Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma

A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.

[1]  G. Peters,et al.  Pretreatment deoxycytidine kinase levels predict in vivo gemcitabine sensitivity. , 2002, Molecular cancer therapeutics.

[2]  Tatjana Crnogorac-Jurcevic,et al.  Expression profiling of microdissected pancreatic adenocarcinomas , 2002, Oncogene.

[3]  J. Abbruzzese.  New applications of gemcitabine and future directions in the management of pancreatic cancer , 2002, Cancer.

[4]  M. Frazier,et al.  Human pancreatic ribonuclease 1 , 2000, Cancer.

[5]  E. Giovannetti,et al.  Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer , 2006, Molecular Cancer Therapeutics.

[6]  M. Monden,et al.  Prognostic Significance of Activated Akt Expression in Pancreatic Ductal Adenocarcinoma , 2004, Clinical Cancer Research.

[7]  R H Hruban,et al.  Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers. , 2001, Cancer research.

[8]  H. Friess,et al.  Enhanced expression of 14-3-3sigma in pancreatic cancer and its role in cell cycle regulation and apoptosis. , 2004, Carcinogenesis.

[9]  R. Hruban,et al.  Characterization of gene expression in mucinous cystic neoplasms of the pancreas using oligonucleotide microarrays , 2004, Oncogene.

[10]  Franco Mosca,et al.  Transcription analysis of human equilibrative nucleoside transporter-1 predicts survival in pancreas cancer patients treated with gemcitabine. , 2006, Cancer research.

[11]  J. Mackey,et al.  cN-II expression predicts survival in patients receiving gemcitabine for advanced non-small cell lung cancer. , 2005, Lung cancer.

[12]  I. Coe,et al.  Differential expression of human nucleoside transporters in normal and tumor tissue. , 2001, Biochemical and biophysical research communications.

[13]  C. W. Lee,et al.  Nitrobenzylthioinosine-binding protein overexpression in human breast, liver, stomach and colorectal tumour tissues. , 1995, Anticancer research.

[14]  N. Hanna,et al.  RRM1 Modulated In Vitro and In Vivo Efficacy of Gemcitabine and Platinum in Non–Small-Cell Lung Cancer , 2008 .

[15]  中村 透.  Genome-wide cDNA microarray analysis of gene-expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection , 2004 .

[16]  A. Jemal,et al.  Cancer Statistics, 2007 , 2007, CA: a cancer journal for clinicians.

[17]  H. Pitt,et al.  Thymidylate synthase expression predicts the response to 5-fluorouracil-based adjuvant therapy in pancreatic cancer. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[18]  K. Yamasawa,et al.  Implication of thymidylate synthase in the outcome of patients with invasive ductal carcinoma of the pancreas and efficacy of adjuvant chemotherapy using 5-fluorouracil or its derivatives , 2002, Anti-cancer drugs.

[19]  R. Wolff,et al.  Pancreatic cancer , 2004, The Lancet.

[20]  Ryuzo Ueda,et al.  Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer , 2004, Cancer science.

[21]  中野 靖弘,et al.  Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells , 2007 .

[22]  M. Talamini,et al.  Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes. , 1997, Annals of surgery.

[23]  D. V. Von Hoff,et al.  Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. , 1997, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  R. Burgemeister.  New Aspects of Laser Microdissection in Research and Routine , 2005, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[25]  G. Fontanini,et al.  Thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer and normal mucosa in patients , 2006, Pharmacogenetics and genomics.

[26]  Bauke Ylstra,et al.  In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant. , 2005, Cancer research.

[27]  R. Abrams,et al.  HUMAN ENT1 IS PREDICTIVE OF RESPONSE IN PATIENTS WITH PANCREATIC CANCER TREATED WITH GEMCITABINE: RESULTS FROM THE RTOG 9704 PROSPECTIVE RANDOMIZED TRIAL , 2007 .

[28]  E. Giovannetti,et al.  Synergistic Cytotoxicity and Pharmacogenetics of Gemcitabine and Pemetrexed Combination in Pancreatic Cancer Cell Lines , 2004, Clinical Cancer Research.

[29]  John R. Mackey,et al.  The Absence of Human Equilibrative Nucleoside Transporter 1 Is Associated with Reduced Survival in Patients With Gemcitabine-Treated Pancreas Adenocarcinoma , 2004, Clinical Cancer Research.

[30]  H. Friess,et al.  Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[31]  M. Saif.  Pancreatic cancer: highlights from the 42nd annual meeting of the American Society of Clinical Oncology, 2006. , 2006, JOP : Journal of the pancreas.

[32]  C. R. Crawford,et al.  Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. , 1998, Cancer research.

[33]  E. Felip,et al.  Gene Expression as a Predictive Marker of Outcome in Stage IIB-IIIA-IIIB Non-Small Cell Lung Cancer After Induction Gemcitabine-Based Chemotherapy Followed By Resectional Surgery , 2004, Clinical Cancer Research.

[34]  Young L. Kim,et al.  Optical Markers in Duodenal Mucosa Predict the Presence of Pancreatic Cancer , 2007, Clinical Cancer Research.

[35]  G. Bepler,et al.  Ribonucleotide Reductase Messenger RNA Expression and Survival in Gemcitabine/Cisplatin-Treated Advanced Non-Small Cell Lung Cancer Patients , 2004, Clinical Cancer Research.

[36]  A. Foulis.  Book Review: AFIP Atlas of Tumot Patholgoy (3rd series, Fascicle 20). Tumours of the Pancreas. Enrico Solcia, Carlo Capella and Gunter Kloppel, Armed Forces Institute of Pathology, Washington, DC , 1998 .

[37]  Takashi Ishikawa,et al.  Human equilibrative nucleoside transporter 1 is associated with the chemosensitivity of gemcitabine in human pancreatic adenocarcinoma and biliary tract carcinoma cells. , 2007, Oncology reports.

[38]  B. Rybarczyk,et al.  Tumors and Fibrinogen , 2001, Annals of the New York Academy of Sciences.

[39]  G. Peters,et al.  Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. , 2002, Biochimica et biophysica acta.

[40]  R. Rosell,et al.  Pharmacogenomics and gemcitabine. , 2006, Annals of oncology : official journal of the European Society for Medical Oncology.

[41]  M. Watson,et al.  Variance in the Expression of 5-Fluorouracil Pathway Genes in Colorectal Cancer , 2005, Clinical Cancer Research.

[42]  B. Sipos,et al.  Tumor Stroma Interactions Induce Chemoresistance in Pancreatic Ductal Carcinoma Cells Involving Increased Secretion and Paracrine Effects of Nitric Oxide and Interleukin-1β , 2004, Cancer Research.