
      • Senn H.-J.
      • Thürlimann B.
      • André F.
      • Baselga J.
      • Bergh J.
      • Bonnefoi H.
      • Brucker S.Y.
      • Cardoso F.
      • Carey L.
      • Ciruelos E.
      • Cuzick J.
      • Denkert C.
      • Di Leo A.
      • Ejlertsen B.
      • Francis P.
      • Galimberti V.
      • Garber J.
      • Gulluoglu B.
      • Goodwin P.
      • Harbeck N.
      • Hayes D.F.
      • Huang C.-S.
      • Huober J.
      • Khaled H.
      • Jassem J.
      • Jiang Z.
      • Karlsson P.
      • Morrow M.
      • Orecchia R.
      • Osborne K.C.
      • Pagani O.
      • Partridge A.H.
      • Pritchard K.
      • Ro J.
      • Rutgers E.J.T.
      • Sedlmayer F.
      • Semiglazov V.
      • Shao Z.
      • Smith I.
      • Toi M.
      • Tutt A.
      • Viale G.
      • Watanabe T.
      • Whelan T.J.
      • Xu B.
      De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen international expert consensus conference on the primary therapy of early breast cancer 2017.
      ]. However, accumulating evidence supports the clinical validity of two biomarkers: intrinsic subtyping and stromal tumor infiltrating-lymphocytes (TILs). In particular, either the HER2-E subtype or high TILs appears to be associated with high response to anti-HER2-based treatments in the neoadjuvant setting [
      • Salgado R.
      • Denkert C.
      • Campbell C.
      • et al.
      Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the NeoALTTO trial.
      ,
      • Denkert C.
      • von Minckwitz G.
      • Brase J.C.
      • et al.
      Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers.
      ,
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. From a prognostic point of view, however, HER2-E subtype is associated with a worse prognosis [
      • Prat A.
      • Carey L.A.
      • Adamo B.
      • et al.
      Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
      ,
      • Conte P.F.
      • Griguolo G.
      • Dieci M.V.
      • et al.
      PAM50 HER2-enriched subtype as an independent prognostic factor in early-stage HER2+ breast cancer following adjuvant chemotherapy plus trastuzumab in the ShortHER trial.
      ] whereas TILs are associated with a better survival outcome [
      • Salgado R.
      • Denkert C.
      • Campbell C.
      • et al.
      Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the NeoALTTO trial.
      ,
      • Loi S.
      • Michiels S.
      • Salgado R.
      • et al.
      Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
      ,
      • Griguolo G.
      • Pascual T.
      • Dieci M.V.
      • Guarneri V.
      • Prat A.
      Interaction of host immunity with HER2-targeted treatment and tumor heterogeneity in HER2-positive breast cancer.
      ]. Unfortunately, the majority of these data were derived from retrospective analyses from individual clinical trials using baseline tumor samples. In addition, most analyses were exploratory and unplanned, and limited by relatively small sample sizes.
      To increase the level of evidence of the association of the HER2-E subtype with the response to anti-HER2--based neoadjuvant regimens, we decided to review the literature and perform a meta-analysis.

      Materials and methods

      Search strategy and selection criteria

      A systematic literature search was performed on 12/February/2019 to identify published observational, phase II and phase III (randomized and non-randomized) neoadjuvant clinical studies involving anti-HER2-based treatments in HER2+ BC, where the association between pathological complete response (pCR) and BC molecular intrinsic subtypes was evaluated. The literature search had no time nor language restriction, however, only clinical studies involving anti-HER2-based neoadjuvant regimens were included, with or without chemotherapy. Additional studies particularly relevant to the topic, for which molecular data had not been published but were available at the Translational Genomic and Targeted Therapeutics in Solid Tumors laboratory of the IDIBAPS (Barcelona, Spain), were also included in the analysis. All pre-clinical studies, phase I trials, non-neoadjuvant trials and neoadjuvant trials without anti-HER2 agents were excluded. The recommendations of the Cochrane Collaboration [

      Higgins J, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [Updated March 2011]. The Cochrane Collaboration http://handbook.cochrane.org.

      ] were followed to identify all relevant studies. For our query, we used a combination of disease characteristics, study design, treatment setting and strategies or drugs. The full query is reported in the Suppl. Materials. Both full articles and studies published in the abstract form were included in the analysis, if odds ratios (OR) data were directly available or computable. The search was conducted on the electronic databases Pubmed and Web of Science®, as well as on San Antonio Breast Cancer Symposiums (SABCS)’s, American Society of Clinical Oncology (ASCO)’s and European Society of Medical Oncology (ESMO)’s annual meetings online archives. Four reviewers (FS, TP, NC and CR) independently evaluated whether each selected randomized clinical trials (RCT) respected the predetermined criteria, and another reviewer (AP) was consulted in case of controversy.

      Data extraction and objectives

      Details on study design, patient/tumor characteristics, interventions and outcome were extracted from each paper. Only the most recent and complete reports were included when duplicate publications were identified. Crude odds ratio (OR) for pCR with their 95% confidence intervals (CI) were extracted or calculated, when necessary, from each published paper or internal datasets. The definition of pCR varied across studies. In 12/16 (75%) studies (2,176/2,703 patients with known PAM50 subtype), pCR was defined as the absence of invasive neoplastic cells at microscopic examination of the primary tumor at surgery in breast and axilla (pCR in-breast and axilla), with remaining in-situ lesions allowed. In 4/16 (25%) studies (527/2703 patients with known molecular subtype), pCR was defined as the absence of tumor cells only in breast, without considering tumor response in axillary lymph nodes (pCR in-breast).
      The primary objective was to compare the association between HER2-E subtype (versus others) and pCR in all patients. Secondary objectives were to:
      • 1.
        compare the association between HER2-E subtype (versus others) and pCR in CT-free studies;
      • 2.
        compare the association between HR-negative disease (versus HR+ ) and pCR in all patients;
      • 3.
        compare the association between HR-negative disease (versus HR+ ) and pCR within HER2-E subtype;
      • 4.
        compare the association between HER2-E subtype (versus others) and pCR within HR+ and HR-negative disease;
      • 5.
        compare the association between each intrinsic subtype (versus the others) and pCR.

      Statistical analyses

      Since a certain degree of heterogeneity was expected, analyses were performed under the Random-Effect Model of DerSimonian and Laird [
      • DerSimonian R.
      • Laird N.
      Meta-analysis in clinical trials.
      ]. Heterogeneity was assessed with Higgin′s I2 index [
      • Higgins J.P.T.
      • Thompson S.G.
      Quantifying heterogeneity in a meta-analysis.
      ]. Pre-planned exploratory subgroup analyses for the primary endpoint were conducted, even if heterogeneity was not relevant. Subgroup analyses of interest were: (1) phase II vs phase III trials, (2) randomized vs. non-randomized trials (3) CT-containing vs. CT-free studies (4) pCR in-breast vs pCR in-breast and axilla. For the primary endpoint, to assess whether the pooled OR estimates were stable or strongly dependent on one or few studies, sensitivity analyses were conducted by interactively recalculating the pooled OR estimates after exclusion of each single study. Publication bias was explored through funnel plot visual inspection and the Egger’s linear regression test for funnel plot asymmetry [
      • Sterne J.A.
      • Egger M.
      Funnel plots for detecting bias in meta-analysis: guidelines on choice of axis.
      ,
      • Egger M.
      • Davey Smith G.
      • Schneider M.
      • Minder C.
      Bias in meta-analysis detected by a simple, graphical test.
      ]. All reported p values were two-sided. All statistical analyses and the generation of forest plots were conducted using R and RevMan [

      R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2017. https://www.R-project.org/.

      ,

      The Cochrane Collaboration. Review Manager (RevMan). Copenhagen: The Nordic Cochrane Centre; 2014.

      ]. The Cochrane risk of bias assessment tool was employed to assess the quality of the data obtained and the risk of bias in each study. Significance was set at p < 0.05, except for Egger’s test, for which significance was set as p < 0.1, as usual. The project was registered in the PROSPERO online database [

      PROSPERO International prospective register of systematic reviews. https://www.crd.york.ac.uk/prospero/.

