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Background: The capsule formulation of CDK4/6 inhibitor palbociclib has reduced solubility at gastric pH > 4.5 and may have decreased activity when used with proton-pump inhibitors (PPI). Herein, we report the effect of PPI on palbociclib capsule activity and safety in the PARSIFAL study., Methods: First-line endocrine-sensitive, hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) patients received palbociclib capsules plus fulvestrant or letrozole. The primary endpoint was progression-free survival (PFS). This post-hoc analysis compared PPI use. Patients were PPI-naïve (N-PPI) if not on PPI during the study, and either early (E-PPI) or long-term PPI (LT-PPI) if on PPI at study entry or for at least ≥⅔ of treatment, respectively. PPI groups were not mutually exclusive., Results: Among 486 patients, 66.9 % were N-PPI, 13.2 % E-PPI, 18.7 % LT-PPI, and 11.5 % of the PPI users were defined as neither. Median PFS (mPFS) was 29.6 months in the study population, 28.7 months in N-PPI, 23.0 months in E-PPI (Hazard Ratio [HR] 1.5; 95%Confidence Interval [CI] 1.1-2.2; p = 0.024), and 23.0 months in LT-PPI (HR 1.4; 95%CI 1.0-1.9; p = 0.035). By landmark analysis, PPI use was associated with poorer mPFS at 3 and 12 months. Grade ≥3 hematological adverse events occurred in 71.7 % of N-PPI, 57.8 % of E-PPI (p = 0.021), and 54.9 % of LT-PPI (p = 0.003). Dose reductions and dosing delays due to hematological toxicity occurred in 70.8 % of N-PPI, 56.3 % of E-PPI (p = 0.018), and 52.7 % of LT-PPI (p = 0.002)., Conclusions: PPI use may reduce palbociclib capsule toxicity, dose modifications, and clinical activity in HR+/HER2- ABC., Competing Interests: Declaration of competing interest SDC reports consulting for MEDSIR and IQVIA and honoraria from Pierre-Fabre, Novartis, and AstraZeneca. JMPG reports consulting for Roche, Eiasi, Daichii Sankyo, AstraZeneca, Gilead, MSD and Seagen and travel expenses from Roche. MB reports consulting fees from Pfizer, Novartis, Lilly, and Steamline-Menarini; honoraria from Pfizer, Novartis, and Lilly; and trave support from Pfizer. FD reports travel grants for Daichii, Novartis, and Pfizer, and participation in monitor/advisory boards for Daiichi, Seagen, Novartis, Gilead, Lilly, and MSD. MGG reports honoraria from Pfizer, Novartis, and AstraZeneca, travel grants from Lilly, Daiichi-Sankyo, and Pfizer, and participation in monitoring/advisory boards for AstraZeneca, Seagen, and Gilead. MRB reports honoraria from Pfizer, Novartis, AstraZenca, and Daiichi Sankyo. JG reports consulting fees from Novartis, Pfizer, AstraZeneca; honoraria from Novartis; and travel support from Roche and AstraZeneca. EAO reports consulting for AstraZeneca, Daichii, Gilead, MSD, and Seagen; honoraria from AstraZeneca, Daichii, Lilly, MSD, and Seagen; travel support from Daichii, Lilly, Gilead, and AstraZeneca; and participation in monitor/advisory boards for Gilead, AstraZeneca, Seagen, and Dachii. PS reports honoraria from Pfizer, AstraZeneca, Novartis, Roche, Merck, and Boehringer Ingelheim; a consulting role for Pfizer, AstraZeneca, Novartis, Roche, Merck, Boehringer Ingelheim, Bayer, Eisai, Celgene, and Puma; and institutional grants from Roche, Genentech, Oncogenex, and Novartis. FM reports grants from Roche, Novartis, AstraZeneca, GSK, MSD, Clovis, Vaccibody, Gilead Sciences, and Esai; consulting fees from AstraZeneca, GSK, and Roche; honoraria from AstraZeneca, MSD, Lilly, Pfizer, Novartis, GSK, Clovis, Myriad, Daiich Sankyo, Seagen, Pierre-Fabre, and Agendia; travel support from AstraZeneca, GSK, Pfizer, and Roche; and participation in advisory boards for Palleos and Amgen. JG reports grant/research support from Roche, Eiasi, and Exact Science; honoraria or consultation fees from AstraZeneca, Daiichi, Eisai, Exact Science, Evapharm, GE Healthcare, Gilead, Lilly, MSD, Novartis, Pfizer, Pierre