Potential biomarkers of long‐term benefit from single‐agent trastuzumab or lapatinib in HER2‐positive metastatic breast cancer

In 2009 a prospective, randomized Phase II trial ({"type":"clinical-trial","attrs":{"text":"NCT00842998","term_id":"NCT00842998"}}NCT00842998) was initiated to evaluate the activity of HER2‐targeting agents without chemotherapy (CT) in HER2‐positive metastatic breast cancer (MBC) patients. The primary tumors of the patients enrolled in this study offered a unique opportunity to identify biomarkers that could predict durable clinical benefit from CT‐free anti‐HER2 therapy.Patients with HER2‐positive MBC were randomized to trastuzumab or lapatinib as first‐line therapy. CT was added to anti‐HER2 therapy in patients failing to achieve tumor regression at the 8‐week evaluation and in those progressing at any time. Expression analysis of 105 selected genes was performed from formalin‐fixed paraffin‐embedded primary tumor samples. The research‐based PAM50 intrinsic subtypes were also identified. Additionally, quantitative HER2 (H2T) and p95HER2 (p95) protein expression were evaluated by HERmark® and VeraTag® assay, respectively. Predictors of persistence on protocol (PP) were studied by Cox univariate and multivariate analysis.Nineteen patients were enrolled. Median overall survival was 43 months and median PP was 3.8 months (0.8–38.8+), with 4 patients (21.1%) persisting on single agent trastuzumab or lapatinib for longer than 12 mo (14.9–38.8 + mo). Seventeen patients were evaluable for PP. Gene expression analysis revealed that high expression of the 17q12‐21 amplicon genes HER2 and GRB7, and the PAM50 HER2‐enriched intrinsic profile, were significantly associated with longer PP. Conversely, high expression of luminal‐related genes such as PGR, MDM2 or PIK3CA, or the PAM50 luminal intrinsic profile correlated with reduced PP. Moreover, increasing H2T/p95 ratio was found to be significantly associated with longer PP (HR 0.56 per 2‐fold increase in H2T/p95, P = 0.0015).Our data suggest that patients belonging to the “HER2‐enriched” subtype and/or having high H2T/p95 protein expression ratio are exquisitely sensitive to anti‐HER2 agents. MBC patients with these tumors could be candidates for studies aimed at establishing chemotherapy‐free regimens.     

Targeting HER2-positive cancer with dolastatin 15 derivatives conjugated to trastuzumab, novel antibody–drug conjugates

Purpose

Targeting tubulin binders to cancer cells using antibody?Cdrug conjugates (ADCs) has great potential to become an effective cancer treatment with low normal tissue toxicity. The nature of the linker used to tether the tubulin binder to the antibody and the conjugation sites on the antibody and the small molecule are important factors in the ADC stability and effectiveness.

Methods

We explored the use of tubulin-targeting dolastatin 15 derivatives (Dol15) tethered covalently to a representative antibody, trastuzumab, via cleavable and non-cleavable linkers at varying antibody reactive sites (i.e., lysine residues, partially reduced hinge region disulfide bonds) and drug coupling sites (i.e., C-terminus, N-terminus), to investigate which constructs were more effective in killing HER2-positive cells in vitro and in vivo.

Results

We found that Dol15 conjugated to trastuzumab via lysine residues at the drug C-terminus using a non-cleavable linker (trastuzumab?Camide?CC-term?CDol15) produced target-dependent growth inhibition of cells endogenously expressing high HER2 levels (i.e., SK-BR-3, SK-OV-3) in vitro. This ADC was effective at varying doses (i.e., 10 and 20?mg/kg) in the SK-OV-3 human ovarian cancer xenograft.

Conclusions

Tethering Dol15 via partially reduced disulfide bonds at the drug C-terminus via a non-cleavable linker (trastuzumab?CMC?CC-term?CDol15) resulted in an equally effective ADC in vitro, showing that site of antibody conjugation did not influence ADC activity. However, tethering Dol15 at the drug N-terminus using non-cleavable and cleavable linkers (trastuzumab?CMC?CN-term?CDol15 and trastuzumab?CMC?CVC?CPABC?CN-term?CDol15, respectively) resulted in ineffective ADCs. Thus, Dol15 tethered at the C-terminus may be a useful tubulin-targeting agent for conjugation at various antibody reactive sites.     