      ], with registration number: CRD42019140902.

      Assessment of risk of bias

      The risk of bias for each trial was assessed by using the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions [

      Higgins J, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [Updated March 2011]. The Cochrane Collaboration http://handbook.cochrane.org.

      ]. Each domain related to a risk of bias was assessed in each included trial, since there is evidence that these issues are associated with biased estimates of treatment effect. The domains were the following: (1) random sequence generation; (2) allocation concealment; (3) blinding of participants and personnel; (4) blinding of outcome assessment; (5) incomplete outcome data; (6) selective reporting; (7) other bias. Review authors’ judgments were categorized as “low risk”, “high risk” or “unclear risk” of bias. Internal validity of eligible studies was assessed according to the Cochrane Collaboration′s ‘Risk of Bias’ tool in Review Manager [

      The Cochrane Collaboration. Review Manager (RevMan). Copenhagen: The Nordic Cochrane Centre; 2014.

      ].

      Results

      Summary of studies and patient characteristics

      A total of 16 studies were included (Table 1, Table 2; Supplementary Tables 1 and 2) [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. From Pubmed and Web of Science® online databases, 2,207 studies were extracted and 10 were included [
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ]. From ASCO, ESMO and SABCS online abstracts books, 4 studies were included [

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. Finally, data from 2 studies (ICO-CLINIC, LPT109096) were available at the Translational Genomic and Targeted Therapeutics in Solid Tumors laboratory at IDIBAPS (Barcelona, Spain) [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. Some data were also retrieved from later-published full articles [
      • Prat A.
      • Pascual T.
      • De Angelis C.
      • et al.
      HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
      ,
      • Pernas S.
      • Petit A.
      • Climent F.
      • et al.
      PAM50 subtypes in baseline and residual tumors following neoadjuvant trastuzumab-based chemotherapy in HER2-positive breast cancer: a consecutive-series from a single institution.
      ]. The selection process is resumed in the PRISMA diagram (Fig. 1). Overall 5 (31.25%) phase III RCT, 5 (31.25%) phase II RCT, 5 (31.25%) non-randomized phase II trials and 1 (6.25%) retrospective observational study were included. All the articles/abstracts containing molecular results have been published between 2014 and 2019.
      Table 1Characteristics of the included randomized phase II and III trials.
      RANDOMIZED TRIALS
      Study nameNOAH*NSABP-B41NeoALTTOCALGB 40,601KRISTINECher-LOBNeoSphereTRYPHAENALPT109096TBCRC023
      PhaseIIIIIIIIIIIIIIIIIIIIIIIII
      RegimenDox + P → P → CMF → T (HER2 positive cohort)AC → P + TAC → P + LAC → P + T + LL + T → PL → PT → PL + T + PL + PT + PTCH + PeT-DM1 + PeL + T → P → FECL → P → FECT → P → FECT + DPe + T + DPe + TPe + DFEC + T + Pe → T + Pe + DFEC → T + Pe + DTCH + PeT + FEC → T + PL + FEC → L + PT + L + FEC → T + L + PL + T +/− Let +/− GnRHa 12 weeksL + T +/− Let +/− GnRHa 24 weeks
      Treatment categoryAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 w/o CT
      N. of evaluable patients/Total of the arm63/117271/529254/455262/305354/44484/121337/417173/22561/100#85/97
      TNM StageIIIII and IIIII and IIIII and IIIII and IIIII and IIIAII and IIIII and IIIII and IIIII and III
      HR statusPos and negPos and negPos and negPos and negPos and negPos and negPos and negPos and negPos and negPos and neg
      HER2E (%)34 (54.0)197 (72.7)110 (43.3)82 (31.3)194 (54.8)22 (26.2)135 (40.1)82 (47.4)41 (67.2)51 (60.0)
      Non-HER2E (%)29 (46.0)74 (27.3)144 (56.7)180 (68.7)160 (45.2)62 (73.8)202 (59.9)91 (52.6)20 (32.8)34 (40.0)
      pCR in HER2E (%)18 (62.1)120 (60.9)57 (51.8)57 (69.5)131 (67.5)11 (50.0)53 (39.3)57 (69.5)30 (73.1)14 (27.5)
      pCR in non-HER2E (%)10 (34.5)19 (25.7)31 (21.5)64 (35.6)47 (29.4)16 (25.8)52 (25.7)46 (50.5)15 (75.0)3 (8.8)
      pCR definitionypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/isypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/is
      Gene expression platformMicroarray-basednCounterRNA seq.RNA seq.nCounterMicroarray-basednCounternCounternCounternCounter
      Data sourcePublished materialPublished materialPublished materialPublished materialPublished materialPublished materialPublished materialPublished materialIDIBAPS labPublished material
      Year of publication20142013/20192012/2016201520172015/20162012/2018201320112019/2019
      First authorPrat ARobidoux A/Swain SMBaselga J/Fumagalli DCarey LPrat AGuarneri V/Dieci MVGianni L/Bianchini GSchneeweiss AHolmes FARimawi MF/Prat A
      Publication formFull articleFull articleFull articleFull articleAbstractFull articleFull article/Abstract/PosterFull articleFull article/internal dataFull article/Full article
      Publication siteClin Can ResLancet Oncol/Breast Can Res TreatLancet/JAMA OncolJ Clin OncolSABCSThe Oncologist/Ann OncolLancet Oncol/ESMO congressAnn OncolBMC Research Notes/Internal dataClin Cancer Res /JNCI
      Legend and footnotes: HER2E = HER2 enriched; non-HER2E = Basal-Like, Luminal A, Luminal B, Normal-like; Pos = positive; Neg = negative; HR = hormone receptors; CT = chemotherapy; N/A = not assessed; pCR = pathologic complete response; AC = doxorubicin + cyclophosphamide; Dox = doxorubicin; CMF = cyclophosphamide + methotrexate + 5-fluorouracil; FEC = 5-fluorouracil + epirubicin + cyclophosphamide; TCH = docetaxel + carboplatin + trastuzumab; P = paclitaxel; D = docetaxel; LD = liposomal doxorubicin; T = trastuzumab; Pe = pertuzumab; L = lapatinib; Let = letrozole; GnRHa = GnRH analogue →= followed by; *:HER2 positive cohort non-treated with trastuzumab and HER2 negative cohort not considered; #: pts with non-available information on pCR excluded; SABCS = San Antonio Breast Cancer Symposium; ASCO = American Society of Clinical Oncology; ESMO = European Society for Medical Oncology.
      Table 2Characteristics of the included non-randomized studies.
      NON-RANDOMIZED STUDIES
      Study nameICO-CLINICBERENICEOpti-HER HEARTPerELISATBCRC006PAMELA
      Study typeRetrospective ObservationalNon-randomized Phase IISingle Arm Phase IINon-Randomized Phase IISingle Arm Phase IISingle Arm Phase II
      RegimenTax+/−Anthra + TddAC → P + T + PeFEC → D + T + PePe + T + LD + PLet + T + PeP + T + PeL + T +/− Let+/−GnRHL + T +/− Let or Tam
      Treatment categoryAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 + CTAnti-HER2 w/o CTAnti-HER2 + CTAnti-HER2 w/o CTAnti-HER2 w/o CT
      N. of evaluable patients/Total of the arm172/173294/40058/8340/4415/1729/65151/151
      TNM StageI–IIIII and IIIII and IIIII and IIIII and IIII - III
      HR statusPos and negPos and negPos and negPosPos and negPos and neg
      HER2E (%)102 (59.3)175 (59.5)30 (51.7)11 (27.5)12 (75.0)22 (75.9)101 (66.9)
      Non-HER2E (%)70 (40.7)119 (40.5)28 (48.3)29 (72.5)3 (25.0)7 (24.1)50 (33.1)
      pCR in HER2E (%)63 (61.8)130 (74.2)25 (83.3)5 (45.5)10 (83.3)6 (20.7)41 (40.6)
      pCR in non-HER2E (%)19 (27.1)52 (43.7)13 (46.4)4 (13.8)2 (66.7)1 (14.3)5 (10.0)
      pCR definitionypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/is ypN0ypT0/isypT0/is
      Gene expression platformnCounternCounternCounternCounternCounternCounter
      Data sourcePublished material/Internal dataPublished materialPublished materialPublished materialPublished materialPublished material
      Year of publication2017/20192017201920192013/20192017
      First authorPernas SSwain SMGavilà JGuarneri VRimawi MF/Prat ALlombart-Cussac A
      Publication formAbstract/Full articleFull articleFull articleFull articleFull articleFull article
      Journal/MeetingSABCS /Front OncolAnn OncolBMC MedicineAnn OncolJ Clin Oncol/JNCILancet Oncol
      Legend and footnotes: HER2E = HER2 enriched; non-HER2E = Basal-Like, Luminal A, Luminal B, Normal-like; Pos = positive; Neg = negative; HR = hormone receptors; CT = chemotherapy; N/A = not assessed; pCR = pathologic complete response; AC = doxorubicin + cyclophosphamide; Dox = doxorubicin; CMF = cyclophosphamide + methotrexate + 5-fluorouracil; FEC = 5-fluorouracil + epirubicin + cyclophosphamide; TCH = docetaxel + carboplatin + trastuzumab; Tax = taxanes; Anthra = anthracyclines; P = paclitaxel; D = docetaxel; LD = liposomal doxorubicin; T = trastuzumab; Pe = pertuzumab; L = lapatinib; Let = letrozole; Tam = tamoxifen; GnRHa = GnRH analogue; → = followed by; dd = dose dense.
      From a total of 3733 patients, PAM50 intrinsic subtype was available for 2703 (72.4%) patients, while HR status was known for 3373 (90.3%) patients. Except for one trial (i.e. PerELISA) which enrolled HR+ tumors-only [
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ], the others included both HR+ and HR-negative tumors. All studies included evaluated anti-HER2-based neoadjuvant regimens with or without CT [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,
      • Gianni L.
      • Pienkowski T.
      • Im Y.-H.
      • et al.
      Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial.
      ], and included tumor stages II or III, except for the PAMELA trial and the retrospective observational study from the Catalan Institute of Oncology and the Hospital Clinic of Barcelona (ICO-CLINIC), which allowed stage I disease [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ]. Various methods for assessing the PAM50 BC intrinsic subtypes were used across all trials (Table 1, Table 2), but all were based upon gene expression data [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ].
      Among the studies included, only the PAMELA single arm phase II trial was specifically designed to prospectively assess PAM50 intrinsic subtypes and test whether patients with the HER2-E subtype benefited more than the other subtypes from a neoadjuvant anti-HER2-based CT-free regimen [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ]. The other studies evaluated PAM50 as an exploratory retrospective analysis; therefore, tumor samples were not always available for all patients included. However, samples were always available for at least half of the population enrolled within each study (Table 1, Table 2). pCR rates in HER2-E subtype were higher than nonHER2-E subtypes in each study, except in the LPT109096 trial. Individual trials′ results are reported in Table 1, Table 2.