Fabre, Roche, Seagen, Sothema and participation in company sponsored speaker's bureaus for Exact Science, Lilly, and Novartis. AS reports grants from Celgene, Roche, Abbvie; travel grants from Celgene, Roche, Pfizer, AstraZeneca, and honoraria from Roche, Celgene, Pfizer, AstraZeneca, Novartis, MSD, Tesaro, Lilly, Seagen, Gilead, GSK, Bayer, Amgen, Pierre Fabre. JA reports trial funding from MEDSIR and consulting for Pfizer, Lilly, and Novartis. PZ reports honoraria from Pfizer, Novartis, Roche, and Pierre Fabre; and travel support from Pfizer, Novartis, Roche, and Lilly. DW reports honoraria from Pfizer, Novartis, AstraZeneca and travel grants from Roche, and Novartis. JC reports consulting for Roche, Celgene, Cellestia, AstraZeneca, Seattle Genetics, Daiichi Sankyo, Erytech, Athenex, Polyphor, Lilly, Merck Sharp&Dohme, GSK, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Expres2ion Biotechnologies; honoraria from Roche, Novartis, Celgene, Eisai, Pfizer, Samsung Bioepis, Lilly, Merck Sharp&Dohme, Daiichi Sankyo.; research funding to the Institution from Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer healthcare, Eisai, F. Hoffman-La Roche, Guardanth health, Merck Sharp&Dohme, Pfizer, Piqur Therapeutics, Puma C, Queen Mary University of London; stock shares for MEDSIR, Nektar Pharmaceuticals, Leuko (relative); Travel, accommodation, expenses from Roche, Novartis, Eisai, Pfizer, Daiichi Sankyo, Astrazeneca, Gilead; and Patents: Pharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent. Javier Cortés Castán, Alejandro Piris Giménez, Violeta Serra Elizalde. WO 2014/199294 A. ISSUED Her2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy. Aleix Prat, Antonio Llombart, Javier Cortés. US 2019/0338368 A1. LICENSED. ALC reports research support from Roche, Agendia, Lilly, Pfizer, Novartis, Merck Sharp & Dohme, Gilead, and Daichii-Sanyo; consulting for Lilly, Roche, Pfizer, and Novartis; participation in speakers' bureaus for Lilly, AstraZeneca, Merck Sharp & Dohme; travel support from Roche, Pfizer, AstraZeneca; and stock or other ownership in MEDSIR and Initia-Research. The remaining authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The combination of trastuzumab plus pertuzumab plus docetaxel as a first-line therapy in patients with HER2-positive metastatic breast cancer has provided significant clinical benefits compared to trastuzumab plus docetaxel alone. However, despite the therapeutic success of existing therapies targeting HER2, tumours invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. It is well known that the tumour microenvironment (TME) has a significant impact on cancer behaviour. Cancer-associated fibroblasts (CAFs) are essential components of the tumour stroma that have been linked to acquired therapeutic resistance and poor prognosis in breast cancer. For this reason, it would be of interest to identify novel biomarkers in the tumour stroma that could emerge as therapeutic targets for the modulation of resistant phenotypes. Conditioned medium experiments carried out in our laboratory with CAFs derived from HER2-positive patients showed a significant capacity to promote resistance to trastuzumab plus pertuzumab therapies in two HER2-positive breast cancer cell lines (BCCLs), even in the presence of docetaxel. In order to elucidate the components of the CAF-conditioned medium that may be relevant in the promotion of BCCL resistance, we implemented a multiomics strategy to identify cytokines, transcription factors, kinases and miRNAs in the secretome that have specific targets in cancer cells. The combination of cytokine arrays, label-free LC-MS/MS quantification and miRNA analysis to explore the secretome of CAFs under treatment conditions revealed several up- and downregulated candidates. We discuss the potential role of some of the most interesting candidates in generating resistance in HER2-positive breast cancer.

HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer., (©2020 American Association for Cancer Research.)

Purpose: Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance., Experimental Design: We have used a platform of HER2-targeted therapy-resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies., Results: We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo , coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo , inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance., Conclusions: We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors., (©2019 American Association for Cancer Research.)

The use of adjuvant pertuzumab in HER2-positive early-stage breast cancer has recently been approved by the EMA on the basis of data from the APHINITY trial. Accordingly, we have produced this opinion article with the aim of putting the study data in perspective against other add-on therapeutic strategies, to clarify methodological or statistical doubts about the study, and to define the population of high-risk patients with hormone receptor-negative breast cancer that we agree, in general, should be treated. With this approval, physicians must be well prepared to place the APHINITY study data in context. It is now up to each country to ratify the EMA-approved indications and to agree on reimbursement, and doctors must optimize their use based on knowledge and discussion with patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: We investigated the value of tumor-infiltrating NK (TI-NK) cells and HLA class I tumor expression as biomarkers of response to neoadjuvant anti-HER2 antibody-based treatment in breast cancer., Experimental Design: TI-NK cells and HLA-I were determined by IHC in pretreatment tumor biopsies from two cohorts of patients with HER2-positive breast cancer [discovery cohort ( n = 42) and validation cohort ( n = 71)]. Tumor-infiltrating lymphocytes (TIL) were scored according to international guidelines. Biomarker association with pathologic complete response (pCR) and disease-free survival (DFS) was adjusted for prognostic factors. Gene set variation analysis was used for determining immune cell populations concomitant to NK-cell enrichment in HER2-positive tumors from the Cancer Genome Atlas ( n = 190)., Results: TI-NK cells were significantly associated with pCR in the discovery cohort as well as in the validation cohort ( P < 0.0001), independently of clinicopathologic factors. A ≥3 TI-NK cells/50x high-power field (HPF) cutoff predicted pCR in the discovery and validation cohort [OR, 188 (11-3154); OR, 19.5 (5.3-71.8)]. Presence of TI-NK cells associated with prolonged DFS in both patient cohorts [HR, 0.07 (0.01-0.6); P = 0.01; HR, 0.3 (0.08-1.3); P = 0.1]. NK-, activated dendritic- and CD8 T-cell gene expression signatures positively correlated in HER2-positive tumors, supporting the value of NK cells as surrogates of effective antitumor immunity. Stratification of patients by tumor HLA-I expression identified patients with low and high relapse risk independently of pCR., Conclusions: This study identifies baseline TI-NK cells as an independent biomarker with great predictive value for pCR to anti-HER2 antibody-based treatment and points to the complementary value of tumor HLA-I status for defining patient prognosis independently of pCR., (©2018 American Association for Cancer Research.)

T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to tumor-associated or tumor-specific antigens. The receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. TCBs targeting HER2 may result in severe toxicities, likely due to the expression of HER2 in normal epithelia. About 40% of HER2-positive tumors express p95HER2, a carboxyl-terminal fragment of HER2. Using specific antibodies, here, we show that p95HER2 is not expressed in normal tissues. We describe the development of p95HER2-TCB and show that it has a potent antitumor effect on p95HER2-expressing breast primary cancers and brain lesions. In contrast with a TCB targeting HER2, p95HER2-TCB has no effect on nontransformed cells that do not overexpress HER2. These data pave the way for the safe treatment of a subgroup of HER2-positive tumors by targeting a tumor-specific antigen., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose To investigate the safety and clinical activity of comprehensive human epidermal growth factor receptor (HER) family receptor inhibition using lumretuzumab (anti-HER3) and pertuzumab (anti-HER2) in combination with paclitaxel in patients with metastatic breast cancer (MBC). Methods This phase Ib study enrolled 35 MBC patients (first line or higher) with HER3-positive and HER2-low (immunohistochemistry 1+ to 2+ and in-situ hybridization negative) tumors. Patients received lumretuzumab (1000 mg in Cohort 1; 500 mg in Cohorts 2 and 3) plus pertuzumab (840 mg loading dose [LD] followed by 420 mg in Cohorts 1 and 2; 420 mg without LD in Cohort 3) every 3 weeks, plus paclitaxel (80 mg/m 2 weekly in all cohorts). Patients in Cohort 3 received prophylactic loperamide treatment. Results Diarrhea grade 3 was a dose-limiting toxicity of Cohort 1 defining the maximum tolerated dose of lumretuzumab when given in combination with pertuzumab and paclitaxel at 500 mg every three weeks. Grade 3 diarrhea decreased from 50% (Cohort 2) to 30.8% (Cohort 3) with prophylactic loperamide administration and omission of the pertuzumab LD, nonetheless, all patients still experienced diarrhea. In first-line MBC patients, the objective response rate in Cohorts 2 and 3 was 55% and 38.5%, respectively. No relationship between HER2 and HER3 expression or somatic mutations and clinical response was observed. Conclusions Combination treatment with lumretuzumab, pertuzumab and paclitaxel was associated with a high incidence of diarrhea. Despite the efforts to alter dosing, the therapeutic window remained too narrow to warrant further clinical development., Trial Registration: on ClinicalTrials.gov with the identifier NCT01918254 first registered on 3rd July 2013.

Background: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy., Objective: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer., Conclusion: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance. Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2 -positive breast cancer explants. Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation. Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006-19. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Biological changes that occur during metastatic progression of breast cancer are still incompletely characterized. In this study, we compared intrinsic molecular subtypes and gene expression in 123 paired primary and metastatic tissues from breast cancer patients. Intrinsic subtype was identified using a PAM50 classifier and χ 2 tests determined the differences in variable distribution. The rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched (HER2-E) tumors, 30.0% in luminal B tumors, and 55.3% in luminal A tumors. In 40.2% of cases, luminal A tumors converted to luminal B tumors, whereas in 14.3% of cases luminal A and B tumors converted to HER2-E tumors. We identified 47 genes that were expressed differentially in metastatic versus primary disease. Metastatic tumors were enriched for proliferation-related and migration-related genes and diminished for luminal-related genes. Expression of proliferation-related genes were better at predicting overall survival in metastatic disease (OSmet) when analyzed in metastatic tissue rather than primary tissue. In contrast, a basal-like gene expression signature was better at predicting OSmet in primary disease compared with metastatic tissue. We observed correlations between time to tumor relapse and the magnitude of changes of proliferation, luminal B, or HER2-E signatures in metastatic versus primary disease. Although the intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquired a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution or acquisition of estrogen independence. Overall, our analysis revealed the value of stratifying gene expression by both cancer subtype and tissue type, providing clinicians more refined tools to evaluate prognosis and treatment. Cancer Res; 77(9); 2213-21. ©2017 AACR ., (©2017 American Association for Cancer Research.)

