title: Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: comparison of real-time reverse transcription-PCR vs. relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products.
abstract: The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.
authors.affiliation: Department of Medical Oncology, City of Hope Comprehensive Cancer Center, Duarte, California 91010, USA. ajuhasz@coh.org
authors.forename: Agnes
authors.initials: A
authors.lastname: Juhasz
authors.forename: Paul
authors.initials: P
authors.lastname: Frankel
authors.forename: Catherine
authors.initials: C
authors.lastname: Cheng
authors.forename: Hector
authors.initials: H
authors.lastname: Rivera
authors.forename: Reena
authors.initials: R
authors.lastname: Vishwanath
authors.forename: Alice
authors.initials: A
authors.lastname: Chiu
authors.forename: Kim
authors.initials: K
authors.lastname: Margolin
authors.forename: Yun
authors.initials: Y
authors.lastname: Yen
authors.forename: Edward M
authors.initials: EM
authors.lastname: Newman
authors.forename: Tim
authors.initials: T
authors.lastname: Synold
authors.forename: Sharon
authors.initials: S
authors.lastname: Wilczynski
authors.forename: Heinz-Josef
authors.initials: HJ
authors.lastname: Lenz
authors.forename: David
authors.initials: D
authors.lastname: Gandara
authors.forename: Kathy S
authors.initials: KS
authors.lastname: Albain
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Longmate
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
mesh_terms: D000465:Algorithms; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001706:Biopsy; D001940:Breast / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002945:Cisplatin / Q000008:administration & dosage; D003844:DCMP Deaminase / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D003842:Deoxycytidine Kinase / Q000235:genetics; D004720:Endonucleases; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D003842:Deoxycytidine Kinase; C071447:ERCC1 protein, human; D004720:Endonucleases; D003844:DCMP Deaminase; D002945:Cisplatin; D000093542:Gemcitabine