Dual targeting of HER1/EGFR and HER2 with cetuximab and trastuzumab in patients with metastatic pancreatic cancer after gemcitabine failure: results of the “THERAPY”phase 1-2 trial

To improve treatment efficacy, we decided to simultaneously target HER1 and HER2 with trastuzumab and cetuximab. Following promising preclinical results, we conducted a phase 1-2 trial in advanced pancreatic cancer patients after first-line gemcitabine-based chemotherapy failure. In this single-arm, non-randomized, multicenter trial, patients received weekly cetuximab (400mg/m², then 250mg/m²). They were sequentially included in two trastuzumab dose levels: 3.0 or 4.0mg/kg, then 1.5 or 2.0mg/kg/weekly. Endpoints were the objective response rate, safety, progression-free (PFS) and overall survival (OS). During phase 1 (n=10 patients), toxicities were evenly distributed except for skin toxicities that frequently caused compliance issues. The higher dose level was defined as the trastuzumab recommended dose. During phase 2 (n=39 patients), toxicities were mainly cutaneous reactions and asthenia. No objective response was observed. Nine patients were stabilized but arrested treatment due to toxicity. Median PFS was 1.8 months (95%CI: 1.7-2.0 months) and median OS was 4.6 months (95%CI: 2.7–6.6 months). Both were positively correlated with skin toxicity severity (P=0.027 and P=0.001, respectively). Conventional phase 1 dose-escalation schedules are unsuitable for targeted therapies because most cutaneous toxicities are not considered dose-limiting toxicities. The compliance issues caused by skin toxicities were particularly detrimental because of the toxicity-response correlation.     

Complete response in HER2+ leptomeningeal carcinomatosis from breast cancer with intrathecal trastuzumab

Trastuzumab, a monoclonal antibody against the HER2 receptor, is a major breakthrough in the treatment of HER2+ breast cancer. However, its high molecular weight precludes it from crossing the intact blood–brain barrier, making the central nervous system a sanctuary to HER2+ breast cancer metastases. We prospectively assessed functional outcome and toxicity of administering trastuzumab directly into the cerebrospinal fluid of a patient with leptomeningeal carcinomatosis (LC) and brain metastases from HER2+ breast cancer that had already been treated with other intrathecal chemotherapy, with no benefit. Upon signed informed consent, weekly lumbar puncture with administration of trastuzumab 25 mg was begun to a 44 year-old women with metastatic breast cancer (lymph node, bone, lung, and liver involvement) previously treated with tamoxifen, letrozole, anthracyclines, taxanes, capecitabine, intravenous trastuzumab, and lapatinib. She received 67 weekly administrations of intrathecal trastuzumab with marked clinical improvement and no adverse events. She survived 27 months after LC diagnosis. A complete leptomeningeal response, with no evidence of leptomeningeal metastasis at necropsy, was achieved. We believe that intrathecal trastuzumab administration should be prospectively evaluated to confirm clinical activity and optimize dose, schedule, and duration of treatment.     

Population pharmacokinetics of trastuzumab emtansine (T-DM1), a HER2-targeted antibody–drug conjugate,in patients with HER2-positive metastatic breast cancer: clinical implications of the effect of covariates

Purpose

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate comprising the humanized monoclonal antibody trastuzumab linked to DM1, a highly potent cytotoxic agent. A population pharmacokinetic (PK) analysis was performed to estimate typical values and interindividual variability of T-DM1 PK parameters and the effects of clinically relevant covariates.

Methods

Serum samples were collected from 671 patients with human epidermal growth factor receptor 2-positive locally advanced or metastatic breast cancer (MBC) who received single-agent T-DM1 in five phase I to phase III studies. Nonlinear mixed-effects modeling with the first-order conditional estimation method was used.

Results

A linear two-compartment model with first-order elimination from the central compartment described T-DM1 PKs in the clinical dose range. T-DM1 elimination clearance was 0.676 L/day, volume of distribution in the central compartment (V _c) was 3.127 L, and terminal elimination half-life was 3.94 days. Age, race, region, and renal function did not influence T-DM1 PK. Given the low-to-moderate effect of all statistically significant covariates on T-DM1 exposure, none of these covariates is expected to result in a clinically meaningful change in T-DM1 exposure.