PAM50/Prosigna gene expression-based assay identifies three categorical risk of relapse groups (ROR-low, ROR-intermediate and ROR-high) in post-menopausal patients with estrogen receptor estrogen receptor-positive (ER+)/ HER2-negative (HER2-) early breast cancer. Low risk patients might not need adjuvant chemotherapy since their risk of distant relapse at 10-years is below 10% with endocrine therapy only. In this study, 517 consecutive patients with ER+/HER2- and node-negative disease were evaluated for Ki67 and Prosigna. Most of Luminal A tumors (65.6%) and ROR-low tumors (70.9%) had low Ki67 values (0-10%); however, the percentage of patients with ROR-medium or ROR-high disease within the Ki67 0-10% group was 42.7% (with tumor sizes ≤2 cm) and 33.9% (with tumor sizes > 2 cm). Finally, we found that the optimal Ki67 cutoff for identifying Luminal A or ROR-low tumors was 14%. Ki67 as a surrogate biomarker in identifying Prosigna low-risk outcome patients or Luminal A disease in the clinical setting is unreliable. In the absence of a well-validated prognostic gene expression-based assay, the optimal Ki67 cutoff for identifying low-risk outcome patients or Luminal A disease remains at 14%.

A subset of HER2+ breast cancer patients manifest clinical resistance to trastuzumab. Recently, miR-26a and miR-30b have been identified as trastuzumab response regulators, and their target gene CCNE2 seems to play an important role in resistance to trastuzumab therapy. Cell viability was evaluated in trastuzumab treated HER2+ BT474 wt (sensitive), BT474r (acquired resistance), HCC1954 (innate resistance), and MDA-MB-231 (HER2-) cell lines, and the expression of miR-26a, miR-30b, and their target genes was measured. BT474 wt cell viability decreased by 60% and miR-26a and miR-30b were significantly overexpressed (~3-fold, p = 0.003 and p = 0.002, respectively) after trastuzumab treatment, but no differences were observed in resistant and control cell lines. Overexpression of miR-30b sensitized BT474r cells to trastuzumab (p = 0.01) and CCNE2, was significantly overexpressed after trastuzumab treatment in BT474r cells (p = 0.032), but no significant changes were observed in sensitive cell line. When CCNE2 was silenced BT474r cell sensitivity to trastuzumab increased (p = 0.03). Thus, the molecular mechanism of trastuzumab action in BT474 cell line may be regulated by miR-26a and miR-30b and CCNE2 overexpression might play an important role in acquired trastuzumab resistance in HER2+ breast cancer given that resistance was diminished when CCNE2 was silenced.

