This study is, to our best knowledge, the first to investigate the prognostic role of tumour-infiltrating CD1a+, CD68+ and CD163+ and MARCO+ immune cells in periampullary adenocarcinoma, with particular reference to morphological type. The results demonstrate that high infiltration of CD1a+ TIDC is an independent predictor of a shorter survival in patients with PB-type tumours, but does not confer any prognostic value in patients with I-type tumours. These findings add to the growing evidence of a dysfunctional inflammatory microenvironment caused by a desmoplastic stroma in these particularly aggressive tumours.
A previous study failed to establish any association between survival and DC infiltration rates in pancreatic cancer, secondary to the scarcity of local homing [17]. The association between high CD1a+ TIDC density and poor prognosis may be explained by their immature state or even the induction of maturation defects in TIDC in situ, initiated by the tumour microenvironment and leading to an immunosuppressive phenotype encouraging tumour tolerance and immune evasion [24]. A study by Yamamoto et al. demonstrated a positive impact of TIDCs on survival in patients with pancreatic cancer [16]. However, in that study, anti-fascin was used as a marker for TIDCs, therefore potentially examining another TIDC population.
In the present study we chose to not investigate mature DCs or to identify functional subtypes of TIDCs. Future studies should however focus not only on the presence of tolerogenic immature CD1a+ DCs in tumour tissue, but also on their functional phenotype, as this may add important information on their potential immune modulatory effect in the inflammatory tumour microenvironment. There has been a steep increase of interest in the field of immunotherapy in recent years and dendritic cell vaccine has shown some promise in combination with conventional chemotherapy in pancreatic cancer [25,26,27]. The findings of the present study indicate that it is important to take morphological type into consideration when evaluating the results from such trials.
Moreover, this study demonstrated an association between high density of CD68+ and CD163+ TAMs with poor prognosis in the whole cohort, but not in strata according to morphological type, and not independent of established clinical prognostic factors. Tumour-educated TAMs facilitate progression of pancreatic cancer and promote angiogenesis, remodelling of stroma, epithelial-mesenchymal transition and extravasation of tumour cells [28, 29]. Previous studies have demonstrated that TAMs are associated with poor prognosis in pancreatic cancer [7, 8], which is in line with the findings of the present study encompassing the full spectrum of periampullary adenocarcinoma. Even though it is mainly the anti-inflammatory subpopulation of TAMs that has been suggested to promote tumour progression, the entire CD68+ TAM population was also found to be associated with poor prognosis in the present study. The reason for this finding is most likely that the predominant subtype of TAM in the tumour microenvironment is leaning towards pro-tumour polarisation [30], thus making up for a large part of the CD68+ TAM population.
Further, the novel macrophage marker MARCO, which has been shown to be a target for immunotherapy [14], was found to be associated with poor prognosis in I-type but not PB-type tumours. The observation that the prognostic impact of MARCO+ cells was particularly evident in patients who received adjuvant chemotherapy, as opposed to patients who did not receive any adjuvant treatment, is noteworthy, despite the lack of a significant treatment interaction. The OS of patient with low MARCO+ TAM infiltration who received adjuvant chemotherapy with I-type morphology had a remarkably better OS than patients that did not receive adjuvant chemotherapy. This result might indicate a potential predictive role of MARCO+ TAM infiltration for chemotherapy response, or high density of MARCO+ TAMs could be a sign of chemotherapy resistance. Further studies are needed to validate these results, especially in intestinal cancers.
Previous research on the role of MARCO in cancer has been scarce. One previous study on hepatocellular cancer showed that decreased expression of MARCO was associated with poor prognosis [31], however, in contrast to the present study, that study did not look at immune cell specific expression of MARCO, but rather at intra-tumoural MARCO expression. The present study found a significant association between MARCO+ cells and CD68+ cells which confirms the results of Sun et al, where MARCO+ cells co-localized with CD68+ macrophages [31]. If the association between high MARCO+ immune cell infiltration and poor survival rates is due to the co-localization of MARCO+ and CD68+ cells, interaction with chemotherapy, or because of the biological mechanisms of MARCO is yet to be determined. Further research into the role of MARCO in periampullary/pancreatic cancer as well as in other intestinal cancer and a broader spectrum of solid cancers is highly warranted.
In a translational context, the findings from the analyses of human tumours in this study are well in line with previous in vitro studies. For instance, Karnevi et al. have shown an intricate interplay between macrophages and tumour cells in vitro, where tumour derived factors drive the differentiation of macrophages into a pro-tumour phenotype [30]. Additionally, the opposite effect has been demonstrated for dendritic cells in vitro, where tumour cell derived factors inhibit and limit the normal anti-tumour function of DCs [32]. Further, in vivo models have shown that macrophage infiltration increases with tumour progression, and that infiltration starts very early before any invasive potential has been developed by the tumour. Collectively, these findings support the conclusions in the present study, wherein dense infiltration of TAMs and DCs are shown to be associated with poor and improved prognosis, respectively, in periampullary adenocarcinoma.
Some subgroup analyses rendered rather small numbers of cases, in particular in strata according to adjuvant or no adjuvant therapy and morphological type. Therefore, the results from the present study need to be validated in additional and preferably larger patient cohorts. However, as about half of the patients in the herein analysed patient cohort received adjuvant chemotherapy and half of the patients did not, it may give some indications to the potential predictive value of the investigated biomarkers, despite the retrospective setting.
Another potential limitation to the study is the use of the TMA technique, and in particular the fact that the tissue cores were primarily sampled from areas with tumour and not the adjacent stroma. However, a large proportion of periampullary cancers have a comparatively high stromal/tumour cell ratio, and three 1 mm cores can be considered a generous sampling. However, validating studies should ideally be specifically designed for a more comprehensive mapping of immune cell signatures. In this context, the TMA technique is likely to be superior to whole tissue section analysis, since it allows for sampling of multiple tissue types from multiple tissue blocks, and thus for a more comprehensive analysis of the inflammatory microenvironment of individual tumours.
