High Hepatocyte Growth Factor Expression in Primary Tumor Predicts Better Overall Survival in Male Breast Cancer

Si-Qi Qiu; Johan van Rooijen; Hilde H. Nienhuis; Bert van der Vegt; Hetty Timmer-Bosscha; Elise van Leeuwen-Stok; Annemiek M. E. Walenkamp; Carolien H. M. van Deurzen; Geertruida H. de Bock; Elisabeth G. E. de Vries; Carolien P. Schröder


Breast Cancer Res. 2020;22(30) 

In This Article



The EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program (NCT01101425) was launched in 2006. This program is a global effort that aims to improve understanding of the biology of male breast cancer and to optimize its clinical management. An important part of this program was to retrospectively analyze male breast cancer tissue of patients diagnosed between 1990 and 2010 in 93 centers in nine countries. A total of 1800 male patients with invasive breast cancer and an age above 18 years at the time of diagnosis, were eligible and enrolled in the main study. The present substudy analyzed the data of the 841 Dutch patients included. This cohort was identified through the Netherlands Cancer Registry. Patient, treatment, and tumor characteristics were collected from the EORTC database. In the tumor, ER, progesterone receptor (PR), androgen receptor (AR), HER2 and Ki67 expression, histological subtype, grade, and lymphovascular invasion had been previously centrally reviewed.[25,26] Definitions for positivity of ER, PR, AR, and HER2, and breast cancer subtype surrogate characterization were reported earlier for all 1800 enrolled patients.[26] Briefly, ER, PR, and AR were reported by Allred scores, with positivity defined as a score ≥ 3 and high positivity as a score of 7 or 8. HER2 status was determined according to the American Society of Clinical Oncology-College of American Pathologist (ASCO-CAP) guidelines.[26] Breast cancer subtype surrogates were characterized according to the 2013 St. Gallen consensus guideline, where the low level of Ki67 expression was reported as the percentage of positive cells < 20% and high level of Ki67 expression as ≥ 20%.[26] The archival tissue of all patients was handled according to the Dutch Code for Proper Use of Human Tissue (www.fedara.org). According to the Dutch Central Committee on Research involving Human Subjects, this retrospective non-interventional study did not require informed consent from these patients.

Tissue Microarray Construction and Immunohistochemistry

Paraffin-embedded primary breast cancer tissue was retrospectively collected by the "Borstkanker Onderzoek Groep" (BOOG). For each formalin-fixed paraffin-embedded (FFPE) block, three representative cores were selected and taken to construct tissue microarrays (TMA) using an Automated Tissue Arrayer ATA-27 (Beecher Instruments, Inc.).[27] Four-micrometer-thick tissue slides were cut from these TMA blocks for immunohistochemical staining of HGF, c-MET, CXCL12, and CXCR4.

Immunohistochemical staining was performed in one batch per marker to prevent intensity differences. Positive control slides determined with primary antibodies and negative control slides with immunoglobulin class-matched control sera were included on the liver for HGF, female breast cancer tissue for c-MET, rectum for CXCL12, and kidney for CXCR4. Besides this, we also included intestine, heart, brain, liver, lung, stomach, kidney, pancreas, placenta, muscle, testis, and tonsil tissues in each of the TMA slides as the internal control. These tissues express different levels of the studied markers, supporting the specificity of the antibodies. Heat-mediated antigen retrieval was performed with a microwave in a citrate buffer (10 mM citrate, pH 6.0) for CXCL12 and Tris-EDTA buffer (pH 8.0) for HGF and c-MET. Antigen retrieval was not performed for CXCR4 staining. Endogenous peroxidase was blocked with 0.3% H202 in phosphate-buffered saline (PBS; Cl2H3K2Na3O8P2, pH 7.4). A specific binding was blocked with human AB serum. Primary antibodies (anti-HGF:[28] AF-294-NA [10 μg/ml], R&D Systems; anti-c-MET:[29] ab51067 [1.269 μg/ml], anti-CXCR4:[30] ab10403 [5 μg/ml], anti-CXCL12:[31] ab25117 [10 μg/ml], all Abcam) were diluted in PBS supplemented with 1% bovine serum albumin. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-goat antibodies (DAKO) were used as secondary and tertiary antibodies respectively for CXCL12 and c-MET staining. HRP-conjugated rabbit anti-goat and HRP-conjugated goat anti-rabbit antibodies were used as secondary and tertiary antibodies (DAKO), respectively, for HGF staining. Staining was visualized using 3,3′-diaminobenzidine and hematoxylin counterstaining.

The immunohistochemistry slides were digitized with a Digital Slide Scanner NanoZoomer and were viewed with NDP software (Hamamatsu, Japan). Only the patients who had two or more cores containing tumor and stromal cells were included for analysis. Two observers, blinded for the clinicopathological characteristics of patients, scored the digitalized images (SQ and JvR) with the supervision of a dedicated breast pathologist (BvdV). CXCL12, HGF, and c-MET staining was scored using a 0–2 scale (0, no staining; 1+, weak staining; 2+, strong staining), as was the percentage of tumor cells stained per intensity. Subsequently, H-scores were calculated for each marker by combining the percentage and intensity (formula used: 1 × percentage of cells with weak staining + 2 × percentage of cells with strong staining). CXCR4 staining was scored as the percentage of tumor cells with a positive nuclear and with a cytoplasm staining, as the intensity of CXCR4 staining was too homogeneous to use the 0–2 scale. The percentages or H-scores from two observers were averaged to obtain the score for each core. In case of discrepancy (defined as > 20% difference in percentage or H-score), a third observer who was blinded to the scores obtained from the two observers re-scored the cores. For these discrepant cores, a consensus score obtained from SQ and JvR was used, based on the scores from the three observers. The average percentage or H-score of replicate cores was used as the final score for each patient. The median percentage or H-score of each studied marker was used as the cutoff to define low and high expression. The studied markers were also expressed in some stromal cells, such as the fibroblasts. However, the staining intensity of the markers in stromal cells was far weaker than their staining intensity in the tumor cells. Therefore, in this study, we did not explore the prognostic value of the studied markers expressed in the stromal cells.

Statistical Analysis

The categorical variables were described by percentages, and continuous variables by median and interquartile range (IQR).

OS was defined as the time between the date of diagnosis and the documented date of death due to any cause. The remaining patients were censored at the last date known to be alive. OS was only defined within the subset of M0 patients at diagnosis, as the sample size of M1 patient group was too small to draw conclusions. Patients with unknown metastastic status (Mx) at the time of diagnosis were also excluded from the OS analysis, as the metastastic status largely influence the patient survival. Only patients with non-missing status/dates of survival were used for the OS analysis. The prognostic value of the markers was determined using univariable and multivariable Cox regression analysis. Variables with a P value of less than 0.1 in the univariable analysis were included in the multivariable analysis. We used the listwise deletion method for handling missing data. In this method, an entire sample was excluded from the analysis if any single value is missing for the variables used in the multivariable Cox regression analysis. For the studied markers which were not associated with patient survival in the univariable analysis, their prognostic values were further investigated in the preplanned subgroup analyses. OS was analyzed using the Kaplan-Meier method, with a log-rank test assessing its difference. All tests and P values tested two-sided were considered significant when ≤ 0.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 19.0 (SPSS. Inc.).