A tumor sorting protocol that enables enrichment of pancreatic adenocarcinoma cells and facilitation of genetic analyses.

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

[1]  R. Hruban,et al.  Genome-Wide Allelotypes of Familial Pancreatic Adenocarcinomas and Familial and Sporadic Intraductal Papillary Mucinous Neoplasms , 2007, Clinical Cancer Research.

[2]  D. Klimstra,et al.  K-ras mutations in pancreatic ductal proliferative lesions. , 1994, The American journal of pathology.

[3]  Luc Girard,et al.  An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays. , 2004, Cancer research.

[4]  C. Chelala,et al.  Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays , 2008, Oncogene.

[5]  R. Hruban,et al.  Genome-wide aberrations in pancreatic adenocarcinoma. , 2005, Cancer genetics and cytogenetics.

[6]  R. Hruban,et al.  BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets. , 2003, The American journal of pathology.

[7]  R. DePinho,et al.  Pancreatic cancer biology and genetics , 2002, Nature Reviews Cancer.

[8]  Robert L. Sutherland,et al.  Cyclins and Breast Cancer , 1996, Journal of Mammary Gland Biology and Neoplasia.

[9]  G. Gaudernack,et al.  Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries. , 2002, Analytical biochemistry.

[10]  P. Malfertheiner,et al.  Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1 , 2002, British Journal of Cancer.

[11]  F. Couch,et al.  BRCA2 and pancreatic cancer , 2002, International journal of gastrointestinal cancer.

[12]  Bert Vogelstein,et al.  Mutations of mitotic checkpoint genes in human cancers , 1998, Nature.

[13]  S. Beghelli,et al.  Genetic abnormalities in pancreatic cancer , 2003, Molecular Cancer.

[14]  J. Bartek,et al.  A series of 14 new monoclonal antibodies to keratins: Characterization and value in diagnostic histopathology , 1991, The Journal of pathology.

[15]  Scott E. Kern,et al.  DPC4, A Candidate Tumor Suppressor Gene at Human Chromosome 18q21.1 , 1996, Science.

[16]  A. Krasinskas,et al.  Loss of Heterozygosity Predicts Poor Survival After Resection of Pancreatic Adenocarcinoma , 2008, Journal of Gastrointestinal Surgery.

[17]  Todd R. Golub,et al.  BRAF mutation predicts sensitivity to MEK inhibition , 2006, Nature.

[18]  Jonathan J Ross,et al.  Targeted therapy in oncology: the agony and ecstasy of personalized medicine , 2001, Expert review of anticancer therapy.

[19]  A. Jemal,et al.  Annual report to the nation on the status of cancer, 1975–2001, with a special feature regarding survival , 2004, Cancer.

[20]  Zhi Hu,et al.  An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. , 2008, Cancer research.

[21]  A. Kallioniemi,et al.  Pancreatic adenocarcinoma—Genetic portrait from chromosomes to microarrays , 2006, Genes, chromosomes & cancer.

[22]  S. Goodman,et al.  Tumor-suppressive pathways in pancreatic carcinoma. , 1997, Cancer research.

[23]  H. Friess,et al.  Application of laser capture microdissection combined with two‐dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma , 2003, Proteomics.

[24]  S. Hirohashi,et al.  Pancreatic adenocarcinomas frequently show p53 gene mutations. , 1993, The American journal of pathology.

[25]  Keith W. Jones,et al.  Whole genome DNA copy number changes identified by high density oligonucleotide arrays , 2004, Human Genomics.

[26]  J. Howe,et al.  A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Yeatman,et al.  Analysis of p53, p21WAF1, and TGF-beta1 in human ductal adenocarcinoma of the pancreas: TGF-beta1 protein expression predicts longer survival. , 1998, American journal of clinical pathology.

[28]  T. Golub,et al.  Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma , 2005, Nature.

[29]  C Caldas,et al.  Allelotype of pancreatic adenocarcinoma. , 1994, Cancer research.

[30]  B. Reid,et al.  Single nucleotide polymorphism array analysis of flow-sorted epithelial cells from frozen versus fixed tissues for whole genome analysis of allelic loss in breast cancer. , 2002, The American journal of pathology.

[31]  B. Ghadimi,et al.  The Genetic Basis of Sporadic Pancreatic Cancer , 2005, Cellular oncology : the official journal of the International Society for Cellular Oncology.