Conclusions

T-DM1 PK properties are consistent and predictable in patients. A further refinement of dose based on baseline covariates other than body weight for the current 3.6 mg/kg regimen would not yield clinically meaningful reductions in interindividual PK variability in patients with MBC.     

Clinical pharmacology of trastuzumab emtansine (T-DM1): an antibody–drug conjugate in development for the treatment of HER2-positive cancer

Purpose

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate comprising trastuzumab and DM1, a microtubule polymerization inhibitor, covalently bound via a stable thioether linker. To characterize the pharmacokinetics (PK) of T-DM1 in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer, data from four studies (TDM3569g, TDM4258g, TDM4374g, and TDM4688g) of single-agent T-DM1 administered at 3.6?mg/kg every 3?weeks (q3w) were assessed in aggregate.

Methods

Multiple analytes—T-DM1, total trastuzumab (TT), DM1, and key metabolites—were quantified using enzyme-linked immunosorbent assays or liquid chromatography tandem mass spectrometry. PK parameters of T-DM1, TT, and DM1 exposure were calculated using standard noncompartmental approaches and correlated to efficacy (objective response rate) and safety (platelet counts, hepatic transaminase concentrations). Immunogenicity was evaluated by measuring anti-therapeutic antibodies (ATA) to T-DM1 after repeated dosing using validated bridging antibody electrochemiluminescence or enzyme-linked immunosorbent assays.

Results

PK parameters for T-DM1, TT, and DM1 were consistent across studies at cycle 1 and steady state. T-DM1 PK was not affected by residual trastuzumab from prior therapy or circulating extracellular domain of HER2. No significant correlations were observed between T-DM1 exposure and efficacy, thrombocytopenia, or increased concentrations of transaminases. Across the studies, ATA formation was detected in 4.5% (13/286) of evaluable patients receiving T-DM1 q3w.

Conclusions

The PK profile of single-agent T-DM1 (3.6?mg/kg q3w) is predictable, well characterized, and unaffected by circulating levels of HER2 extracellular domain or residual trastuzumab. T-DM1 exposure does not correlate with clinical responses or key adverse events.     

Antitumor activity of combinations of anti-HER-2 antibody trastuzumab and oral fluoropyrimidines capecitabine/5′-dFUrd in human breast cancer models

The anti-HER-2 antibody, trastuzumab (Herceptin), and the oral fluoropyrimidine, capecitabine (Xeloda), are both effective in breast cancer with different modes of action and toxicity profiles. Therefore, the efficacy in combination therapy of these agents for the treatment of HER-2-overexpressing breast cancer was of interest. An antagonistic interaction in vitro between trastuzumab and 5-fluorouracil (5-FUra) in combination has previously been reported. In the same study, the in vivo antitumor activity of this combination was investigated. However, the results were inconclusive since 5-FUra at the dose used had sufficient antitumor efficacy as a single agent and therefore it was not possible to clarify the potential difference between 5-FUra alone and the combination. In the present study, we investigated the efficacy of trastuzumab in combination with capecitabine or its intermediate metabolite 5'-dFUrd in HER-2-overexpressing human breast cancer cell lines and xenograft models. In vivo antitumor activity of the combination was at least additive, in terms of tumor growth inhibition and tumor growth delay, in human breast cancer models KPL-4 and BT-474. The combination treatment in vivo was superior to the treatment with either single agent alone, even though in vitro treatment with trastuzumab and 5'-dFUrd/5-FUra showed antagonistic antiproliferative activity in these cell lines. The reason for the discrepancy between the in vivo and in vitro results was not clarified. However, observed additive in vivo antitumor activity clearly indicates that the clinical efficacy of the combination of trastuzumab and capecitabine/5'-dFUrd against HER-2-overexpressing breast cancer is worth investigating.     

