Immunohistochemical Features Associated with Sensitivity to Lapatinib-plus-Capecitabine and Resistance to Trastuzumab in HER2-positive Breast Cancer

Patients and Methods

Patients and treatment. The study cohort comprised of patients with advanced breast cancer enrolled in the GlaxoSmithKline Lapatinib Expanded Access Program (LEAP) at Seoul National University Hospital between February 2007 and April 2008 and who agreed to participate in the pharmacogenomics study. The inclusion criteria were ≥18 years of age with a life expectancy of eight weeks or more; locally advanced or MBC that had progressed after treatment regimens consisting of an anthracycline, a taxane, and trastuzumab; HER2 overexpression [IHC 3+ or gene amplification by fluorescence in situ hybridization (FISH)]; Eastern Cooperative Oncology Group performance status of 0-2; cardiac ejection fraction within the normal range; adequate renal, hepatic, and hematologic function; no contraindication to lapatinib plus capecitabine; and at least one measurable lesion. Patients previously treated with lapatinib were excluded, whereas prior exposure to capecitabine was permitted.

Treatment consisted of 1,250 mg/day lapatinib and 2000 mg/m² capecitabine in two divided doses on days 1 to 14 of a 21-day cycle. Treatment was continued until disease progression, intolerable toxicity, or patient consent withdrawal. Treatment response was evaluated every six weeks according to the Response Evaluation Criteria in Solid Tumors (version 1.0) (5). Complete or partial responses (PRs) were confirmed by computed tomographic scans taken at least four weeks apart. Brain metastasis was evaluated using magnetic resonance imaging. Clinical data were collected and responses were evaluated prospectively. Written informed consent for LEAP and the pharmacogenomics study was obtained from all patients before study entry. The study protocol was reviewed and approved by the Institutional Review Board at Seoul National University Hospital (H-0701-065-196). Declaration of Helsinki recommendations for biomedical research involving human subjects were also followed.

Collection of tumor tissues. Pre-treatment biopsies of trastuzumab-resistant tumours (tumours that had progressed during trastuzumab therapy) were performed on patients who agreed to undergo the procedure, and collected tissues were used for the biomarker study. For the other patients, archival tumour tissues were used. In patients with multiple archival tumour tissues, the most recent tissue sample was selected. All patients were investigated for the presence of additional paired trastuzumab-resistant and trastuzumab-sensitive (tissues collected before initiation of trastuzumab) tumour tissues. To identify the IHC features associated with trastuzumab resistance, all tumour tissues collected were classified as trastuzumab-resistant and sensitive and IHC staining was compared between them.

IHC staining and interpretation. Hematoxylin and eosin staining was performed to confirm the presence of tumour. Antigen retrieval was performed using the appropriate method for each antibody. Samples were immunostained with the following antibodies according to the manufacturers' instructions: anti-ER (1:100; 1D5; Dako, Glostrup, Denmark), anti-PR (1:100; 636; Dako), anti-EGFR (1:100; 31G7; Zymed, San Francisco, CA, USA), anti-HER2 (1:200; A0485; Dako), anti-HER3 (1:30; C-17; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pHER3 (1:30; 21D3; Cell Signaling, Beverly, MA, USA), anti-PTEN (1:100; 6H2.1, Dako), anti-TYMS (1:60; TYMS106, Thermo Fisher, Fremont, CA, USA), and anti-TYMP (1:60; PGF.44C; Thermo Fisher).

IHC staining was scored and confirmed by two pathologists who were blinded to the clinical information. Positive ER and PR expression were defined as nuclear staining in 10% or more of tumour cells. EGFR overexpression was classified as weak to moderate or strong membranous staining in more than 10% of tumour cells. HER2 membranous staining was scored on a scale of 0 to 3+ according to the HercepTest protocol (6). For tissue samples with a HER2 staining score of 2+, additional HER2 FISH testing was performed. HER2 status was considered positive when the IHC score was 3+ or the gene copy ratio of HER2/CEP17 by FISH was 2.2 or higher. For HER3, pHER3 and PTEN, the intensity (0, none; 1, weak; 2, moderate; 3, strong) and extent (0%-100% of tumour cells) of IHC staining in both the cytoplasm and membrane were determined. For HER3 and pHER3, a staining intensity score of 2 or more was defined as positive. Cytoplasmic PTEN expression was scored semiquantitatively using the immunoreactive score (IRS) as described elsewhere (7). An IRS of 0-3 was defined as low PTEN expression. Vascular endothelium, which is known to express normal PTEN, was used as a positive internal control. For TYMS and TYMP, intensity (0-3) and extent of staining in both the cytoplasm and nucleus were scored. Low expression of TYMS and TYMP was defined as cytoplasmic or nuclear staining with an intensity score of 0 or 1, whereas high expression was defined as cytoplasmic or nuclear staining with an intensity score of 2 or 3.

Statistical analyses. Categorical variables were compared using Pearson's χ² test or Fisher's exact test, where appropriate. TTP and OS were defined as the time from treatment initiation to the time of disease progression and death from any cause, respectively. The Kaplan–Meier method was used to estimate the median TTP and OS. TTP and OS comparisons were performed using the log-rank test. Multivariate analyses of TTP and OS were conducted using the backward stepwise Cox proportional hazard regression model after adjusting for age, hormone receptor status, number of prior chemotherapies (<4 vs. ≥4), and prior exposure to capecitabine. All p-values were two-tailed, and p<0.05 was considered statistically significant.