Prognostic relevance of molecular subtypes and master regulators in pancreatic ductal adenocarcinoma

Rekin's Janky, Maria Mercedes Binda, Joke Allemeersch, Anke Van den Broeck, Olivier Govaere, Johannes V Swinnen, Tania Roskams, Stein Aerts, Baki Topal, Rekin's Janky, Maria Mercedes Binda, Joke Allemeersch, Anke Van den Broeck, Olivier Govaere, Johannes V Swinnen, Tania Roskams, Stein Aerts, Baki Topal

Abstract

Background: Pancreatic cancer is poorly characterized at genetic and non-genetic levels. The current study evaluates in a large cohort of patients the prognostic relevance of molecular subtypes and key transcription factors in pancreatic ductal adenocarcinoma (PDAC).

Methods: We performed gene expression analysis of whole-tumor tissue obtained from 118 surgically resected PDAC and 13 histologically normal pancreatic tissue samples. Cox regression models were used to study the effect on survival of molecular subtypes and 16 clinicopathological prognostic factors. In order to better understand the biology of PDAC we used iRegulon to identify transcription factors (TFs) as master regulators of PDAC and its subtypes.

Results: We confirmed the PDAssign gene signature as classifier of PDAC in molecular subtypes with prognostic relevance. We found molecular subtypes, but not clinicopathological factors, as independent predictors of survival. Regulatory network analysis predicted that HNF1A/B are among thousand TFs the top enriched master regulators of the genes expressed in the normal pancreatic tissue compared to the PDAC regulatory network. On immunohistochemistry staining of PDAC samples, we observed low expression of HNF1B in well differentiated towards no expression in poorly differentiated PDAC samples. We predicted IRF/STAT, AP-1, and ETS-family members as key transcription factors in gene signatures downstream of mutated KRAS.

Conclusions: PDAC can be classified in molecular subtypes that independently predict survival. HNF1A/B seem to be good candidates as master regulators of pancreatic differentiation, which at the protein level loses its expression in malignant ductal cells of the pancreas, suggesting its putative role as tumor suppressor in pancreatic cancer.

Trial registration: The study was registered at ClinicalTrials.gov under the number NCT01116791 (May 3, 2010).

Keywords: HNF1A/B; Master regulators; Molecular subtypes; Pancreatic ductal adenocarcinoma.

Figures

Fig. 1
Fig. 1
Expression heatmap for merged data. a Heatmap for 56 PDAssign genes vs 184 PDAC samples (+13 histologically normal pancreatic tissue samples as “Control” samples in grey). Samples are ordered and clustered by NMF clusters obtained from the NMF clustering of the merged PDAC data. Genes are clustered by hierarchical clustering using Pearson correlation distance (complete linkage). Sample legends show the sample clustering of the published subtypes (for the UCSF and GSE15471 tumors), but also the different predicted clusters from NMF of our 118 PDAC data (k3) and the predicted K-Ras dependency (kras) (see also Additional file 2: Figure S1 and Additional file 3: Figure S2). b Comparison of the predicted subtypes and known subtypes at the sample levels
Fig. 2
Fig. 2
Disease-free (DFS) and overall survival (OS) of patients according to molecular subtypes of PDAC. Molecular subtypes are predicted by using the published PDassign genes as a classifier of our PDAC samples. Survival according to 2 molecular subtypes (k2) classification: a DFS is significantly better for k2.cl1 (red line) than that for k2.cl2 (blue line) (p = 0.035). b No statistically significant difference in OS is observed between k2.cl1 (red line) vs. k2.cl2 (blue line) (p = 0.081). Survival according to 3 molecular subtypes (k3) classification: c DFS is significantly better for k3.cl1 (magenta line) than that for k3.cl2 (blue line) (p = 0.026). d No statistically significant difference in OS is observed between the 3 subtypes separately (p = 0.193); k3.cl1 (magenta line), k3.cl2 (blue line), k3.cl3 (orange line). Tables 1 and 2 provide more information on these survival curves
Fig. 3
Fig. 3
Master regulators in PDAC vs Control (histologically normal pancreatic tissue samples). a Result summary of the regulatory analysis with iRegulon on 2640 up regulated genes. b Venn diagram of the predicted up-regulated targets from AP1, ETS and IRF. c Results of the regulatory analysis with iRegulon on 1325 down-regulated genes. d Venn diagram of the predicted down-regulated targets from HNF1A/B and Nuclear Receptors. Raw results of the analysis are presented in Additional file 4: Table S3 and Additional file 5: Figure S4
Fig. 4
Fig. 4
Immunohistochemistry for HNF1β. a Strong nuclear expression in normal acinar parenchyma and normal ducts (upper part) while the expression is completely lost in a poorly differentiated PDAC (lower part) (Magnification 50x). b IHC shows a lower expression in high-grade dysplasia (upper part) compared to normal duct (arrow) (Magnification 100x). c IHC for HNF1β shows reduced expression in a well to moderately differentiated PDAC compared to a non-neoplastic duct (asterisk) (Magnification 200x). Histograms showing the histoscores corresponding to the left (a) (b) (c). Asterisk on the histogram indicates that the differences with each of the other categories are significant (Mann Whitney test, p < = 0.0294). d Normal pancreas (positive control) showing a strong staining in ducts and in the acinar parenchyma (Magnification 40x)

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