Robert S. Warren

Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction

Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Microsatellite Instability Predicts Improved Response to Adjuvant Therapy With Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer: Cancer and Leukemia Group B Protocol 89803

PURPOSE Colon cancers exhibiting DNA mismatch repair (MMR) defects demonstrate distinct clinical and pathologic features, including better prognosis and reduced response to fluorouracil (FU) -based chemotherapy. This prospective study investigated adjuvant chemotherapy containing FU and irinotecan in patients with MMR deficient (MMR-D) colon cancers. PATIENTS AND METHODS Cancer and Leukemia Group B 89803 randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly bolus FU/leucovorin (LV) or weekly bolus irinotecan, FU, and LV (IFL). The primary end point was overall survival; disease-free survival (DFS) was a secondary end point. Tumor expression of the MMR proteins, MLH1 and MSH2, was determined by immunohistochemistry (IHC). DNA microsatellite instability was also assessed using a panel of mono- and dinucleotide markers. Tumors with MMR defects were those demonstrating loss of MMR protein expression (MMR-D) and/or microsatellite instability high (MSI-H) genotype. RESULTS Of 723 tumor cases examined by genotyping and IHC, 96 (13.3%) were MMR-D/MSI-H. Genotyping results were consistent with IHC in 702 cases (97.1%). IFL-treated patients with MMR-D/MSI-H tumors showed improved 5-year DFS as compared with those with mismatch repair intact tumors (0.76; 95% CI, 0.64 to 0.88 v 0.59; 95% CI, 0.53 to 0.64; P = .03). This relationship was not observed among patients treated with FU/LV. A trend toward longer DFS was observed in IFL-treated patients with MMR-D/MSI-H tumors as compared with those receiving FU/LV (0.57; 95% CI, 0.42 to 0.71 v 0.76; 95% CI, 0.64 to 0.88; P = .07; hazard ratio interaction between tumor status and treatment, 0.51; likelihood ratio P = .117). CONCLUSION Loss of tumor MMR function may predict improved outcome in patients treated with the IFL regimen as compared with those receiving FU/LV.

Induction of Vascular Endothelial Growth Factor by Insulin-like Growth Factor 1 in Colorectal Carcinoma

Vascular endothelial growth factor (VEGF) is an angiogenic hormone that is produced by and supports the growth of many types of malignancies. The present study shows that insulin-like growth factor 1 (IGF-I), a mitogen that promotes the propagation of cancers through autocrine and paracrine mechanisms, increases the expression of mRNA for VEGF and production of VEGF protein by COLO 205 colon carcinoma cells. IGF-I also induces expression of VEGF mRNA in SW620, LSLiM6, and HCT15 colon carcinoma cells showing that this is a common response to IGF-I. Whereas IGF-I induced VEGF mRNA in each cell line examined (2.3-12-fold), it induced proliferation only in COLO 205 and LSLiM6 cells. Thus, the proliferative response induced by IGF-I and its ability to induce VEGF occur through distinguishable mechanisms. IGF-I increases the cellular content of VEGF mRNA by increasing the rate of transcription (5-fold after 4 h) and also by increasing the half-life of VEGF mRNA (0.6 ± 0.07 h in control cells to 2.0 ± 0.37 h in IGF-I-treated cells). Monoclonal antibody (αIR3) directed against the type 1 IGF receptor significantly attenuated the ability of IGF-I to promote expression of VEGF mRNA. Interestingly, by itself αIR3 acted as a weak agonist and induced a modest increase in the cellular content of VEGF mRNA. αIR3 also promoted tyrosine phosphorylation of the β subunit of the IGF-I receptor, and the magnitude of this response was comparable with that induced by IGF-I. These observations point to a nonlinear relationship between activation of the IGF-I receptor and induction of VEGF mRNA. Thus, in addition to its direct, growth stimulatory effect on transformed cells, IGF-I induces the expression of VEGF which can promote the progression of cancer by regulating the development of new blood vessels.