      pCR and HER2-E subtype

      The HER2-E subtype was significantly associated with pCR compared to others (OR = 3.50, 95% CI 2.79 – 4.39, p < 0.001, I2 = 33%, Fig. 2). The funnel plot suggested the absence of publication bias (Suppl. Fig. 1), confirmed by a non-significant Egger′s test (p = 0.48). The influential analysis showed consistent results when omitting a single trial with an I2 range varying from 3.4% (omitting the NeoSphere trial) [
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ] to 37.7% (omitting the TBCRC023 trial) [

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. Full results of the influential analysis are reported in Table 3. Considering the absence of significant heterogeneity, an exploratory, non-preplanned analysis done with the fixed-effect model [
      • Peto R.
      Why do we need systematic overviews of randomized trials?.
      ] was performed with a similar result (OR = 3.51, 95% CI: 2.96–4.16, p < 0.001, I2 = 33%).
      Figure thumbnail gr2
      Fig. 2Forest Plots comparing the association with pCR between the HER2-E and the other intrinsic subtypes in the overall population.
      Table 3Influential analyses concerning the primary end-point.
      Study omittedOR95% CIpI2
      BERENICE3.472.70–4.47<0.000137.6%
      CALGB406013.442.68–4.40<0.000136.7%
      Cher-LOB3.522.78–4.48<0.000137.4%
      ICO-CLINIC3.432.69–4.38<0.000136.6%
      KRISTINE3.342.63–4.23<0.000128.9%
      LPT1090963.622.95–4.45<0.000121.3%
      NeoALTTO3.452.69–4.43<0.000137.3%
      NEOSPHERE3.853.18–4.66<0.00013.4%
      NOAH3.572.82–4.50<0.000135.2%
      NSABP-B413.412.67–4.35<0.000135.6%
      Opti-HER-HEART3.442.72–4.35<0.000135.9%
      PAMELA3.412.71–4.30<0.000134.1%
      Per-ELISA3.412.72–4.28<0.000132.3%
      TBCRC0063.512.78–4.43<0.000137.4%
      TBCRC0233.482.75–4.42<0.000137.7%
      TRYPHAENA3.642.88–4.59<0.000131.2%
      Legend: OR = odds ratio; CI = confidence intervals.
      There were no statistically significant differences in terms of association with pCR for all the subgroups considered for the preplanned sensitivity analyses, namely randomized vs. non-randomized studies (p = 0.46), phase II vs. phase III studies (p = 0.13), CT-containing vs. CT-free studies (p = 0.30), pCR in-breast vs pCR in-breast + axilla (p = 0.32). Compared to other intrinsic subtypes, the HER2-E subtype was significantly associated with pCR compared to Basal-like (OR = 2.50, 95% CI 1.78–3.52, p < 0.001, I2 = 0%, Suppl. Fig. 2A), Luminal A (OR = 4.81, 95% CI 3.16 – 7.33, p < 0.001, I2 = 55%, Suppl. Fig. 2B), Luminal B (OR = 3.82, 95% CI 2.97–4.91, p < 0.001, I2 = 0%, Suppl. Fig. 2C) and Luminal A/B (OR = 4.36, 95% CI 3.17–6.00, p < 0.001, I2 = 52%, Suppl. Fig. 2D) subtypes. Other comparisons among intrinsic subtypes can be found in the Suppl. Materials.

      pCR, HR status and HER2-E subtype

      Fifteen of the 16 trials were used to assess the association between HR status and pCR. HR-negative disease was significantly associated with pCR compared to HR+ disease (OR = 2.41, 95% CI 2.00 – 2.92, p < 0.001, I2 = 30%, Fig. 3A). The inspection of the funnel plot (Suppl. Fig. 3), as well as the result of the Egger’s test (p = 0.68), did not reveal a significant publication bias. The HER2-E subtype was significantly associated with pCR within both HR-negative disease (OR = 2.28, 95% CI 1.21 – 4.29, p = 0.01, I2 = 47%, Fig. 3B) and HR+ disease (OR = 3.61, 95% CI 2.61 – 5.00, p < 0.001, I2 = 1%, Fig. 3C). Similar to what was observed for the general population, HR-negative disease was significantly associated with pCR compared to HR+ disease within the HER2-E subtype (OR = 1.76, 95% CI 1.30 – 2.38, p < 0.001, I2 = 0%, Fig. 3D).
      Figure thumbnail gr3
      Fig. 3A-D. Forest Plots comparing the association with pCR between HR-positive and HR-negative tumors (A) in the overall population; the association with pCR between the HER2-E and the other intrinsic subtypes within the HR-negative (B) and HR-positive (C) disease, and the association of pCR between HR-positive and HR-negative tumors within the HER2-E subtype (D).

      pCR, HR status and HER2-E subtype in the absence of CT

      A total of 5 studies evaluated dual HER2 blockade in the absence of CT [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ], although for one of these (i.e. NeoSphere), data for the CT-free arm were not available separately from the other CT-containing arms’ data [
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ]. In CT-free regimens, HER2-E subtype was significantly associated with pCR compared to the other subtypes (OR = 5.52, 95% CI 2.89–10.54, p < 0.001, I2 = 0%, Fig. 4A), while there was no apparent difference between HR-negative vs. HR+ disease (OR = 1.49, 95% CI 0.44–5.03, p = 0.52, I2 = 76%, Fig. 4B). When considering HR status, the HER2-E subtype was found to be significantly associated with pCR within HR+ disease (OR = 4.08, 95% CI: 1.76 – 9.46, p = 0.001, I2 = 0%, Suppl. Fig. 4A), but not within HR-negative disease (OR = 2.18, 95% CI: 0.66 – 7.26, p = 0.20, I2 = 0%, Suppl. Fig. 4B). Conversely, in patients with HER2-E subtype, HR status was not significantly associated with pCR (OR = 1.30, 95% CI 0.67 – 2.52, p = 0.44, I2 = 0%, Suppl. Fig. 5).
      Figure thumbnail gr4
      Fig. 4Forest Plots comparing the association with pCR between the HER2-E and the other subtypes (A), and between HR-negative and HR-positive tumors (B) in CT-free trials.

