When identifying new prognostic biomarkers it is of great importance to decide which tumour site to examine, as the expression might differ in primary tumour and metastases. In this study we have analyzed PODXL expression in 31 primary tumours and a total number of 140 corresponding lymph node metastases, thus providing a thorough characterization of PODXL expression in both settings. Moreover, we have examined the potential effect of radiation therapy on PODXL expression in rectal cancer patients.
PODXL has previously been found to correlate with a poor prognosis in CRC [7, 8]. So far, all results are derived from studies based on TMAs with retrospectively collected tumour samples. In this study, we used full-face sections, one from each case, to determine PODXL expression in 73 CRC patients. Our results indicate that by use of full-face section analysis, a larger proportion of tumours are identified as being PODXL positive, i.e. having membranous expression, compared to TMA-based analyses (24,7% vs 8-13%) [7, 8]. These findings are not unexpected, since PODXL is, in the vast majority of cases, overexpressed in a heterogenous fashion, preferrably at the invasive tumour front. Therefore, use of the TMA-technique will most likely lead to an underestimation of positive cases. Moreover, no tumour in this cohort had membranous staining in more than 50% of the tumour cells (i.e. category 4), which may be explained by the size of the cohort, but could also be attributed to the tumour area selected for sampling. The number of cases denoted as having > 50% cells with membranous PODXL ex-pression in previous studies is however negligible, further supporting that using a cutoff based on the presence or absence of membranous staining should be sufficient for prognostication purposes. Even if recognition of membranous PODXL expression is fairly straightforward, and the mere presence rather than the quantity seems to be of prognostic importance, it would however be of interest to compare visual scoring and automated analysis in future studies [13, 14].
For characterization of key molecular alterations and expression of investigative biomarkers in tumours from large patient cohorts, whether retrospectively or prospectively defined, the TMA-technology is indispensable . For prospective biomarker studies and in clinical use, however, analysis of full-face sections should be the most convenient and applicable method.
Prognostic biomarkers in CRC are routinely analyzed in the primary tumor, whereas tumor cells in lymph node metastases are not characterized. Previous studies on KRAS expression have shown a discrepancy between the primary tumour and corresponding lymph node metastases [16, 17], whereas the expression of other biomarkers, e.g. ER and HER-2 in breast cancer have been demonstrated to be highly concordant . Of note, while most studies related to the concordance between primary and metastatic lesions have only examined a few lymph nodes (typically two per patient), we have in this study strived to examine all metastatic lymph nodes.
Although there was a discordance of PODXL expression between primary tumours and lymph node metastases in some cases, this was limited to a few cases with PODXL positive primaries where a clonal distribution of PODXL expression was observed in the metastatic lymph nodes. These results reflect the fact that the small proportion of cells in a positive primary tumour displaying membranous PODXL expression are highly prone to metastasize. The excellent concordance between primary tumour and lymph node metastases demonstrate that assessment of the primary tumour is sufficient to determine if a patient has a PODXL positive tumour. The expression of PODXL in lymph node metastases can however provide prognostic information when no primary tumour is available for analysis. Moreover, in future studies, it would be of interest to perform in-depth analyses of other molecular characteristics, and drivers of the metastatic phenotype, that may differ between PODXL negative and positive lymph node metastases in individual cases.
The significant associations between PODXL expression and several unfavourable clinicopathological characteristics (e.g. TNM stage) that have been demonstrated in our previous studies [7, 8], did not reach statistical significance in this study, most likely due to the small sample size. Nevertheless, despite the small number of patients, a statistically significant relationship between PODXL expression and differentiation grade and mucinous histology was seen. Moreover, there was an overweight of PODXL positive tumours among patients who received adjuvant treatment, an indirect measurement of more aggressive tumours. The significant association of PODXL expression with female gender has not been observed in previous studies and is likely attributable to the small size of the cohort.
It is well known that approximately half of the patients with CRC stage III disease will relapse, and that adjuvant chemotherapy reduces the risk of recurrence with 20-30%. Our previous study has shown that patients with PODXL positive tumours within this group benefit from adjuvant chemotherapy irrespective of treatment regime . While the majority of patients with stage III disease in this study received adjuvant treatment, a few patients were not considered candidates for chemotherapy due to old age or comorbidity. In cases of doubt whether adjuvant treatment should be given, assessment of PODXL expression may be a useful prognostic tool. Moreover, the finding of a higher proportion of PODXL positive tumours in full-face sections is of particular clinical relevance in stage II disease, where it is of uttermost importance to identify patients with high-risk disease who would benefit from adjuvant treatment. Previous studies have shown that adjuvant chemotherapy could improve survival for this group of patients, but that the incremental benefits are small . Therefore, further prognostic tools are needed to better guide treatment decisions in this patient category. As PODXL expression has been demonstrated to have a prognostic value in patients with stage II disease in retrospective analysis  this association warrants further study in the prospective setting.
In biomarker studies it is important to consider the effect of neoadjuvant treatment on biomarker expression. In this study we found an excellent concordance between PODXL expression in rectal tumours before and after neoadjuvant irradiation, suggesting that PODXL expression is not affected by radiation therapy.
In conclusion, the results from this study suggest that PODXL expression in CRC is concordant in primary tumours and corresponding lymph node metastases in individual patients, and also remains unaffected by neoadjuvant radiation therapy. The results further support the clinical utility of PODXL as a biomarker for risk assessment in CRC, even in cases where no primary tumour is available for analysis, and irrespective of histopathological response to neoadjuvant treatment.