Hepatic Arterial Infusion Versus Systemic Therapy for Hepatic Metastases From Colorectal Cancer: A Randomized Trial of Efficacy, Quality of Life, and Molecular Markers (CALGB 9481)

PURPOSE Hepatic metastases derive most of their blood supply from the hepatic artery; therefore, for patients with hepatic metastases from colorectal cancer, hepatic arterial infusion (HAI) of chemotherapy may improve outcome. METHODS In a multi-institutional trial, 135 patients were randomly assigned to receive HAI versus systemic bolus fluorouracil and leucovorin. The primary end point was survival; secondary end points were response, recurrence, toxicity, quality of life, cost, and the influence of molecular markers. RESULTS Overall survival was significantly longer for HAI versus systemic treatment (median, 24.4 v 20 months; P = .0034), as were response rates (47% and 24%; P = .012) and time to hepatic progression (THP; 9.8 v 7.3 months; P = .034). Time to extrahepatic progression (7.7 v 14.8 months; P = .029) was significantly shorter in the HAI group. Quality-of-life measurements showed improved physical functioning in the HAI group at the 3- and 6-month follow-up assessments. Toxicity included grade > or = 3 neutropenia (2% and 45%; P < .01), stomatitis (0% and 24%; P < .01), and bilirubin elevation (18.6% and 0; P < .01) in the HAI and systemic treatment groups, respectively. A greater proportion of men versus women receiving HAI experienced biliary toxicity (37% and 15%, respectively; P = .05). For HAI patients with thymidylate synthase levels in tumor less than or > or = 4, the median survival was 24 and 14 months, respectively (P = .17). CONCLUSION HAI therapy increased overall survival, response rate, THP, and was associated with better physical functioning compared with systemic therapy. Additional studies need to address the overall benefit and cost of new chemotherapy agents versus HAI alone or the combination of HAI with new agents.

Safety and feasibility of injection with an E1B-55 kDa gene-deleted, replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase I trial.

Novel therapies are needed for locally advanced pancreatic carcinoma. ONYX-015 (dl1520) is an E1B-55 kDa region-deleted adenovirus that selectively replicates in and lyses tumor cells with abnormalities in p53 function (eg gene mutation). We carried out a phase I dose escalation study of ONYX-015 in patients with unresectable pancreatic cancer. ONYX-015 was administered via CT-guided injection (n = 22 patients) or intraoperative injection (n = 1) into pancreatic primary tumors every 4 weeks until tumor progression. Interpatient dose escalation was carried out with at least three patients per dose level from 108 p.f.u. up to the 1011 p.f.u. dose level (two patients treated at this dose). The majority of patients had abnormally low cellular immunity (CD4 counts and hypersensitivity skin testing). Injection of ONYX-015 into pancreatic carcinomas was well-tolerated. Mild, transient pancreatitis was noted in only one patient. Dose-escalation proceeded to the highest dose level. Neutralizing antibodies rose post-treatment in all patients. After injection, ONYX-015 was detectable in the blood 15 min later, but not between 1 and 15 days later. Viral replication was not documented, however, in contrast to trials in other tumor types. No objective responses were demonstrated. Intratumoral injection of an E1B-55 kDa region-deleted adenovirus into primary pancreatic tumors was feasible and well-tolerated at doses up to 1011 p.f.u. (2 × 1012particles), but viral replication was not detectable.

