Mena^calc, a quantitative method of metastasis assessment, as a prognostic marker for axillary node-negative breast cancer

The characteristics of a prospectively ascertained consecutive series of 1561 ANN cases enrolled from eight Toronto hospitals between September 1987 and March 1993 and clinically followed for recurrence and death have been described previously [30, 31]. In brief, all women who had ANN invasive breast cancer pathologically confirmed at the participating centers were potentially eligible. The pathology report was used to determine the initial eligibility (which required clear resection margins and at least four lymph nodes sampled), pathologic size of the invasive component (centrally reviewed at Mount Sinai Hospital, Toronto, ON, Canada), presence of vascular or lymphatic invasion by tumor cells, estrogen receptor (ER) status, progesterone receptor (PgR) status, nuclear grading, histologic grading, and histologic subtype of the invasive and intraductal components (if present). Imaging (bone scan and abdominal ultrasound or abdominal computed tomography scan) and chest x-ray were required for patients with T2 tumors. If the patient was eligible on the basis of pathology, staging and age (between 18 and 75 years inclusive), the surgeon invited the patient to participate and provided a signed consent form. Patients were excluded from recruitment into this ANN cohort if (1) No tumor specimen was provided for analysis, (2) no axillary dissection was performed as part of surgical management, (3) less than four lymph nodes were biopsied and analyzed, (4) pathology revealed that the patient was diagnosed with carcinoma-in-situ disease (i.e., no invasive component), (5) the patient had distant metastases at the time of diagnosis, (6) the patient had synchronous primary breast tumors, (7) the patient had a previous breast malignancy, and (8) the patient had a secondary malignancy other than non-melanoma of the skin and carcinoma-in-situ of the cervix. Charts were reviewed every 3 months in the first 2 years after diagnosis, every 6 months until 5 years after diagnosis, and annually thereafter. Patient status on July 10, 2002 was used to determine survival times and censoring status using clinical follow-up data.

Approval of the study protocol was obtained from the research ethics boards of Mount Sinai Hospital (#01-0313-U) and the University Health Network (#02-0881-C), Toronto. Written-informed consent was received from all study participants. In the preparation of this paper, we used the reporting recommendations for tumor marker prognostic studies (REMARK) to present our results [32].

Tissue microarrays (TMAs) were constructed from tumors of 888 women and biomarker status was determined as described below. Areas of invasive carcinoma were selected from a hematoxylin and eosin-stained section of each tumor and two 0.6-mm cores of tissue were taken from the corresponding areas of the paraffin block. The selected donor cores were embedded in a paraffin block and 4-μm sections were cut from this recipient block and used in series for immunohistochemical (IHC) staining. Microwave antigen retrieval was carried out in a Micromed T/T Mega Microwave Processing Lab Station (ESBE Scientific). Slides were pretreated at 115 °C for 12 minutes in TTMega Tris (pH 9.0) and incubated with antibodies to ER (clone 6 F11, 1:75 dilution, Vector, Burlington, ON, Canada), PgR (clone PgR1294, 1:1000 dilution, DAKO, Glostrup, Denmark), p53 (clone D.07, 1:400 dilution, ID Lab), or CK5 (clone XM26, 1:400 dilution, Vector, Burlington, ON, Canada). Alternatively, slides were pretreated with pepsin at 37 °C for 10 minutes and then incubated with antibodies to EGFR (clone 31G7, 1:25 dilution, Zymed, South San Francisco, CA, U.S.A.) or HER-2 (clone CB11/TAB250 (cocktail), 1:300 dilution, Novocastra, Newcastle upon Tyne, U.K. and Zymed, South San Francisco, CA, U.S.A.). Sections were developed with diaminobenzidine tetrahydrochloride and counterstained in Mayer’s hematoxylin.