      Risk of bias analysis

      With respect to the risk of bias, as defined by the Cochrane’s manual for systematic reviews [

      Higgins J, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [Updated March 2011]. The Cochrane Collaboration http://handbook.cochrane.org.

      ], the risk of selection bias for random sequence generation and allocation concealments was present in the 6/16 (37.5%) of the studies in both cases, with an unclear risk in 1/16 (6.25%) studies included, concerning the random sequence generation selection bias (Fig. 5 and Suppl. Fig. 6). The performance bias due to blinding of participants and personnel was present in 12/16 (75%) of cases, with an unclear risk in 3/16 (18.75%) of the studies included. No detection bias related to the blinding of outcome assessment, attrition bias due to incomplete outcome data and selective reporting bias were observed. Concerning the last two, an unclear risk was present in 1/16 (6.25%) cases. Finally, we accounted for a 6.25% high risk of other bias related to the ICO-CLINIC study, due to its retrospective and non-trial design.

      Discussion

      The development of effective drugs against HER2+ BC has been particularly successful in the last few years [
      • Slamon D.J.
      • Leyland-Jones B.
      • Shak S.
      • et al.
      Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
      ,
      • Swain S.M.
      • Kim S.-B.
      • Cortés J.
      • et al.
      Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic breast cancer (CLEOPATRA study): overall survival results from a randomised, double-blind, placebo-controlled, phase 3 study.
      ,
      • Martin M.
      • Holmes F.A.
      • Ejlertsen B.
      • et al.
      Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial.
      ,
      • Geyer C.E.
      • Forster J.
      • Lindquist D.
      • et al.
      Lapatinib plus capecitabine for HER2-positive advanced breast cancer.
      ,
      • Verma S.
      • Miles D.
      • Gianni L.
      • et al.
      Trastuzumab emtansine for HER2-positive advanced breast cancer.
      ]. Since the introduction of trastuzumab [
      • Slamon D.J.
      • Leyland-Jones B.
      • Shak S.
      • et al.
      Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
      ], other effective and tolerable anti-HER2 drugs (i.e. lapatinib, pertuzumab, neratinib and T-DM1) have been introduced in the metastatic and/or early disease settings, contributing to important improvements in survival outcomes [
      • Mounsey L.A.
      • Deal A.M.
      • Keith K.C.
      • et al.
      Changing natural history of HER2-positive breast cancer metastatic to the brain in the era of new targeted therapies.
      ,
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
      • McGuire W.L.
      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ]. However, HER2+ disease is clinically and biologically heterogeneous and not all patients benefit to the same extend from current treatments. Thus, better identification of patients using biomarkers should allow the design of prospective trials aiming to improve precision medicine in HER2+ BC.
      Among the different biomarkers evaluated in HER2+ disease over the last decade [
      • Prat A.
      • Carey L.A.
      • Adamo B.
      • et al.
      Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
      ,
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ,
      • Salgado R.
      • Denkert C.
      • Campbell C.
      • et al.
      Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the NeoALTTO trial.
      ,
      • Denkert C.
      • von Minckwitz G.
      • Brase J.C.
      • et al.
      Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers.
      ,
      • Loi S.
      • Michiels S.
      • Salgado R.
      • et al.
      Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
      ,
      • Loibl S.
      • Majewski I.
      • Guarneri V.
      • et al.
      PIK3CA mutations are associated with reduced pathological complete response rates in primary HER2-positive breast cancer: pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab.
      ,
      • Baselga J.
      • Cortés J.
      • Im S.-A.
      • et al.
      Biomarker analyses in CLEOPATRA: a phase III, placebo-controlled study of pertuzumab in human epidermal growth factor receptor 2-positive, first-line metastatic breast cancer.
      ,
      • Cortazar P.
      • Zhang L.
      • Untch M.
      • et al.
      Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis.
      ], the HER2-E subtype has been proposed to identify patients whose HER2+ tumors are HER2 “addicted” (meaning driven primarily by signaling via the HER2 pathway). Retrospective analysis of the HER2-E subtype, mostly exploratory and unplanned, using baseline tumor samples from individual clinical trials have linked this phenotype with high rates of pathological complete response following neoadjuvant anti-HER2-based therapies [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ]. However, to date, no combined analysis or meta-analysis has been performed and analyses within all of those studies were limited by relatively small sample sizes. Here, we performed a trial-level meta-analysis of 16 neoadjuvant studies and 2703 patients to evaluate the association of the HER2-E subtype with pCR. In particular, we confirmed that the HER2-E subtype is a consistent biomarker to identify patients with a higher likelihood of achieving a pCR following anti-HER2-based therapy with or without cytotoxic therapy. Importantly, the association of the HER2-E subtype with pCR appeared to be independent of HR status, which is the only biomarker with clinical utility in HER2+ disease. Additionally, our results confirm the ability of HR status to predict pCR by itself and within the HER2-E subtype, although we could not demonstrate this in the CT-free setting, which had substantially fewer contributing trials.
      We adopted pCR as our clinical endpoint for this meta-analysis. This is because numerous studies have demonstrated a favorable prognostic role in early stage HER2+ BC [
      • Cortazar P.
      • Zhang L.
      • Untch M.
      • et al.
      Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis.
      ,
      • Von Minckwitz G.
      • Untch M.
      • Blohmer J.-U.
      • et al.
      Definition and impact of pathologic complete response on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes.
      ,
      • Nekljudova V.
      • Loibl S.
      • von Minckwitz G.
      • et al.
      Trial-level prediction of long-term outcome based on pathologic complete response (pCR) after neoadjuvant chemotherapy for early-stage breast cancer (EBC).
      ,
      • Broglio K.R.
      • Quintana M.
      • Foster M.
      • et al.
      Association of pathologic complete response to neoadjuvant therapy in HER2-positive breast cancer with long-term outcomes: a meta-analysis.
      ,

      Spring LM, Fell G, Arfe A, et al. Pathological complete response after neoadjuvant chemotherapy and impact on breast cancer recurrence and mortality, stratified by breast cancer subtypes and adjuvant chemotherapy usage: Individual patient-level meta-analyses of over 27,000 patients [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 GS2-03.

      ] so its use as primary endpoint in neoadjuvant trials has been increasing over the years and has also been endorsed for regulatory purposes by regulatory agencies such as US Food and Drug Administration (FDA), for accelerated approval of neoadjuvant treatments in high risk early-stage BC [

      FDA Guidance for Industry. Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early-Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval. Available at: https://www.fda.gov/media/83507/download. Last accessed: 02/12/2020.

      ]. Furthermore, the FDA recently approved the use of adjuvant T-DM1 (in HER2+ BC) or capecitabine (in HER2-negative BC) in case of no achievement of pCR following standard neoadjuvant systemic therapy and surgery, making of pCR a fundamental tool in therapeutic decision-making in non-metastatic BC for escalating treatment strategies. At the same time, there is also an increasing use of pCR as a tool to identify potentially effective and safe de-escalating therapeutic approaches in HER2+ BC [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Rimawi MF, Niravath P, Wang T, et al. TBCRC023: A Randomized Phase II Neoadjuvant Trial of Lapatinib Plus Trastuzumab Without Chemotherapy for 12 vs. 24 Weeks in Patients with HER2-positive Breast Cancer. Clin Cancer Res. 2019; doi: 10.1158/1078-0432.CCR-19-0851 [Epub ahead of print].