Background: Trastuzumab emtansine (T-DM1) exhibited enhanced antitumor activity when combined with docetaxel or pertuzumab in preclinical studies. This phase Ib/IIa study assessed the feasibility of T-DM1 + docetaxel in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) and T-DM1 + docetaxel ± pertuzumab in patients with HER2-positive locally advanced breast cancer (LABC)., Patients and Methods: Phase Ib (part 1) explored dose escalation, with T-DM1 + docetaxel administered for greater than or equal to six cycles in patients with MBC. Phase Ib (part 2) began with the maximum tolerated dose (MTD) identified in part 1. Patients with LABC were administered less than or equal to six cycles of T-DM1 + docetaxel or T-DM1 + docetaxel + pertuzumab. Phase IIa explored the MTDs identified in phase Ib., Results: Administered with T-DM1 (3.6 mg/kg), the docetaxel MTD was 60 mg/m(2) in MBC. In LABC, the MTD was 100 mg/m(2) docetaxel in combination with T-DM1 (3.6 mg/kg), given with granulocyte colony-stimulating factor (G-CSF). Administered with T-DM1 (3.6 mg/kg) + pertuzumab (840 mg, cycle 1; 420 mg, subsequent cycles), the docetaxel MTD in LABC was 75 mg/m(2) with G-CSF support. Neutropenia was the most common grade 3-4 adverse event (AE; MBC, 72% and LABC, 29%). In total, 48% (12/25) of MBC patients and 47% (34/73) of LABC patients experienced AEs requiring dose modification. In MBC (median prior systemic agents = 5), the objective response rate was 80.0% (20/25; 95% confidence interval [CI] 59.3-93.2) and the median progression-free survival was 13.8 months (range, 1.6-33.5). The pathologic complete response (ypT0/is, ypN0) rate in LABC was 60.3% (44/73; 95% CI 48.1-71.5). Pharmacokinetic analyses indicated a low risk of drug-drug interaction between T-DM1 and docetaxel., Conclusions: T-DM1 combined with docetaxel ± pertuzumab appeared efficacious in MBC or LABC; however, nearly half of patients experienced AEs requiring dose reductions with these T-DM1 combinations., Clinicaltrialsgov Identifier: NCT00934856., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Trastuzumab emtansine (T-DM1), a new agent developed for the treatment of HER2-positive breast cancer, is an antibody-drug conjugate with a complex compound obtained by the conjugation of trastuzumab, a stable thioether linker, and the potent cytotoxic drug maytansine-derivate(DM1), which inhibits cell division and induces cell death., Field of Study: PubMed database, ESMO, ASCO, San Antonio Breast Cancer Symposium Meeting abstracts and clinicaltrials.gov were searched using the terms "Anti-HER2 treatment breast cancer and trastuzumab emtansine (T-DM1) "; papers considered relevant for the aim of this review were selected., Findings/results: The phase I trials have determined the safe dosing range of T-DM1, established at 3.6 mg/kg every 3 weeks. The phase III randomized EMILIA and TR3RESA trials have shown that T-DM1 provides objective tumor responses and significantly improves progression free survival and overall survival in HER2-positive metastatic breast cancer patients previously treated with anti-HER2-based regimens. The ongoing phase III trials KAITLIN and KATHERINE will give us further information about the place T-DM1 should occupy in the treatment of patients with early stage HER2-positive breast cancer. In this review we analyze the most relevant clinical trials conducted with T-DM1 and the role of this compound in the management of advanced breast cancer., Conclusion: T-DM1 has shown clinically relevant activity in the treatment of HER2-positive breast cancer patients after progression on trastuzumab and taxane based therapy, both in the second line treatment setting and after early relapse on adjuvant trastuzumab therapy. This is accompanied by a favorable safety and tolerability profile., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aim: Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies., Methods: We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells., Results: In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis., Conclusion: Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The addition of trastuzumab (T) and lapatinib (L) to neoadjuvant chemotherapy increases the pathological complete response (pCR) rate in patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We investigated the efficacy of T or L with neoadjuvant chemotherapy and specific efficacy biomarkers., Methods: Patients with stages I-III (including inflammatory) HER2-positive breast cancer were randomised to receive epirubicin (E) plus cyclophosphamide (C) × 4 cycles followed by docetaxel (D) plus either T (EC-DT) or L (EC-DL). End points included pCR (primary), clinical response, toxicity, and pCR-predictive biomarkers., Results: We randomised 102 patients to EC-DT (50) and EC-DL (52). Median age was 48, 56% were premenopausal and 58% had oestrogen receptor (ER)-positive tumours. Pathological complete response in breast was 52.1% (95% CI:38.0-66.2%) for EC-DT and 25.5% (95% CI:13.5-37.5%) for EC-DL (P=0.0065). Pathological complete response in breast and axilla was 47.9% for EC-DT and 23.5% for EC-DL (P=0.011). Grade 3-4 toxicity did not differ across treatments, except for diarrhoea (2% in EC-DT vs 13.5% in EC-DL, P=0.030). Multivariate analyses showed that treatment (P=0.036) and ER (P=0.014) were the only predictors of pCR in both groups., Conclusion: EC-DT exhibited higher efficacy and lower toxicity than EC-DL. Of the different biomarkers studied, only the absence of ER expression was associated with increased pCR.

Purpose: The combination of pertuzumab and trastuzumab resulted in a clinical benefit rate (CBR) of 50% in patients with human epidermal growth factor receptor 2 (HER2) -positive breast cancer whose disease progressed during prior trastuzumab-based therapy. To define whether this previously observed encouraging activity was a result of the combination of pertuzumab and trastuzumab or of pertuzumab alone, we recruited a third cohort of patients who received pertuzumab without trastuzumab. We then investigated the impact of reintroducing trastuzumab to patients whose disease progressed on pertuzumab monotherapy., Patients and Methods: Twenty-nine patients with HER2-positive breast cancer whose disease progressed during prior trastuzumab-based therapy received pertuzumab (840 mg loading dose, then 420 mg every 3 weeks) until progressive disease or unacceptable toxicity. Seventeen patients with disease progression continued to receive pertuzumab (at the same dose), with the addition of trastuzumab (4 mg/kg loading dose and then 2 mg/kg weekly or 8 mg/kg loading dose and then 6 mg/kg every 3 weeks)., Results: All 29 patients enrolled for pertuzumab monotherapy experienced disease progression. The objective response rate (ORR) and CBR were 3.4% and 10.3%, respectively, during pertuzumab monotherapy. With the addition of trastuzumab, the ORR and CBR were 17.6% and 41.2%, respectively. Progression-free survival was longer with combination therapy than pertuzumab monotherapy (17.4 v 7.1 weeks, respectively). Treatment was well tolerated with minimal cardiac dysfunction., Conclusion: Although pertuzumab has some activity in patients with HER2-positive breast cancer that progressed during therapy with trastuzumab, the combination of pertuzumab and trastuzumab seems to be more active than monotherapy.

