Volume 155, Issue 5, Pages 1625-1639.e2 (November 2018) Immune Cell and Stromal Signature Associated With Progression-Free Survival of Patients With Resected Pancreatic Ductal Adenocarcinoma  Ujjwal Mukund Mahajan, Eno Langhoff, Elisabetta Goni, Eithne Costello, William Greenhalf, Christopher Halloran, Steffen Ormanns, Stephan Kruger, Stefan Boeck, Silvia Ribback, Georg Beyer, Frank Dombroswki, Frank-Ulrich Weiss, John P. Neoptolemos, Jens Werner, Jan G. D’Haese, Alexandr Bazhin, Julian Peterhansl, Svenja Pichlmeier, Markus W. Büchler, Jörg Kleeff, Paula Ganeh, Matthias Sendler, Daniel H. Palmer, Thomas Kohlmann, Roland Rad, Ivonne Regel, Markus M. Lerch, Julia Mayerle  Gastroenterology  Volume 155, Issue 5, Pages 1625-1639.e2 (November 2018) DOI: 10.1053/j.gastro.2018.08.009 Copyright © 2018 AGA Institute Terms and Conditions

Gastroenterology 2018 155, 1625-1639. e2DOI: (10. 1053/j. gastro. 2018 Copyright © 2018 AGA Institute Terms and Conditions

Figure 1 Low expression levels of tumor-infiltrate leukocytes correlate with poor PFS across resected PDAC. (A) CONSORT diagram. 5FU/FA, 5-fluorouracil/folinic acid; FFPE, formalin-fixed paraffin-embedded. (B) Representative image showing analysis of immune infiltration (number of cells/mm2) using an algorithm developed for NIH ImageJ software. (C) The Kaplan-Meier survival analysis shows that patients with low CD3 expression are burdened with decreased PFS time in comparison with high CD3 expression. Gastroenterology 2018 155, 1625-1639.e2DOI: (10.1053/j.gastro.2018.08.009) Copyright © 2018 AGA Institute Terms and Conditions

Figure 2 Contribution of multifaceted stromal composition in defining PFS. (A) Representative images of α-SMA and collagen-I staining characterizing fibrogenic, inert, dormant, and fibrolytic stroma. (B) Kaplan-Meier survival analysis of patients with resected PDAC in respect to stroma subtype with median survival for inert stroma (light green) as 13.76 (95% CI 10.94–16.65), dormant stroma (orange) as 12.81 (95% CI 9.88–16.06), fibrogenic stroma (light blue) as 14.09 (95% CI 11.99–20.10), and fibrolytic stroma (red) as 11.03 (95% CI 8.87–12.74). (C) Correlation matrixes demonstrating results of the correlation analyses followed by unsupervised hierarchical clustering between leukocyte subpopulations and corresponding stromal composition. (D) Box and whiskers plot comparing the differential distribution of leukocyte subpopulations in different stromal composition with P values from Kruskal-Wallis testing with Dunn post hoc. Gastroenterology 2018 155, 1625-1639.e2DOI: (10.1053/j.gastro.2018.08.009) Copyright © 2018 AGA Institute Terms and Conditions

Figure 3 Inferred leukocyte subtype frequencies associated with prognostic association in determining PFS corresponding to stromal composition. (A) Random forest iterations determine “variable of importance (VIMP)” and minimal depth of the prognostic variables in determining PFS. Longer VIMP bars with shorter minimal depth bars indicate a higher effect of the variable. Area shaded in gray depicts the prognostic window for the prognostic variables. (B) Multivariate recursive partitioning for discrete-time survival tree for PFS depicting prognostic signatures amalgamating 2 or more markers with median PFS varying between 7.95 months and 16.60 months with a relative error of prediction given with 0.08 (X-error −0.02). (C) Waterfall plot of each prognostic signature as delineated from the terminal nodes illustrating the relative prognostic index of each signature calculated using Cox multivariate proportional hazards. (D) Alluvial plot depicting prediction accuracy of response (predicted m[pfst] per subcohort) in comparison with actual response (actual m[pfst] per subcohort) in the validation cohort. Left side of the alluvial plot depicts predicted response per node and right depicts actual response per node of the validation cohort. The blue area connecting left to the right graph represent perfect match, whereas the red area represents mismatch in the response. The thickness of the color areas depicts the number of subjects. P < .05 is considered as significant. Gastroenterology 2018 155, 1625-1639.e2DOI: (10.1053/j.gastro.2018.08.009) Copyright © 2018 AGA Institute Terms and Conditions