      ]. In fact, identification of effective de-escalating treatment strategies to spare toxicity and financial costs to patients is a main focus of the research community nowadays [
      • Dieci M.V.
      • Vernaci G.
      • Guarneri V.
      Escalation and de-escalation in HER2 positive early breast cancer.
      ,
      • Veeraraghavan J.
      • De Angelis C.
      • Reis-Filho J.S.
      • et al.
      De-escalation of treatment in HER2-positive breast cancer: determinants of response and mechanisms of resistance.
      ]. In adjuvant setting, several prospective trials of early stage HER2+ BC have attempted to demonstrate that de-escalating strategies based on a shorter duration of adjuvant trastuzumab provided similar benefits as the conventional 1 year; however, the results using non-inferiority trial designs were mixed [
      • Pondé N.
      • Gelber R.D.
      • Piccart M.
      PERSEPHONE.
      ]. On the contrary, a single-arm trial from a single institution (i.e. the APT trial) evaluating 12 doses of adjuvant weekly paclitaxel and 1-year of trastuzumab in largely HR+ stage I disease significantly impacted on daily clinical practice after showing extraordinary DFS and OS rates at 7-years [
      • Tolaney S.M.
      • Guo H.
      • Pernas S.
      • et al.
      Seven-year follow-up analysis of adjuvant paclitaxel and trastuzumab trial for node-negative, human epidermal growth factor receptor 2-positive breast cancer.
      ]. In this scenario, at least 3 critical questions remain to be answered regarding de-escalation of systemic therapy in early HER2+ disease: (1) who can be treated with less or even no adjuvant trastuzumab after surgery? (2) who does not need (neo)adjuvant pertuzumab in stage II and III disease? (3) can we decrease the amount of chemotherapy? In fact, immunohistochemically HER2+/non-HER2-E tumors might be poorly dependent, if not totally independent, from the HER2-signaling pathway and not gain any benefit from adjuvant trastuzumab following previous neoadjuvant therapy and surgery. At the same time some HER2+ tumors might be “HER2 addicted” enough not to need chemotherapy at all or to require a shortened adjuvant trastuzumab duration and/or no adjuvant dual blockade therapy. To address these questions, well-designed clinical trials integrating clinical variables (such as tumor dimension and axillary nodes involvement), response data and biomarkers such as the HER2-E subtype, TILs, intra-tumor heterogeneity [

      Metzger Filho O, Viale G, Trippa L, et al. HER2 heterogeneity as a predictor of response to neoadjuvant T-DM1 plus pertuzumab: Results from a prospective clinical trial. J Clin Oncol. 2019;37(15_suppl):502-502. doi:10.1200/JCO.2019.37.15_suppl.502.

      ] and PIK3CA status are needed.
      This meta-analysis has several limitations. First, some secondary end-points were affected by discrete levels of heterogeneity (I2 ≥ 75% and p heterogeneity < 0.05, results in Fig. 4B and Suppl. Materials). This was mostly attributable to the paucity of molecular data from some trials and differences in the effects observed, preventing them from being fully reliable, regardless of the analytical model applied. However, this consideration doesn’t apply to the main result of the study. Second, although several studies correlated pCR with long-term survival outcomes (EFS/DFS and OS) in the context of HER2+ BC [
      • Cortazar P.
      • Zhang L.
      • Untch M.
      • et al.
      Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis.
      ,
      • Von Minckwitz G.
      • Untch M.
      • Blohmer J.-U.
      • et al.
      Definition and impact of pathologic complete response on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes.
      ,
      • Nekljudova V.
      • Loibl S.
      • von Minckwitz G.
      • et al.
      Trial-level prediction of long-term outcome based on pathologic complete response (pCR) after neoadjuvant chemotherapy for early-stage breast cancer (EBC).
      ,
      • Broglio K.R.
      • Quintana M.
      • Foster M.
      • et al.
      Association of pathologic complete response to neoadjuvant therapy in HER2-positive breast cancer with long-term outcomes: a meta-analysis.
      ,

      Spring LM, Fell G, Arfe A, et al. Pathological complete response after neoadjuvant chemotherapy and impact on breast cancer recurrence and mortality, stratified by breast cancer subtypes and adjuvant chemotherapy usage: Individual patient-level meta-analyses of over 27,000 patients [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 GS2-03.

      ], others failed to demonstrate its role as an efficient surrogate endpoint for survival [
      • Berruti A.
      • Amoroso V.
      • Gallo F.
      • et al.
      Pathologic complete response as a potential surrogate for the clinical outcome in patients with breast cancer after neoadjuvant therapy: a meta-regression of 29 randomized prospective studies.
      ,
      • Korn E.L.
      • Sachs M.C.
      • McShane L.M.
      Statistical controversies in clinical research: assessing pathologic complete response as a trial-level surrogate end point for early-stage breast cancer.
      ]. Additionally, there is a specific lack of survival data related to intrinsic subtypes within the clinical trials included in this study. Therefore, no claims regarding the association of the HER2-E subtype with t patients' survival outcome can be made based on this meta-analysis. Moreover, 4/16 trials reported data regarding in-breast pCR, which has not been recognized by the FDA as a validated endpoint for drug approval in neoadjuvant setting [

      FDA Guidance for Industry. Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early-Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval. Available at: https://www.fda.gov/media/83507/download. Last accessed: 02/12/2020.

      ]. Third, the methods used to apply the PAM50 algorithm varied across trials. For example, 13 studies used the nCounter platform [
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ], 2 studies used RNA-seq data [
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ] and 2 studies used microarray-based data [
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ]. Finally, we were only able to perform a study-level meta-analysis instead of a patient-level meta-analysis, which would have increased precision and homogeneity and enabled thorough exploration of potential effect moderators.
      To conclude, our results demonstrate that the HER2-E subtype is a consistent biomarker of response following neoadjuvant anti-HER2-based regimens, with and without CT and beyond HR status. This biomarker, along with TILs and other biomarkers, such as PIK3CA mutations [
      • Loibl S.
      • Majewski I.
      • Guarneri V.
      • et al.
      PIK3CA mutations are associated with reduced pathological complete response rates in primary HER2-positive breast cancer: pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab.
      ,
      • Baselga J.
      • Cortés J.
      • Im S.-A.
      • et al.
      Biomarker analyses in CLEOPATRA: a phase III, placebo-controlled study of pertuzumab in human epidermal growth factor receptor 2-positive, first-line metastatic breast cancer.
      ,
      • Kim S.-B.
      • Wildiers H.
      • Krop I.E.
      • et al.
      Relationship between tumor biomarkers and efficacy in TH3RESA, a phase III study of trastuzumab emtansine (T-DM1) vs. treatment of physician’s choice in previously treated HER2-positive advanced breast cancer.
      ,
      • Rimawi M.F.
      • De Angelis C.
      • Contreras A.
      • et al.
      Low PTEN levels and PIK3CA mutations predict resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2 over-expressing breast cancer.
      ], either alone or in combination [
      • Veeraraghavan J.
      • De Angelis C.
      • Mao R.
      • et al.
      A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.
      ], should be routinely incorporated in future prospective clinical trials designed to implement strategies to escalate and/or de-escalate systemic therapies [
      • Prat A.
      • Pascual T.
      • De Angelis C.
      • et al.
      HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
      ,
      • Llombart-Cussac A.
      • Cortés J.
      • Paré L.
      • et al.
      HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
      ,
      • Carey L.A.
      • Berry D.A.
      • Cirrincione C.T.
      • et al.
      Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
      ,
      • Prat A.
      • Bianchini G.
      • Thomas M.
      • et al.
      Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
      ,

      Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

      ,
      • Fumagalli D.
      • Venet D.
      • Ignatiadis M.
      • et al.
      RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
      ,

      Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

      ,
      • Bianchini G.
      • Pusztai L.
      • Pienkowski T.
      • et al.
      Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
      ,
      • Dieci M.V.
      • Prat A.
      • Tagliafico E.
      • et al.
      Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
      ,
      • Ignatiadis M.
      • Van den Eynden G.
      • Roberto S.
      • et al.
      Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
      ,
      • Holmes F.A.
      • Espina V.
      • Liotta L.A.
      • et al.
      Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
      ,
      • Swain S.M.
      • Ewer M.S.
      • Viale G.
      • et al.
      Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
      ,
      • Gavilá J.
      • Oliveira M.
      • Pascual T.
      • et al.
      Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
      ,

      Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      ,

      Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

      ,
      • Guarneri V.
      • Dieci M.V.
      • Bisagni G.
      • et al.
      De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
      ,
      • Rimawi M.F.
      • Mayer I.A.
      • Forero A.
      • et al.
      Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
      ,

      Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      ,

      Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

      ].

