A comparative study of two PODXL antibodies in 840 colorectal cancer patients

The study population comprised 840 consecutive colorectal cancer patients operated in 1983–2001 at the Department of Surgery, Helsinki University Central Hospital. The Finnish Population Register Centre provided follow-up vital status data needed to compute survival statistics, and Statistics Finland provided cause of death for all those deceased. Median age at diagnosis was 66, with a median follow-up of 5.1 years (range 0–25.8). The 5-year disease-specific survival rate was 58.9% (95% Cl 55.0-62.8%). This study was approved by the Surgical Ethics Committee of Helsinki University Central Hospital (Dnro HUS 226/E6/06, extension TMK02 §66 17.4.2013) and the National Supervisory Authority of Welfare and Health (Valvira Dnro 10041/06.01.03.01/2012).

Formalin-fixed and paraffin-embedded tumour samples came from the archives of Department of Pathology, Helsinki University Central Hospital. Representative areas of tumour samples on haematoxylin- and eosin-stained tumour slides were marked by an experienced pathologist. Three 1.0-mm-diameter punches taken from each sample were mounted on recipient paraffin block with a semiautomatic tissue microarray instrument (TMA) (Beecher Instruments, Silver Spring, MD, USA) as described [22].

The monoclonal in-house antibody (HES9) recognises amino acid residues 189–192 of PODXL. The polyclonal antibody (HPA 2110, Atlas Antibodies, Stockholm, Sweden) recognises amino acid residues 278–415 of PODXL. Both epitopes are in the extracellular part of PODXL. Of four protein coding PODXL splice variants, the epitope sequence of the pAb matches three with 100% (PODXL 001, 005, and 201, The Human Protein Atlas). The fourth splice variant matches with 87% (PODXL 202). The epitope sequence of the mAb HES9 matches all splice variants with 100%. The antibodies have been described in detail [21, 23, 24].

TMA-blocks were freshly cut into 4-μm sections. After deparaffinization in xylene and rehydration through a gradually decreasing concentration of ethanol to distilled water, slides were treated in a PreTreatment module (Lab Vision Corp., Fremont, CA, USA) in Tris–HCl (pH 8.5) buffer for 20 minutes at 98°C for antigen retrieval. For the staining procedure by the Dako REAL EnVision Detection system, Peroxidase/DAB+, Rabbit/Mouse (Dako, Glostrup, Denmark) an Autostainer 480 (Lab Vision) was used. Tissues were incubated with the mAb (dilution 1:500 = 5 μg/ml) or pAb (dilution 1:250) for one hour at room temperature. In every staining series renal tissue served as positive control.

As reported PODXL expression by the HES9 mAb was cytoplasmic and often granular. Positivity in tumour cells was uniform, with no nuclear expression [21]. By the pAb PODXL expression was cytoplasmic, with no nuclear expression. In some cases there were a distinct membranous positivity, even with weak cytoplasmic positivity. For the mAb negative cytoplasmic staining was scored as 0, weakly positive as 1, moderately positive as 2, and strongly positive as 3. For the pAb cytoplasmic staining was scored 0–2 (negative-moderate-strong) and in case of distinct membranous staining as 3, regardless of the intensity of the cytoplasmic staining [17]. Stainings were scored independently by T.K. and J.H., who were blinded to clinical data and outcome. Differences in scoring were discussed until consensus.