The TMAs were deparaffinized by melting at 60 °C in an oven equipped with a fan for 20 minutes followed by 2x xylene treatment for 20 minutes. Slides were then rehydrated and antigen retrieval was done in citrate buffer (pH 6.0) at 97 °C for 20 minutes in a PT module (Labvision, Kalamazoo, MI, U.S.A.). Endogenous peroxidase was blocked by using 0.3 % hydrogen peroxide in methanol followed by incubation of slides in a blocking buffer (0.3 % bovine serum albumin in TBST (0.1 mol/L of TRIS-buffered saline (pH 7.0) containing 0.05 % Tween-20)) for 30 minutes at room temperature. Slides were incubated with a cocktail of mouse anti-pan-Mena (1:1000 dilution, BD Biosciences, San Jose, CA, U.S.A., catalog number 610693) mixed with rabbit anti-Mena11a (1:500 dilution of 1 mg/ml stock, generated in the lab of FG) in the blocking buffer overnight at 4 °C. After washing away the primary antibodies, slides were incubated with secondary antibody (goat anti-rabbit conjugated to horseradish peroxidase, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, U.S.A.) to target Mena11a for one hour. After washing, slides were incubated with biotinylated tyramide (Perkin Elmer, Waltham, MA, U.S.A.) diluted at 1:50 in amplification buffer for 10 minutes. After washing, peroxidase activity was quenched by 2x treatment with benzoic hydrazide (100 mM in PBS) with 50 mM hydrogen peroxide for seven minutes each. After washing, slides were incubated for an hour with goat anti-mouse envision (DAKO, Carpinteria, CA, U.S.A.) followed by treatment with a chicken anti-Pan cytokeratin (1:100 dilution, generated in house) for 2 hours at room temperature. Slides were washed and then incubated with goat anti-chicken conjugated to Alexa546 (Invitrogen, Grand Island, NY, U.S.A.) to visualize cytokeratin and streptavidin conjugated to CY7 (750 nm, Invitrogen, Grand Island, NY, U.S.A.) to visualize Mena11a for an hour. After washing, slides were treated with CY5 conjugated tyramide (1:50 dilution; Perkin Elmer, Waltham, MA, U.S.A.) in amplification buffer for 10 minutes to visualize pan-Mena. Slides were mounted with ProLong gold mixed with DAPI (Molecular Probes, Grand Island, NY, U.S.A.). Serial sections of the index array used for assay standardization [33] were stained alongside each cohort to assess the assay reproducibility. An additional serial section of the index array was stained with each experiment with no primary antibodies as a negative control.

Of the 888 tumors submitted for Mena multiplex immunofluorescence staining, 403 had sufficient tumor material to permit reliable interpretation of pan-Mena and Mena11a.

The automated quantitative analysis (AQUA) technology (HistoRx, Branford, CT, U.S.A.) allows quantitative measurement of biomolecules in subcellular compartments as described previously [34, 35]. Briefly, a series of monochromatic images for each histospot was captured using a PM-2000 microscope (HistoRx, Branford, CT, U.S.A.) equipped with an automated stage. A binary ‘tumor mask’ was created using cytokeratin staining of the histospot representing only epithelial cells and excluding stromal features. AQUA scores for both pan-Mena and Mena11a were calculated by dividing the signal intensity by the area of the specific compartment (in this case within the tumor mask area). Normalized AQUA scores for both targets (pan-Mena and Mena11a) were used to calculate the Mena^calc fraction for each histospot by subtracting the z score of Mena11a from the z score of pan-Mena as described previously [29]. At the end of this procedure, Mena^calc was obtained for 403 tumors from the ANN cohort.

Pearson’s correlation coefficient (R) was used to assess the reproducibility of the multiplexed assay between near-serial sections of the index assay as described previously [29]. Pan-Mena and Mena11a AQUA scores, and Mena^calc values from two independent cores for each histospot were averaged and the averages were used for final analysis. Mena^calc scores were categorized as scores that were at or above the median (Mena^calc high) or below the median (Mena^calc low). This median cutoff was selected because of the division noted in the Kaplan Meier (K-M) survival curves generated from quartile groups. The chi-square test or Fisher’s exact test was used to analyze the Mena^calc marker associations with clinical-pathological tumor variables. Analysis of the association of overall survival (OS) with marker status was performed using K-M survival curves and the univariate Cox proportional hazards (PH) model with the log-rank or the likelihood ratio test. Multivariate analyses by the Cox PH model were conducted to assess the contribution of Mena^calc, in addition to HER2 status (using IHC data), hormone receptors (using IHC data) and other clinical-pathological tumor variables. Hazard ratios (HRs) and 95 % confidence intervals (CI) were also estimated, with Firth’s bias corrected penalized Cox regression method [39] applied for subgroup analyses with a small number of events. A test with a P-value < 0.05 was considered statistically significant. All tests were two-sided. P-values were not adjusted for multiple testing. All statistical analyses were performed using SAS 9.1 software (SAS Institute, Inc.). Survival curves were plotted using R statistical software, version 2.15.0 (http://​www.​r-project.​org/​).