      Declaration of Competing Interest

      FS has declared travel and accommodation expenses paid by Roche, Pfizer and Celgene. SDP has declared honoraria from Roche, Pfizer, Astra-Zeneca, Novartis, Celgene, Eli Lilly, Amgen and Eisai. AP has declared an immediate family member being employed by Novartis, personal honoraria from Pfizer, Novartis, Roche, MSD Oncology, Lilly and Daiichi Sankyo, travel, accommodations and expenses paid by Daiichi Sankyo, research funding from Roche and Novartis, consulting/advisory role for NanoString Technologies, Amgen, Roche, Novartis, Pfizer and Bristol-Myers Squibb and patent PCT/EP2016/080056: HER2 AS A PREDICTOR OF RESPONSE TO DUAL HER2 BLOCKADE IN THE ABSENCE OF CYTOTOXIC THERAPY. OTHER AUTHORS CoI. PFC had declared consultant role for Novartis, Eli Lilly, Astra Zeneca and Tesaro, honoraria from BMS, Roche, Eli Lilly, Novartis and AstraZeneca, research funding from Novartis, Roche, BMS, Merck-KGa, Italian Ministry of Health, Veneto Secretary of Health and University of Padova. CMP is an equity stock holder and consultant of BioClassifier LLC and is also listed an inventor on patent applications on the Breast PAM50. LAC has declared that Companies who have provided funds to her institution in the past 1–2 years either for her service on advisory/consultative programs or sponsored research were Genentech, Roche, Novartis, Seattle Genetics, G1 Therapeutics, Immunomedics and Innocrin. SP has received honoraria for talks and travel grants from Roche outside of the submitted work and serves as an advisor/consultant to Polyphor. RS has declared research funding from AstraZeneca, GlaxoSmithKline, Gilead Sciences, and PUMA Biotechnology, and consulting/advisory role with compensation for Macrogenics, and Eli Lilly. CKO has declared research funding from AstraZeneca and GlaxoSmithKline, advisory boards for Tolmar Pharmaceuticals, Genentech, and AstraZeneca, DMC for Eli Lilly and stockholder of GeneTex.
      MFR has declared research funding from GlaxoSmithKline and Genentech. JCB reports employment and stocks with Novartis. The other authors have nothing to declare.

      Acknowledgements

      We are grateful to Dario Bruzzese (associate professor of Statistics at the University of Naples Federico II) for his methodological suggestions.

      Funding

      Instituto de Salud Carlos III - PI16/00904 (to AP), Pas a Pas (to AP), Save the Mama (to AP), Breast Cancer Now - 2018NOVPCC1294 (to AP). Fundación Científica Asociación Española Contra el Cáncer – Ayuda Postdoctoral AECC 2017 (to FB-M). Fundación SEOM , Becas FSEOM para Formación en Investigación en Centros de Referencia en el Extranjero 2016 (to AF-M). NCI Breast SPORE program P50-CA58223 (to CMP) and Susan G. Komen SAC-160074 (to CMP ), and NCI 1R01CA229409 (to LAC). The US Department of Defense grants W81XWH-17-1-0579 (to MFR) and W81XWH-17-1-0580 (to RS); NIH: SPORE Grant P50 CA186784 (to RS, CKO, and MFR); Cancer Center Grants ( P30 CA125123 ); research grant from the Breast Cancer Research Foundation BCRF- 18-145 (to RS and CKO).

      Authors contributions

      FS, AP, LAC and CMP conceived the study. FS, TP, NC and CR performed the systematic review of the literature and AP was consulted for a final decision in case of controversy. FS performed the statistical analyses. FS, TP and AP wrote the first draft of the article. All authors contributed in interpreting the data, writing and correcting the manuscript drafts and approved the final version.

      Appendix A. Supplementary material

      The following are the Supplementary data to this article:

      References

        • Cronin K.A.
        • Harlan L.C.
        • Dodd K.W.
        • Abrams J.S.
        • Ballard-Barbash R.
        Population-based estimate of the prevalence of HER-2 positive breast cancer tumors for early stage patients in the US.
        Cancer Invest. 2010; 28: 963-968https://doi.org/10.3109/07357907.2010.496759
        • Wolff A.C.
        • Hammond M.E.H.
        • Allison K.H.
        • et al.
        Human Epidermal growth factor receptor 2 testing in breast cancer: american society of clinical oncology/college of american pathologists clinical practice guideline focused update.
        J Clin Oncol Off J Am Soc Clin Oncol. 2018; 36: 2105-2122https://doi.org/10.1200/JCO.2018.77.8738
        • Slamon D.J.
        • Leyland-Jones B.
        • Shak S.
        • et al.
        Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
        N Engl J Med. 2001; 344: 783-792https://doi.org/10.1056/NEJM200103153441101
        • Swain S.M.
        • Kim S.-B.
        • Cortés J.
        • et al.
        Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic breast cancer (CLEOPATRA study): overall survival results from a randomised, double-blind, placebo-controlled, phase 3 study.
        Lancet Oncol. 2013; 14: 461-471https://doi.org/10.1016/S1470-2045(13)70130-X
        • Martin M.
        • Holmes F.A.
        • Ejlertsen B.
        • et al.
        Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial.
        Lancet Oncol. 2017; 18: 1688-1700https://doi.org/10.1016/S1470-2045(17)30717-9
        • Geyer C.E.
        • Forster J.
        • Lindquist D.
        • et al.
        Lapatinib plus capecitabine for HER2-positive advanced breast cancer.
        N Engl J Med. 2006; 355: 2733-2743https://doi.org/10.1056/NEJMoa064320
        • Verma S.
        • Miles D.
        • Gianni L.
        • et al.
        Trastuzumab emtansine for HER2-positive advanced breast cancer.
        N Engl J Med. 2012; 367: 1783-1791https://doi.org/10.1056/NEJMoa1209124
        • Mounsey L.A.
        • Deal A.M.
        • Keith K.C.
        • et al.
        Changing natural history of HER2-positive breast cancer metastatic to the brain in the era of new targeted therapies.
        Clin Breast Cancer. 2018; 18: 29-37https://doi.org/10.1016/j.clbc.2017.07.017
        • Prat A.
        • Pineda E.
        • Adamo B.
        • et al.
        Clinical implications of the intrinsic molecular subtypes of breast cancer.
        Breast Edinb Scotl. 2015; 24: S26-S35https://doi.org/10.1016/j.breast.2015.07.008
        • Prat A.
        • Carey L.A.
        • Adamo B.
        • et al.
        Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer.
        J Natl Cancer Inst. 2014; 106https://doi.org/10.1093/jnci/dju152
        • Prat A.
        • Pascual T.
        • De Angelis C.
        • et al.
        HER2-enriched subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 blockade.
        J Natl Cancer Inst. 2019; 30https://doi.org/10.1093/jnci/djz042
        • Cancer Genome Atlas Network
        Comprehensive molecular portraits of human breast tumors.
        Nature. 2012; 490: 61-70https://doi.org/10.1038/nature11412
        • Prat A.
        • Perou C.M.
        Deconstructing the molecular portraits of breast cancer.
        Mol Oncol. 2011; 5: 5-23https://doi.org/10.1016/j.molonc.2010.11.003
        • Llombart-Cussac A.
        • Cortés J.
        • Paré L.
        • et al.
        HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.
        Lancet Oncol. 2017; 18: 545-554https://doi.org/10.1016/S1470-2045(17)30021-9
        • Carey L.A.
        • Berry D.A.
        • Cirrincione C.T.
        • et al.
        Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III Trial of paclitaxel plus trastuzumab with or without lapatinib.
        J Clin Oncol Off J Am Soc Clin Oncol. 2016; 34: 542-549https://doi.org/10.1200/JCO.2015.62.1268
        • Prat A.
        • Bianchini G.
        • Thomas M.
        • et al.
        Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study.
        Clin Cancer Res Off J Am Assoc Cancer Res. 2014; 20: 511-521https://doi.org/10.1158/1078-0432.CCR-13-0239
      1. Swain SM, Tang G, Lucas PC, et al. Intrinsic subtypes of HER2-positive breast cancer and their associations with pathologic complete response (pCR) and outcomes: Findings from NSABP B-41, a randomized neoadjuvant trial. J Clin Oncol. 2018;36(15_suppl):580-580. doi:10.1200/JCO.2018.36.15_suppl.580.