Objectives: ERBB2 is an oncogene with prognostic and predictive value. Topoisomerase IIalpha is an enzyme encoding close to the ERBB2 oncogene, that represents a molecular target for anthracyclines. An indirect mechanism of increasing ERBB2 and topoisomerase IIalpha gene copy number is chromosome 17 polysomy. The aim of the present study was to clarify the implication of polysomy 17 in ERBB2 and topoisomerase IIalpha expression. In addition, we assessed the relation of ERBB2 and topoisomerase IIalpha gene dosage to mRNA and protein levels., Methods: We selected 83 cases diagnosed as invasive breast cancer. We analysed ERBB2 and topoisomerase IIalpha genes, mRNA and protein by fluorescence in situ hybridisation, real-time reverse-transcription polymerase chain reaction and immunohistochemistry., Results: We observed a progressive increase in mRNA expression from 0+ to 3+ and also a significant difference in the ERBB2 RNA levels between normal and amplified cases. We found that polysomy of chromosome 17 does not affect the ERBB2 expression and that topoisomerase IIalpha mRNA expression is not related to gene status., Conclusions: Our results demonstrate that polysomy of chromosome 17 is not related to ERBB2 expression. Thereby, it is important to use centromeric probes to clearly discriminate between true ERBB2 gene amplification and polysomy of chromosome 17.

Background: The HER-2 receptor undergoes a proteolytic cleavage generating an NH(2)-terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated. We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer., Methods: Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival., Results: The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004)., Conclusions: p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

Purpose: Combining trastuzumab with doxorubicin and paclitaxel (AT) is attractive because of the activity of AT and survival improvements observed when trastuzumab is added to either agent in HER2-positive metastatic breast cancer. This pilot study evaluates the efficacy and cardiac tolerability of AT followed by paclitaxel with trastuzumab started with AT or paclitaxel alone and investigates pharmacokinetic interactions., Experimental Design: Two cohorts of 16 patients were enrolled. Cohort 1 received three cycles of AT (60/150 mg/m2) plus trastuzumab (4 mg/kg initial dose followed by 2 mg/kg), initiated concomitantly with doxorubicin, followed by nine cycles of paclitaxel (80 mg/m2) plus trastuzumab and then trastuzumab alone. Cohort 2 was treated with the same regimen, but trastuzumab was initiated with paclitaxel after AT. Cardiac function, pharmacokinetic interactions, and efficacy were evaluated., Results: Median baseline left ventricular ejection fraction (LVEF) was 62% (range, 57-74%) and 66% (range, 57-77%) in cohorts 1 and 2, respectively. Most patients had an absolute decrease in LVEF. Congestive heart failure was not observed. LVEF in three patients decreased to <50% but recovered despite continued treatment. Response rates were 87.5% in both cohorts (cohort 1:2 complete response, 12 partial response; cohort 2:3 complete response, 11 partial response). No unexpected side effects were observed. Pharmacokinetics of paclitaxel and its metabolites and of doxorubicin were similar without and with trastuzumab., Conclusions: Trastuzumab administered with AT followed by weekly paclitaxel alone is highly active whether trastuzumab is initiated with AT or paclitaxel. Congestive heart failure was not observed, and LVEF decreases were reversible. Further studies of this regimen are warranted.

