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Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease with a 5-year survival rate of 4%. A key hallmark of PDAC is extensive stromal involvement, which makes capturing precise tumor-specific molecular information difficult. Here we have overcome this problem by applying blind source separation to a diverse collection of PDAC gene expression microarray data, including data from primary tumor, metastatic and normal samples. By digitally separating tumor, stromal and normal gene expression, we have identified and validated two tumor subtypes, including a 'basal-like' subtype that has worse outcome and is molecularly similar to basal tumors in bladder and breast cancers. Furthermore, we define 'normal' and 'activated' stromal subtypes, which are independently prognostic. Our results provide new insights into the molecular composition of PDAC, which may be used to tailor therapies or provide decision support in a clinical setting where the choice and timing of therapies are critical.

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Figure 1: Successful deconvolution of normal tissue with NMF.
Figure 2: The dual action of stroma is described by distinct gene expression patterns, which are not present in PDAC cell lines.
Figure 3: Tumor-specific gene expression suggests two subtypes of PDAC with similarities to other tumor types.
Figure 4: Multivariate survival analysis of tumor and stromal subtypes.
Figure 5: Associations between tumor and stromal subtypes, PDX tumors, KRAS mutations and SMAD4 expression.
Figure 6: Overcoming limited tumor cellularity shows true heterogeneity among matched primary and metastatic sites.

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Gene Expression Omnibus

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Acknowledgements

We thank the University of North Carolina (UNC) Tissue Procurement, Translational Pathology Laboratory, Animal Studies, Animal Histopathology, and Center for Gastrointestinal Biology and Disease Histopathology core facilities (NIH grant P30-DK034897), the PDX Program and Department of Pharmacology for tremendous technical support. We thank the patients and their families who generously donated their samples to research and in particular to the University of Nebraska Medical Center Rapid Autopsy Pancreatic Program and the Johns Hopkins Gastrointestinal Cancer Rapid Medical Donation Program. This work was partially supported by grant R01-CA140424 from the US National Institutes of Health (NIH) (J.J.Y.), the Kimmel Foundation (J.J.Y.), the American College of Surgeons (J.J.Y.), the University Cancer Research Fund (J.J.Y.) and UNC Lineberger Comprehensive Cancer Center Postdoctoral Training Grant T32-CA009156 from the US NIH (R.A.M.).

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Authors and Affiliations

Authors

Contributions

Designed the study and wrote the manuscript: R.A.M. and J.J.Y. Performed experiments and collected data: R.A.M., R.M., E.L.F., S.G.H.L., L.A.W., S.C.E., A.H.C. and J.K.S. Pathologist: K.E.V. Performed data analysis: R.A.M., R.M., J.J.Y., K.A.H. and N.U.R. Provided samples: J.M.A., H.J.K., D.J.B., M.S.T., C.A.I.-D., M.A.H. and J.J.Y. Performed project oversight: J.J.Y.

Corresponding author

Correspondence to Jen Jen Yeh.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Representative H&E staining of patient tumor samples.

(a) Liver metastases showing regions of tumor and normal tissue. (b) Primary pancreatic tumor sample showing normal pancreatic tissue and tumor cells in the same field. (c) Primary pancreatic tumor with high tumor cellularity. (d) Primary pancreatic tumor with abundant tumor stroma. (e) Percentage of tumor in primary pancreatic tumors in the UNC and ICGC cohorts. Black arrowheads show areas of tumor stroma. Black arrows show areas of tumor. White arrowheads show normal tissue. Scale bars, 200 μm.

Supplementary Figure 2 Deconvolution of a large cohort of PDAC reveals distinct gene expression patterns from multiple tissue types.

Solid color bars above the heat map show the tissue of origin and tumor status of the samples, which were used to order the samples horizontally. Factor weights derived by NMF for selected factors are shown as grayscale bars. Heat maps show the z-normalized gene expression of exemplar genes from each factor. All tumors, cell lines and adjacent normal tissues from our cohort are shown.

Supplementary Figure 3 Correlation of pathology assessments of tumor with factor weights in normal pancreas and primary tumors.

Horizontal axes all show tumor cellularity, while vertical axes show factor weight. Red dashed lines show best linear fits. P values are given for each R2.

Supplementary Figure 4 Primary tumors have higher expression of stroma genes than metastatic samples.

(a) Normalized average gene expression of the 25 exemplar activated stroma genes is higher in primary tumor samples than in metastases (P < 0.001). (b) The average expression of the 25 exemplar normal stroma genes is slightly higher in primary tumors than in metastases (P = 0.031).