        • Fumagalli D.
        • Venet D.
        • Ignatiadis M.
        • et al.
        RNA Sequencing to predict response to neoadjuvant anti-HER2 therapy: a secondary analysis of the NeoALTTO randomized clinical trial.
        JAMA Oncol. 2017; 3: 227-234https://doi.org/10.1001/jamaoncol.2016.3824
      2. Bianchini G, Parker JS, Carey LA, et al. Research-based PAM50 predicts risk of relapse in residual disease after anti-HER2 therapies. Ann Oncol. 2018; 29(8_suppl):viii58–viii86.

        • Bianchini G.
        • Pusztai L.
        • Pienkowski T.
        • et al.
        Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.
        Ann Oncol Off J Eur Soc Med Oncol. 2015; 26: 2429-2436https://doi.org/10.1093/annonc/mdv395
        • Dieci M.V.
        • Prat A.
        • Tagliafico E.
        • et al.
        Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial.
        Ann Oncol. 2016; 27: 1867-1873https://doi.org/10.1093/annonc/mdw262
        • Ignatiadis M.
        • Van den Eynden G.
        • Roberto S.
        • et al.
        Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy: a TRYPHAENA substudy.
        J Natl Cancer Inst. 2019; 111: 69-77https://doi.org/10.1093/jnci/djy076
        • Holmes F.A.
        • Espina V.
        • Liotta L.A.
        • et al.
        Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling.
        BMC Res Notes. 2013; 6: 507https://doi.org/10.1186/1756-0500-6-507
        • Swain S.M.
        • Ewer M.S.
        • Viale G.
        • et al.
        Pertuzumab, trastuzumab, and standard anthracycline- and taxane-based chemotherapy for the neoadjuvant treatment of patients with HER2-positive localized breast cancer (BERENICE): a phase II, open-label, multicenter, multinational cardiac safety study.
        Ann Oncol Off J Eur Soc Med Oncol. 2018; 29: 646-653https://doi.org/10.1093/annonc/mdx773
        • Gavilá J.
        • Oliveira M.
        • Pascual T.
        • et al.
        Safety, activity, and molecular heterogeneity following neoadjuvant non-pegylated liposomal doxorubicin, paclitaxel, trastuzumab, and pertuzumab in HER2-positive breast cancer (Opti-HER HEART): an open-label, single-group, multicenter, phase 2 trial.
        BMC Med. 2019; 17: 8https://doi.org/10.1186/s12916-018-1233-1
      3. Pernas S, Petit A, Climent F, et al. Abstract P2-09-11: PAM50 intrinsic subtyping as a predictor of pathological complete response to neoadjuvant trastuzumab-based chemotherapy in early HER2-positive breast cancer. Cancer Res. 2018;78(4 Supplement):P2-09-11. doi:10.1158/1538-7445.SABCS17-P2-09-11.

      4. Prat A, Slamon D, Hurvitz S, et al. Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC). Cancer Res. 2018; 78 (4 Supplement):PD3-06. doi:10.1158/1538-7445.SABCS17-PD3-06.

        • Guarneri V.
        • Dieci M.V.
        • Bisagni G.
        • et al.
        De-escalated therapy for HR+/HER2+ breast cancer patients with Ki67 response after 2 weeks letrozole: results of the PerELISA neoadjuvant study.
        Ann Oncol. 2019; 30: 921-926https://doi.org/10.1093/annonc/mdz055
        • Rimawi M.F.
        • Mayer I.A.
        • Forero A.
        • et al.
        Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.
        J Clin Oncol Off J Am Soc Clin Oncol. 2013; 31: 1726-1731https://doi.org/10.1200/JCO.2012.44.8027
      5. Prat A, De Angelis C, Pascual T, et al. Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer. Cancer Res. 2018; 78(4 Supplement):P2-09-12. doi:10.1158/1538-7445.SABCS17-P2-09-12.

      6. Prat A, De Angelis C, Pascual T, et al. HER2-enriched subtype and ERBB2 mRNA as predictors of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer: A combined analysis of TBCRC006/023 and PAMELA trials. J Clin Oncol. 2018; 36(15_suppl):509–509. doi:10.1200/JCO.2018.36.15_suppl.509.

        • Cejalvo J.M.
        • Pascual T.
        • Fernández-Martínez A.
        • et al.
        Clinical implications of the non-luminal intrinsic subtypes in hormone receptor-positive breast cancer.
        Cancer Treat Rev. 2018; 67: 63-70https://doi.org/10.1016/j.ctrv.2018.04.015
        • Curigliano G.
        • Burstein H.J.
        • Winer E.P.
        • Gnant M.
        • Dubsky P.
        • Loibl S.
        • Colleoni M.
        • Regan M.M.
        • Piccart-Gebhart M.
        • Senn H.-J.
        • Thürlimann B.
        • André F.
        • Baselga J.
        • Bergh J.
        • Bonnefoi H.
        • Brucker S.Y.
        • Cardoso F.
        • Carey L.
        • Ciruelos E.
        • Cuzick J.
        • Denkert C.
        • Di Leo A.
        • Ejlertsen B.
        • Francis P.
        • Galimberti V.
        • Garber J.
        • Gulluoglu B.
        • Goodwin P.
        • Harbeck N.
        • Hayes D.F.
        • Huang C.-S.
        • Huober J.
        • Khaled H.
        • Jassem J.
        • Jiang Z.
        • Karlsson P.
        • Morrow M.
        • Orecchia R.
        • Osborne K.C.
        • Pagani O.
        • Partridge A.H.
        • Pritchard K.
        • Ro J.
        • Rutgers E.J.T.
        • Sedlmayer F.
        • Semiglazov V.
        • Shao Z.
        • Smith I.
        • Toi M.
        • Tutt A.
        • Viale G.
        • Watanabe T.
        • Whelan T.J.
        • Xu B.
        De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen international expert consensus conference on the primary therapy of early breast cancer 2017.
        Ann Oncol. 2017; 28: 1700-1712https://doi.org/10.1093/annonc/mdx308
        • Salgado R.
        • Denkert C.
        • Campbell C.
        • et al.
        Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the NeoALTTO trial.
        JAMA Oncol. 2015; 1: 448-454https://doi.org/10.1001/jamaoncol.2015.0830
        • Denkert C.
        • von Minckwitz G.
        • Brase J.C.
        • et al.
        Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers.
        J Clin Oncol Off J Am Soc Clin Oncol. 2015; 33: 983-991https://doi.org/10.1200/JCO.2014.58.1967
        • Conte P.F.
        • Griguolo G.
        • Dieci M.V.
        • et al.
        PAM50 HER2-enriched subtype as an independent prognostic factor in early-stage HER2+ breast cancer following adjuvant chemotherapy plus trastuzumab in the ShortHER trial.
        J Clin Oncol. 2019; 37: 544
        • Loi S.
        • Michiels S.
        • Salgado R.
        • et al.
        Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
        Ann Oncol Off J Eur Soc Med Oncol. 2014; 25: 1544-1550https://doi.org/10.1093/annonc/mdu112
        • Griguolo G.
        • Pascual T.
        • Dieci M.V.
        • Guarneri V.
        • Prat A.
        Interaction of host immunity with HER2-targeted treatment and tumor heterogeneity in HER2-positive breast cancer.
        J ImmunoTher Cancer. 2019; 7https://doi.org/10.1186/s40425-019-0548-6
        • Loibl S.
        • Majewski I.
        • Guarneri V.
        • et al.
        PIK3CA mutations are associated with reduced pathological complete response rates in primary HER2-positive breast cancer: pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab.
        Ann Oncol Off J Eur Soc Med Oncol. 2016; 27: 1519-1525https://doi.org/10.1093/annonc/mdw197
        • Baselga J.
        • Cortés J.
        • Im S.-A.
        • et al.
        Biomarker analyses in CLEOPATRA: a phase III, placebo-controlled study of pertuzumab in human epidermal growth factor receptor 2-positive, first-line metastatic breast cancer.
        J Clin Oncol Off J Am Soc Clin Oncol. 2014; 32: 3753-3761https://doi.org/10.1200/JCO.2013.54.5384
        • Kim S.-B.
        • Wildiers H.
        • Krop I.E.
        • et al.
        Relationship between tumor biomarkers and efficacy in TH3RESA, a phase III study of trastuzumab emtansine (T-DM1) vs. treatment of physician’s choice in previously treated HER2-positive advanced breast cancer.
        Int J Cancer. 2016; 139: 2336-2342https://doi.org/10.1002/ijc.30276
        • Rimawi M.F.
        • De Angelis C.
        • Contreras A.
        • et al.
        Low PTEN levels and PIK3CA mutations predict resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2 over-expressing breast cancer.
        Breast Cancer Res Treat. 2018; 167: 731-740https://doi.org/10.1007/s10549-017-4533-9
        • Veeraraghavan J.
        • De Angelis C.
        • Mao R.
        • et al.
        A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.
        Ann Oncol Off J Eur Soc Med Oncol. 2019; https://doi.org/10.1093/annonc/mdz076
      7. Higgins J, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [Updated March 2011]. The Cochrane Collaboration http://handbook.cochrane.org.