The HER family of transmembrane tyrosine kinase receptors is composed of four members, BER1 to HER4. HER2 is a ligand-orphan receptor expressed in many human tumors and overexpressed in 25-30% of breast cancers. HER2 amplifies the signal provided by other receptors of the HER family by forming heterodimers. The essential role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In particular, the humanized MAb trastuzumab (Herceptin) has antitumor activity against HER2-overexpressing human breast tumor cells and is widely used for the treatment of women with HER2 overexpressing breast cancers. Trastuzumab induces HER2 receptor downmodulation and, as a result, inhibits critical signalling pathways (i.e. ras-Raf-MAPK and PI3K/Akt) and blocks cell cycle progression by inducing the formation of p27/Cdk2 complexes. Trastuzumab also inhibits HER2 cleavage, preceding antibody-induced receptor downmodulation, and this effect might contribute to its antitumor activity in some cancers. In vivo, trastuzumab inhibits angiogenesis and induces antibody-dependent cellular cytotoxicity. A limitation of trastuzumab is that its activity is largely restricted to breast cancers with the highest level of HER2 overexpression or HER2 gene amplification. However, there is a large population of breast cancers and of many other tumors that have low or moderate HER2 expression. In such tumors, HER2 functions as a preferred coreceptor to form heterodimers with HER1 (EGFR), HER3 or HER4. For this reason, a humanized monoclonal antibody, called 2C4, that targets the role of HER2 as a coreceptor is under active development. 2C4 binds to a different epitope of HER2 ectodomain than trastuzumab and sterically hinders HER2 recruitment in heterodimers with other HER receptors. This results in the inhibition of signalling by HER2-based heterodimers both in cells with low and high HER2 expression. In vitro and in vivo antitumor activity has been reported in a range of breast and prostate tumor models. Therefore, 2C4 may have potential against a wide variety of solid tumors. Phase I trials are underway.

Background: The full-length receptor p185HER-2 undergoes a metalloprotease-dependent cleavage producing a membrane-associated fragment (p95HER-2) in cultured breast cancer cells. P95HER-2 has potentially enhanced signaling activity, but its expression and role in human breast cancer is poorly characterized., Purpose: The purpose of this project was to characterize the expression of p95HER-2 in primary breast cancers and nodal metastasis, and to study association with clinicopathological factors., Experimental Design: P95HER-2 and p185HER-2 were examined in 337 primary breast tumors and 81 metastatic lymph nodes by Western blot analysis, and tested for associations with other clinicopathological factors., Results: P95HER-2 was present in 20.9% of primary tumors from node-negative patients, in 29.1% from patients with one to three metastatic nodes, and in 36.7% from patients with four or more metastatic nodes (P = 0.027). Whereas p185HER-2 overexpression was unrelated to nodal disease (P = 0.63), the odds of lymph node metastasis were enhanced 2.9-fold by the presence of p95HER-2 (48.8% of node-negative versus 73.5% of node-positive patients; P = 0.03; odds ratio = 2.9). P95HER-2 was more frequent in metastatic lymph nodes than in primary tumors (45.7% versus 26.7%; P = 0.0009), whereas p185HER-2 overexpression was similar in both (22.3% versus 23.5%; P = 0.933). P95HER-2 did not significantly correlate with patient age, tumor size, stage, histotype, or hormone receptor status., Conclusions: P95HER-2 in primary tumors was related to extent of lymph node involvement and was enhanced in nodal tissue suggesting an important role as a marker or cause in breast cancer metastasis. Examination of the prognostic value of p95HER-2 in breast cancer and its coexpression with metalloprotease activity seem warranted.

HER2 is a ligand-less tyrosine kinase receptor of the ErbB family that is frequently overexpressed in breast cancer. It undergoes proteolytic cleavage that results in the release of the extracellular domain and the production of a truncated membrane-bound fragment, p95. We show that HER2 shedding is activated by 4-aminophenylmercuric acetate (APMA), a well-known matrix metalloprotease activator, in HER2-overexpressing breast cancer cells. The HER2 p95 fragment, which appears after APMA-induced cleavage, is phosphorylated. We analyzed 24 human breast cancer specimens, and a phosphorylated M(r) 95,000 HER2 band could be detected in some of them, which indicated that the truncated receptor is also present in vivo. The activation of HER2 shedding by APMA in cells was blocked with batimastat, a broad-spectrum metalloprotease inhibitor. Trastuzumab (Herceptin; Genentech, San Francisco, CA), a humanized monoclonal antibody directed at the HER2 ectodomain, which has been shown to be active in patients with HER2-overexpressing breast cancer, inhibited basal and induced HER2 cleavage and, as a consequence, the generation of phosphorylated p95. This inhibitory effect of trastuzumab was not shared by 2C4, an antibody against a different epitope of the HER2 ectodomain. The inhibition of basal and APMA-induced cleavage of HER2 by trastuzumab preceded antibody-induced receptor down-modulation, which indicated that the effect of trastuzumab on cleavage was not attributable to a decrease in cell-surface HER2 induced by trastuzumab. We propose that the inhibition of HER2 cleavage and prevention of the production of an active truncated HER2 fragment represent a novel mechanism of action of trastuzumab.

