Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue

Abstract

Background: There is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status.

Methods: Fresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection.

Results: Fresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity.

Conclusions: The preservation of the patient's tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

Figure 1

Histological resemblance between patient tumor and resulting tumorgraft following sequential grafting into immunocompromised mice. Representative sections stained with hematoxylin and eosin of indicated tumor samples (Scale bars indicate 100 μm). A) Adenocarcinoma of pancreas with desmoplastic stromal reaction in original patient tumor. 1^st generation tumorgraft (total time in vivo, 217 days) with preservation of atypical glandular structures with inspissated mucinous material surrounded by fibrous stroma. 2^nd generation tumorgraft (303 days) with similar histology. B) Adenocarcinoma of colon with numerous atypical glands containing mucinous material and necrotic debris with surrounding fibrovascular connective tissue. 1^st generation tumorgraft after 101 days reveals dilated atypical ducts with mucin and necrotic debris in lumens comprised of multiple columnar layers of atypical epithelium surrounded by fibrovascular stroma. 2^nd generation graft after 177 days with similar histology. C) Lung carcinoma with prominent fibrovascular connective tissue in original patient neoplasm that becomes progressively overgrown by malignant cells in 1^st (216 days) and 2^nd (286 days) generation tumor grafts. D) Cervical carcinoma with fibromucinous stroma in original patient neoplasm, with expansion of large islands of malignant cells surrounded by fibrovascular stroma in 1^st (282 days) and 2^nd (345 days) generation tumorgrafts.

Figure 2

Unsupervised cluster analysis of genomes of tumorgrafts and originating patient tumors. Unsupervised clustering was performed on genomes amplified by A) Affymetrix method, 28 genomes total; and B) NuGEN method, 20 genomes total. Clustering was performed using programming language R version 2.11.1 on normalized gene expression data that was pre-processed using the Affymetrix MAS5 algorithm to an average intensity target of 500. Other than a few highlighted outliers discussed in the text, patient tumors and their derived tumorgrafts are closely related at the gene expression level.

Figure 3

Enrichment of canonical pathway maps in genes downregulated in tumorgrafts compared to their donor tumors.A) Scatter plot of expression of gene probes (25,806) graphically depicting the up-regulated (red circle) and down-regulated (green circle) 2-fold or greater (t-test, p ≤ 0.05) in tumorgrafts relative to the originating patient tumors. B) Ontology enrichment analysis was performed using the MetaCore™ pathway analysis suite (GeneGo-Thomson Reuters, St. Joseph, MI). Eight of the top ten canonical pathways enriched in the genes down-regulated in the tumorgrafts, relative to the originating patient tumors, were immune-related, consistent with immunodeficient status of the murine host.

Figure 4

Enrichment of canonical pathway maps in genes up and downregulated in tumorgraft forming tumors compared to non-tumorgraft forming tumors. Ontology enrichment analysis was performed using the MetaCore™ pathway analysis suite (GeneGo-Thomson Reuters, St. Joseph, MI). A) The top ten canonical pathway maps enriched in the genes up-regulated (n = 491) in the tumors that formed tumorgrafts included cell signalling and cell cycle-related pathways. B) Eight of top ten canonical pathway maps enriched in the genes down-regulated (n = 691) in the tumorgrafts that formed tumorgrafts were immune-related pathways.

Figure 5

WNT signalling pathway map enriched with genes up-regulated in tumorgraft-forming tumors. The MetaCore™ pathway analysis suite (GeneGo-Thomson Reuters, St. Joseph, MI) identified a pathway map for WNT signalling. VEGF-A, Frizzled, β-catenin, FAK-1, c-Myc, Laminin 1, XIAP, p21, MELP, and 4E-BP1 present in this pathway were all up-regulated in tumorgraft forming tumors.

Figure 6

Immune response pathway map enriched with genes down-regulated in the tumorgraft forming patient tumors. The MetaCore™ pathway analysis suite (GeneGo-Thomson Reuters, St. Joseph, MI) identified a pathway map for an immune response involving integrins in NK cell cytotoxicity that was enriched for several ligands down-regulated in tumors that did not form tumorgrafts (Target cell denotes tumor cells).