        • DerSimonian R.
        • Laird N.
        Meta-analysis in clinical trials.
        Control Clin Trials. 1986; 7: 177-188
        • Higgins J.P.T.
        • Thompson S.G.
        Quantifying heterogeneity in a meta-analysis.
        Stat Med. 2002; 21: 1539-1558https://doi.org/10.1002/sim.1186
        • Sterne J.A.
        • Egger M.
        Funnel plots for detecting bias in meta-analysis: guidelines on choice of axis.
        J Clin Epidemiol. 2001; 54: 1046-1055
        • Egger M.
        • Davey Smith G.
        • Schneider M.
        • Minder C.
        Bias in meta-analysis detected by a simple, graphical test.
        BMJ. 1997; 315: 629-634https://doi.org/10.1136/bmj.315.7109.629
      8. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2017. https://www.R-project.org/.

      9. The Cochrane Collaboration. Review Manager (RevMan). Copenhagen: The Nordic Cochrane Centre; 2014.

      10. PROSPERO International prospective register of systematic reviews. https://www.crd.york.ac.uk/prospero/.

        • Pernas S.
        • Petit A.
        • Climent F.
        • et al.
        PAM50 subtypes in baseline and residual tumors following neoadjuvant trastuzumab-based chemotherapy in HER2-positive breast cancer: a consecutive-series from a single institution.
        Front Oncol. 2019; 9https://doi.org/10.3389/fonc.2019.00707
        • Gianni L.
        • Pienkowski T.
        • Im Y.-H.
        • et al.
        Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial.
        Lancet Oncol. 2012; 13: 25-32https://doi.org/10.1016/S1470-2045(11)70336-9
        • Peto R.
        Why do we need systematic overviews of randomized trials?.
        Stat Med. 1987; 6: 233-244
        • Slamon D.J.
        • Clark G.M.
        • Wong S.G.
        • Levin W.J.
        • Ullrich A.
        • McGuire W.L.
        Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
        Science. 1987; 235: 177-182https://doi.org/10.1126/science.3798106
        • Cortazar P.
        • Zhang L.
        • Untch M.
        • et al.
        Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis.
        Lancet Lond Engl. 2014; 384: 164-172https://doi.org/10.1016/S0140-6736(13)62422-8
        • Von Minckwitz G.
        • Untch M.
        • Blohmer J.-U.
        • et al.
        Definition and impact of pathologic complete response on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes.
        J Clin Oncol. 2012; 30: 1796-1804
        • Nekljudova V.
        • Loibl S.
        • von Minckwitz G.
        • et al.
        Trial-level prediction of long-term outcome based on pathologic complete response (pCR) after neoadjuvant chemotherapy for early-stage breast cancer (EBC).
        Contemp Clin Trials. 2018; 71: 194-198
        • Broglio K.R.
        • Quintana M.
        • Foster M.
        • et al.
        Association of pathologic complete response to neoadjuvant therapy in HER2-positive breast cancer with long-term outcomes: a meta-analysis.
        JAMA Oncol. 2016; 2: 751-776
      11. Spring LM, Fell G, Arfe A, et al. Pathological complete response after neoadjuvant chemotherapy and impact on breast cancer recurrence and mortality, stratified by breast cancer subtypes and adjuvant chemotherapy usage: Individual patient-level meta-analyses of over 27,000 patients [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 GS2-03.

      12. FDA Guidance for Industry. Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early-Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval. Available at: https://www.fda.gov/media/83507/download. Last accessed: 02/12/2020.

      13. Rimawi MF, Niravath P, Wang T, et al. TBCRC023: A Randomized Phase II Neoadjuvant Trial of Lapatinib Plus Trastuzumab Without Chemotherapy for 12 vs. 24 Weeks in Patients with HER2-positive Breast Cancer. Clin Cancer Res. 2019; doi: 10.1158/1078-0432.CCR-19-0851 [Epub ahead of print].

        • Dieci M.V.
        • Vernaci G.
        • Guarneri V.
        Escalation and de-escalation in HER2 positive early breast cancer.
        Curr Opin Oncol. 2019; 31: 35-42https://doi.org/10.1097/CCO.0000000000000492
        • Veeraraghavan J.
        • De Angelis C.
        • Reis-Filho J.S.
        • et al.
        De-escalation of treatment in HER2-positive breast cancer: determinants of response and mechanisms of resistance.
        Breast Edinb Scotl. 2017; 34: S19-S26https://doi.org/10.1016/j.breast.2017.06.022
        • Pondé N.
        • Gelber R.D.
        • Piccart M.
        PERSEPHONE.
        NPJ Breast Cancer. 2019; 5: 1https://doi.org/10.1038/s41523-018-0098-y
        • Tolaney S.M.
        • Guo H.
        • Pernas S.
        • et al.
        Seven-year follow-up analysis of adjuvant paclitaxel and trastuzumab trial for node-negative, human epidermal growth factor receptor 2-positive breast cancer.
        J Clin Oncol Off J Am Soc Clin Oncol. 2019; 37: 1868-1875https://doi.org/10.1200/JCO.19.00066
      14. Metzger Filho O, Viale G, Trippa L, et al. HER2 heterogeneity as a predictor of response to neoadjuvant T-DM1 plus pertuzumab: Results from a prospective clinical trial. J Clin Oncol. 2019;37(15_suppl):502-502. doi:10.1200/JCO.2019.37.15_suppl.502.

        • Berruti A.
        • Amoroso V.
        • Gallo F.
        • et al.
        Pathologic complete response as a potential surrogate for the clinical outcome in patients with breast cancer after neoadjuvant therapy: a meta-regression of 29 randomized prospective studies.
        J Clin Oncol. 2014; 32: 3883-3891https://doi.org/10.1200/JCO.2014.55.2836
        • Korn E.L.
        • Sachs M.C.
        • McShane L.M.
        Statistical controversies in clinical research: assessing pathologic complete response as a trial-level surrogate end point for early-stage breast cancer.
        Ann Oncol. 2016; 27: 10-15https://doi.org/10.1093/annonc/mdv507