For statistical purposes, categories of PODXL expression were dichotomised into low (0–2) and high (3) for the mAb and into non-membranous (0–2) and membranous (3) for the pAb. To study the two antibodies together a categorization with three classes was created; low (mAb: low and pAb: non-membranous), moderate (either mAb: high or pAb: membranous), and high (mAb: high and pAb: membranous). The antibodies were also categorized as weak (mAb: low and pAb: non-membranous) and strong (mAb: high and/or pAb: membranous). Evaluation of the association between PODXL expression and clinicopathological parameters was done by the Fisher exact-test or the linear-by-linear association test for ordered parameters. Kappa-value was used for testing the concordance of PODXL expression according to mAb and pAb. Disease-specific overall survival was counted from date of surgery to date of death from colorectal cancer, or until end of follow-up. Survival analysis was done by the Kaplan-Meier method and compared by the log rank test. The Cox regression proportional hazard model served for uni- and multivariable survival analysis, adjusted for sex, age, Dukes classification, and differentiation. Testing of the Cox model assumption of constant hazard ratios over time involved the inclusion of a time-dependent covariate separately for each testable variable. Hazard ratios of differentiation and Dukes class D were analyzed in two periods (0 to 1.25 and 1.25 to 5 years) in order to meet the assumptions of the Cox model, and the time-dependent Cox model was used. Interaction terms were considered, but no significant interactions were found. All tests were two-sided. A p-value of 0.05 was considered significant. All statistical analyses were done with SPSS version 20.0 (IBM SPSS Statistics, version 20.0 for Mac; SPSS, Inc., Chicago, IL, USA, an IBM Company).

PODXL expression by the pAb was cytoplasmic in tumour cells, but in some cases a distinct membranous expression was seen, which did not always correlate with intensity of cytoplasmic expression. Such distinct membranous staining was not seen with the mAb HES9 [21].

Of 840 tumours represented in the TMA, PODXL staining with pAb could be evaluated in 780 (92.6%); 46 (5.9%) had no cytoplasmic positivity, 322 (41.2%) showed moderate cytoplasmic staining, 349 (44.7%) strong cytoplasmic staining, and 63 (8.1%) positive staining with distinctive membranous staining. Representative images of pAb stainings are shown in Figure 1. Comparative images of high cytoplasmic staining by the mAb and membranous staining by the pAb in same tumours are shown in Figure 2. The staining results by the mAb HES9 have been described previously [21].

There was a strong association between membranous PODXL expression and poor differentiation (p < 0.0001) and advanced stage (p < 0.001). Membranous PODXL expression did not associate with age, gender, tumour location (right vs left hemicolon or colon vs rectum), or tumour histology (Table 1). The corresponding results for the mAb HES9 have been described [21].

The agreement of expression of the two antibodies across cases was low (kappa-value = 0.219, standard error 0.060, p < 0.0001) using dichotomous values for both antibodies. Of the distinctive strong staining by mAb (n = 44) and membranous staining by pAb (n = 63) only 14 tumours were shared.

Analysis of combined PODXL expression (low-moderate-high categories) with clinicopathological parameters showed significant associations between high PODXL expression and poor differentiation (p < 0.0001), advanced stage (p < 0.0001), and tumour side (p = 0.003). High expression did not associate with age, gender, tumour location (colon vs rectum), nor with tumour histology (Table 2). Association of the clinicopathological parameters with the strong PODXL expression (weak-strong categories) were similar (data not shown).

For colorectal cancer patients with membranous PODXL expression by polyclonal antibody disease-specific survival (DSS) was significantly poorer (p = 0.001). Five-year DSS was 40.5% (95% CI 27.4-53.6%) for patients with membranous PODXL expression compared to 60.0% (95% CI 56.3-63.7%) for non-membranous expression (Figure 3). Results were similar for the combination of pAb and mAb (weak-strong), but the group with poor prognosis was larger (n = 93 vs 63) (data not shown).Combination (low-moderate-high) of mAb and pAb showed a significantly poorer DSS for colorectal cancer patients with high expression compared to low expression (p < 0.0001) or moderate expression (p < 0.0001). No statistically significant difference in DSS was seen between those with low and moderate expression (p = 0.24). Five-year DSS for CRC patients with low expression was 60.3% (95% CI 56.6-64.0%), for those with moderate expression 52.8% (95% CI 41.0-64.6%),and for those with high expression 8.3% (95% CI −7.4-24.0%) (Figure 4).

Cox regression univariable analysis confirmed these results. In multivariable survival analyses adjusted for age, gender, Dukes classification, and differentiation grade, membranous PODXL expression by the pAb remained statistically significant. Also the combined high expression of PODXL using mAb and pAb remained statistically significant in multivariable analysis (Table 3).