Supplementary Figure 5 Immunofluorescence staining of cancer-associated fibroblasts (CAFs).

(a) EpCAM, (b) vimentin and (c) SMAα. Staining of T3M4 pancreatic cancer cells as a (d) positive control for EpCAM, (e) negative control for vimentin and (f) negative control for SMAα. Scale bars, 50 μm.

Supplementary Figure 6 Hierarchical clustering of the Spearman correlation of samples from the UNC, TCGA bladder cancer and Perou data sets shows similarities among basal-like samples.

The color bars above the heat map show subtype, either from the original publication (known tumor subtype) or from our cross-platform classifier (pan-platform classification).

Supplementary Figure 7 Comparison to the subtypes from Collisson et al.

(a) Consensus-clustered heat map of normalized data from UNC and Collisson et al. using the gene sets from Collisson et al. Primary tumors, normal pancreas and cell lines are shown. Samples from Collisson et al. were previously classified as exocrine-like (magenta), classical (cyan) and quasimesenchymal (yellow). (b) Kaplan-Meier plots of UNC samples classified by PAM into the subtypes from Collisson et al. (c) Mouse- and human-specific gene expression of the gene lists from Collisson et al. in PDXs shown in log2 (1 + RPKM). Genes from the classical subtype are expressed by tumor cells, genes from the quasimesenchymal subtype are expressed by a mixture of human and mouse cells, and genes from the exocrine-like subtype are weakly expressed throughout.

Supplementary Figure 8 Collagen I staining of mouse stroma in PDX tumors.

(a) Staining for mouse collagen I of stroma in a representative PDX tumor. (b) Corresponding H&E staining of an adjacent section. Staining for mouse collagen I of (c) mouse skin and (d) human skin. Black arrowheads show areas of tumor stroma. Black arrows show areas of tumor. Scale bars, 200 μm.

Supplementary Figure 9 Tumor gene expression in PDX models.

(a) Mouse- and human-specific gene expression of gene lists from the basal-like and classical subtypes in 37 PDX tumors shown in log2 (1 + RPKM). Both gene sets are robustly expressed by the human (tumor) but not the mouse (stroma) cells in PDX samples. (b) Consensus clustering of these PDX tumors using the gene lists from the basal-like and classical subtypes divides the samples into two groups.

Supplementary Figure 10 SMAD4 staining of representative patient and matched PDX tumors.

Positive SMAD4 staining of a (a) patient adenocarcinoma and (b) corresponding PDX at passage 4. SMAD4 loss in a (c) patient adenocarcinoma and (d) corresponding PDX at passage 2. SMAD4 staining of control tissues: (e) human skin, (f) mouse skin and (g) human normal pancreas. Scale bars, 200 μm.

Supplementary Figure 11 Consensus-clustered heat map of ICGC data for which genetic information was available.

The color bars above the heat map show the subtypes and genetic alterations for key genes in PDAC. The heat maps show the z-normalized gene expression of genes from the basal-like and classical tumor subtypes.

Supplementary Figure 12 Gene signature scores by subtype.

Scores are normalized across the cohort and are calculated as the mean expression across a panel of genes obtained from MSigDB. (a) The basal-like subtype shows downregulation of GATA6. (b) Tumors from the classical subtype are enriched in genes associated with mucinous ovarian cancer. (c) Tumors from the basal-like subtype are enriched in genes related to KRAS activation and STK11 loss.

Supplementary Figure 13 Differences in extracellular mucin in tumors from the classical and basal-like subtypes.

(a) Number of samples with low (<10%) compared to high (10%) extracellular mucin content. (b) Representative H&E staining of samples with a low degree and (c) high degree of extracellular mucin content. Scale bars, 200 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Table 3 and Supplementary Note. (PDF 5214 kb)

Supplementary Data Set

Gene expression of samples derived from RNA-seq data and KRAS mutation status. Expression is given as FPKM after processing with Xenome to sort human- and mouse-specific expression. (TXT 18938 kb)

Supplementary Table 1

List of the gene sets enriched in each factor. (XLSX 449 kb)

Supplementary Table 2

List of gene weights for each factor. (XLSX 2551 kb)

Supplementary Table 4

Overlap of the samples with those from Stratford et al. (XLSX 13 kb)

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Moffitt, R., Marayati, R., Flate, E. et al. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat Genet 47, 1168–1178 (2015). https://doi.org/10.1038/ng.3398

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