Additive impact of HER2‐/PTK6‐RNAi on interactions with HER3 or IGF‐1R leads to reduced breast cancer progression in?vivo

The human epidermal growth factor receptor 2 (HER2) and the protein tyrosine kinase 6 (PTK6) are often co‐ and over‐expressed in invasive breast cancers. At early diagnosis, only distinct groups, such as HER2‐or hormone receptor‐positive benefit from a targeted therapy. However, a part of these tumours develops resistance within a year of administration of the drug but the majority of the patients depends on general therapies with severe side effects. A PTK6‐directed approach does not yet exist.In our present study, we successfully demonstrate, in vitro and in vivo, a significantly additive reduction of tumourigenesis of breast cancer cells simultaneously depleted of both HER2 and PTK6. In comparison with single RNAi approaches, the combined RNAi (co‐RNAi) led to a stronger reduced phosphorylation of tumour‐promoting proteins. Moreover, the co‐RNAi additively decreased cell migration as well as two and three dimensional cell proliferation in vitro. The in vivo experiments showed an additive reduction (p < 0.00001) in the growth of xenografts due to the co‐RNAi compared with HER2 or PTK6 RNAi alone. Interestingly, the complexes of HER2 or PTK6 with tumour‐relevant interaction partners, such as HER3 or the insulin‐like growth factor receptor 1 (IGF‐1R), respectively, were also reduced in xenografts although their protein expression levels were not affected following the co‐RNAi of HER2 and PTK6. Our present study reveals the potential of using combined HER2‐ and PTK6‐ knockdown as a powerful strategy for the treatment of breast cancers. Therefore, the combined inhibition of these proteins may represent an attractive tool for efficient therapy of breast cancers.     

Population pharmacokinetics and exposure–response analyses of trastuzumab in patients with advanced gastric or gastroesophageal junction cancer

Purpose

The aim of this study was to characterize trastuzumab population pharmacokinetics (PKs) in patients with human epidermal growth factor receptor 2-positive advanced gastric or gastroesophageal junction cancer and the relationship of trastuzumab PK with patient response.

Methods

A nonlinear mixed effects PK model was built using data from the ToGA study. Patients were randomized to intravenous trastuzumab plus chemotherapy or chemotherapy alone. The influence of demographic, laboratory, and disease characteristics on PK parameters was assessed. An exploratory exposure–response analysis compared various PK parameters at steady state with best overall tumor response and overall survival (OS).

Results

Trastuzumab PK was best described by a two-compartment model with parallel linear and nonlinear (Michaelis–Menten) elimination from the central compartment. Total clearance (and half-life) of trastuzumab was concentration-dependent. Body weight, prior gastrectomy, and serum albumin had the greatest influence on trastuzumab PK; increasing weight and decreasing albumin levels were associated with increased clearance, while prior gastrectomy correlated with decreased clearance. Median values for AUC, C _max, and C _min were lower in patients with progressive disease (PD) than other response categories, although the 1.5 interquartile ranges overlapped. Patients with the lowest C _min had the highest PD rate and a shorter OS.

Conclusions

In the advanced gastric cancer population, trastuzumab PK was best described by a two-compartment model with parallel linear and nonlinear elimination. Predicted PK exposure was lower than previously reported for breast cancer. Patients with the lowest C _min had a shorter OS and the highest PD rate, but a distinct correlation was not observed for tumor response.     

Combination of microtubule associated protein-tau and β-tubulin III predicts chemosensitivity of paclitaxel in patients with advanced gastric cancer

AimTo investigate the role of microtubule associated protein-tau (MAP-tau) and β-tubulin III (TUBB3) in predicting the chemosensitivity of paclitaxel in patients with advanced gastric cancer (GC).MethodsMAP-tau and TUBB3 expressions were detected using immunohistochemistry in 244 advanced GC patients prior to chemotherapy. The associations of MAP-tau and TUBB3 expressions with paclitaxel sensitivity were assessed using in vitro and in vivo xenograft analysis.ResultsA total of 149 patients receiving paclitaxel plus capecitabine (cohort 1) and 95 patients receiving cisplatin plus capecitabine (cohort 2) were included in this study. In cohort 1, the clinical benefit rate (CBR), median progression-free survival (PFS) and overall survival (OS) were found to be significantly higher in patients with low levels of MAP-tau and TUBB3 expressions and were significantly higher than those in patients with high levels of MAP-tau and TUBB3 expressions (CBR: 72.2% versus 35.9%; PFS: 271 versus 102 days; OS: 394 versus 173 days; all P < 0.05). This was not observed in cohort 2. In in vitro studies, the sensitivity of paclitaxel in human gastric cancer cells was inversely correlated with the expression levels of MAP-tau and TUBB3, as in in vivo animal xenografts.ConclusionsThe combination of MAP-tau and TUBB3 was found to predict chemosensitivity to paclitaxel in gastric cancer in vitro and in vivo. This merits further study and may help guide individual therapy.