The search for new methods of treating cancer, combined with advances in our understanding of carcinogenesis, molecular biology and technology, has resulted in the development of novel biologic agents with proven clinical efficacy. One such agent is trastuzumab (Herceptin), a humanized monoclonal antibody that targets the human epidermal growth factor receptor-2 (HER2). HER2 is a member of a family of receptors that interact with each other and various ligands to stimulate various intracellular signal transduction pathways involved in cell growth control. HER2 is overexpressed in 20%-30% of women with breast cancer and is associated with aggressive tumor characteristics and poor prognosis. Trastuzumab is the first humanized monoclonal antibody to be approved for therapeutic use and the first oncogene-targeted treatment with proven survival benefit in women with HER2-positive metastatic breast cancer. However, its mechanism of action has not been fully characterized and appears to be complex. This paper reviews current knowledge of the mechanism of action of trastuzumab, including HER2 protein downregulation, prevention of HER2-containing heterodimer formation, initiation of G1 arrest and induction of p27, prevention of HER2 cleavage, inhibition of angiogenesis, and induction of immune mechanisms. The significance of these mechanisms for selection of concomitant chemotherapy is also considered.

Breast carcinomas express high levels of ErbB receptors and their ligands, and their overexpression has been associated with a more aggressive clinical behavior. For these reasons therapies directed at these receptors have the potential to be useful anti-cancer treatments. A series of monoclonal antibodies (MAbs)3 directed against the EGF (ErbB1) receptor and the closely related HER2/Neu (ErbB2) receptor are currently under evaluation. These MAbs have shown promising preclinical activity and "chimeric" and "humanized" MAbs have been produced in order to obviate the problem of host immune reactions. These antibodies are currently being tested in clinical trials either alone or in combination with chemotherapeutic agents. Clinical activity with one of these antibodies, trastuzumab, a humanized anti-ErbB2 MAb, has been documented in patients with breast cancer in a series of clinical trials and has recently been approved for the therapy of patients with metastatic ErbB2 overexpressing breast cancer. In addition to antibodies, compounds that inhibit receptor tyrosine kinases have shown significant preclinical activity and are currently being evaluated in the clinic.

HER2/neu, a Mr 185,000 tyrosine kinase receptor that is overexpressed in breast cancer, undergoes proteolytic cleavage of its extracellular domain (ECD). In contrast with other membrane-bound proteins, including growth factor receptors, that are cleaved by a common machinery system, we show that HER2 cleavage is a slow process and is not activated by protein kinase C. Pervanadate, a general inhibitor of protein-tyrosine phosphatases, induces a rapid and potent shedding of HER2 ECD. The shedding of HER2 ECD is inhibited by the broad-spectrum metalloprotease inhibitors EDTA, TAPI-2, and batimastat. The tissue inhibitor of metalloproteases-1; an inhibitor of matrix metalloproteases that does not inhibit cleavage by the general protein kinase C-dependent shedding machinery, also inhibited HER2 ECD shedding, whereas tissue inhibitor of metalloproteases-2 did not. These data suggest that HER2 cleavage is a process regulated by an as-yet-unidentified distinct protease.

Recombinant humanized anti-HER2 antibody, rhuMAb HER2, inhibits the growth of breast cancer cells overexpressing HER2 and has clinical activity. We explored in preclinical models its capacity to enhance the tumoricidal effects of paclitaxel and doxorubicin. In cultures of naturally HER2-overexpressing cancer cells, rhuMAb HER2 inhibited growth and enhanced the cytotoxic effects of paclitaxel. Treatment of well established BT-474 breast cancer xenografts overexpressing HER2 in athymic mice with rhuMAb HER2 resulted in a dose-dependent antitumor activity. In combination studies, treatment with paclitaxel and rhuMAb HER2 or doxorubicin and rhuMAb HER2 resulted in greater inhibition of growth than that observed with any agent alone. The combination of paclitaxel and rhuMAb HER2 resulted in the highest tumor growth inhibition and had a significantly superior complete tumor regression rate when compared with either paclitaxel or rhuMAb HER2 alone. Clinical trials that are built on these results are under way.

Background: The contribution of p53 and HER-2/neu to the management of node-negative breast cancer (NNBC) could be improved by combining their results., Material and Methods: We studied paraffin-embedded primary tumors for p53 (BP-53-12-1) (n=57) and HER2/neu (pAB1) (n=63) from NNBC patients. The results were grouped in a negative (p53(-)/neu(-)) versus a positive group (one or both overexpressed). The association between both groups (negative and positive) and clinicopathologic parameters, S-phase fraction and DNA ploidy, and patients' outcome, was analyzed., Results: In 28% of the tumors p53 was overexpressed, and HER2/neu in 11%. Sixty-five percent (37 out of 57) were p53(-)/neu(-), and 35% overexpressed one (31.5%) or both (3.5%) oncoproteins. Significant correlations were found between p53(-)/neu(-) tumors and age greater than 50 (p=0.003), S-phase fraction lower than 7 (p=0.03), and positive estrogen receptor contents (p=0.049). Actuarial 5-year disease-free and overall survival for p53(-)/neu(-) tumors were 88% and 97%, respectively, versus 50% and 66%, for tumors overexpressing one or both oncoproteins (p=0.004).