The leukocyte complexity and mutational landscape of periampullary adenocarcinoma. Morphology matters.

Background: Periampullary adenocarcinomas are a heterogenous group of tumours with poor prognosis that hasnot improved considerably the last decades. Tumour morphology, i.e. intestinal type (I-type) or pancreatobiliarytype (PB-type), has been demonstrated to be a more relevant prognostic factor than anatomical origin. Tumourinfiltrating immune cells are vital in shaping the natural progress of cancer. The aim of this thesis was tocharacterise the landscape of immune cells and common mutations in the entire spectrum of periampullaryadenocarcinoma, with particular reference to morphology and clinical outcome.Methods: All studies are based on tumours from a retrospective, consecutive cohort of 175 patients withperiampullary adenocarcinoma who underwent surgical resection in Skåne University Hospital between 2001 and2011. The infiltration of several immune cell populations was analysed by single-marker immunohistochemistry(Paper I-II) and multiplexed immunofluorescence (Paper IV and V) on tissue microarrays. Targeted nextgeneration DNA seqencing (Paper III) was applied to analyse mutations in 70 common cancer-associated genesin tumours from 102 cases.Results: Paper I shows that high infiltration of natural killer NK/NKT cells was associated with prolonged overallsurvival (OS), particularly in patients with I-type tumours. In patients with PB-type tumours, high infiltration ofNK/NKT cell infiltration was only prognostic in cases who did not receive adjuvant chemotherapy, and, notably,there was a significant negative interaction between adjuvant chemotherapy and NK/NKT cell density in relation toOS. Paper II shows that high infiltration of immature dendritic cells was an independent factor of poor prognosis inpatients with PB-type tymours. High infiltration of CD68+ and CD163+ macrophages was assocated with reducedOS in the entire cohort, whereas high infiltration of MARCO+ macrophages was a negative prognostic factor onlyin patients who recived adjuvant chemotherapy, but there was no signficant treatment interaction. Paper IIIdemonstrated that APC and ERBB3 mutations were more common in I-type tumours while CDKN2A mutationswere more common in PB-type tumours. KRAS mutations were associated with reduced OS in patients with I-typetumours. in patients with PB-type tumours, SMARCA4 mutation was a negative prognostic factor in patients notreceiving adjuvant chemotherapy and there was a positive interaction between high expression of BRG1, theprotein encoded by SMARCA4, and adjuvant chemotherapy in relation to OS. Paper IV demonstrates that theprognostic impact of different lymphocyte subsets, including signatures thereof, differ by morphology, and thathigh levels of CD8+ T cells interacting with cancer cells and CD4+ T cells, respectively, were associated with aprolonged OS. Moreover, immune cell density differed by the mutational status of several genes. Paper V providesfurther validation of the beneficial prognostic impact of NK/NKT cells, also in the context of interaction withmacrophages. In addition, several prognostic innate immune cell subsets and signatures were defined, thatdiffered by tissue compartment and morphology.Conclusions: The prognostic and potential predictive impact of tumour infiltrating immune cells and commonmutations in periampullary adenocarcinoma differs by morphology, thus highlighting the importance of takingmorphology into account in the quest for complementary biomarkers. Moreover, the prognostic value of tumourinfiltrating immune cells can be futher refined by analyses of their spatial and compartmental distribution