Intravascular adenoviral agents in cancer patients: Lessons from clinical trials

A large number of adenoviral agents are being developed for the treatment of cancer. However, the treatment-related death of a patient with ornithine transcarbamylase deficiency following adenovirus administration by hepatic artery has led to serious concerns regarding the safety of intravascular adenovirus. Both replication-incompetent (rAd.p53, e.g., SCH58500) and replication-selective (dl1520, aka Onyx-015; CG7870) oncolytic adenoviruses, by intravascular administration, are in clinical trials. We review Phases I and I/II results from these clinical trials. dl1520 and rAd.p53 were well-tolerated following hepatic artery infusion at doses of up to 2×1012 and 2.5×1013 particles, respectively. At a dose of 7.5×1013 particles, rAd.p53 was associated with dose-limiting cardiac output suppression; dl1520 dose escalation did not proceed higher than 2×1012. Intravenous (i.v.) infusions of dl1520 and CG7870 have been well tolerated by i.v. infusion at doses of 2×1013 and 6×1012, respectively, without identification of a maximally tolerated dose to date. Mild/moderate transaminitis was demonstrated in some patients on both the hepatic arterial and i.v. trials at doses ≥1012 particles. Interleukin (IL)-6 and IL-10 were induced in a dose-dependent manner in most patients, but significant interpatient and intrapatient (on repeat doses) variabilities were demonstrated. Evidence of p53 gene expression (Ad.p53) or viral replication (dl1520) was demonstrated in the majority of patients receiving ≥1012 particles. Over 100 cancer patients have been treated with intravascular adenovirus constructs to date with an acceptable toxicity profile; further clinical trial testing appears appropriate in cancer patients.

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen I, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Chromosome arm 20q gains and other genomic alterations in colorectal cancer metastatic to liver, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization

Comprehensive information about the molecular cytogenetic changes in metastases of colorectal cancer is not yet available. To define such changes in metastases, we measured relative DNA sequence copy numbers by comparative genomic hybridization (CGH). Samples from 27 liver metastases and 6 synchronous primary tumors were analyzed. An average of 9.9 aberrations per tumor was found in the metastases. Gains of chromosome arms 20q (85%), 13q (48%), 7p (44%), and 8q (44%) and losses of chromosome arms 18q (89%), 8p (59%), 1p (56%), and 18p (48%) were detected most frequently. Chromosomes 14 and 15 were lost in 26% and 30% of the metastases, respectively. No consistent differences were observed between primary tumors and synchronous metastases. Fluorescence in situ hybridization (FISH) was used for further characterization of gains of chromosome arm 20q. Touch preparations of 13 tumors that had demonstrated 20q gain with CGH were examined with FISH by use of a set of probes mapping to different parts of 20q. A probe for 20p was used as a reference. FISH showed relative gain of at least one 20q locus in 12 of the tumors. High‐level gains were detected in 38% of the tumors, preferentially for probes mapping to band 20q13. Our CGH data indicate that colorectal metastases show chromosomal changes similar to those that have been reported for primary tumors. Chromosomal losses were seen at higher frequency, particularly for chromosomes 14 and 15. By FISH, we identified subregions on chromosome arm 20q that are frequently involved in DNA amplifications in colorectal cancer and that may harbor candidate proto‐oncogenes. Genes Chromosomes Cancer 25:82–90, 1999.

Prognostic and Predictive Biomarkers in Resected Colon Cancer: Current Status and Future Perspectives for Integrating Genomics into Biomarker Discovery

In this article, the authors review the current status of biomarker research in the adjuvant treatment of colon cancer, drawing on their experiences and considering future strategies for biomarker discovery in the postgenomic era.

Prognostic Impact of Deficient DNA Mismatch Repair in Patients With Stage III Colon Cancer From a Randomized Trial of FOLFOX-Based Adjuvant Chemotherapy

PURPOSE The association of deficient DNA mismatch repair (dMMR) with prognosis in patients with colon cancer treated with adjuvant fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy remains unknown. PATIENTS AND METHODS Resected, stage III colon carcinomas from patients (N = 2,686) randomly assigned to FOLFOX ± cetuximab (North Central Cancer Treatment Group N0147 trial) were analyzed for mismatch repair (MMR) protein expression and mutations in BRAF(V600E) (exon 15) and KRAS (codons 12 and 13). Association of biomarkers with disease-free survival (DFS) was determined using Cox models. A validation cohort (Cancer and Leukemia Group B 88903 trial) was used. RESULTS dMMR was detected in 314 (12%) of 2,580 tumors, of which 49.3% and 10.6% had BRAF(V600E) or KRAS mutations, respectively. MMR status was not prognostic overall (adjusted hazard ratio [HR], 0.82; 95% CI, 0.64 to 1.07; P = .14), yet significant interactions were found between MMR and primary tumor site (P(interaction) = .009) and lymph node category (N1 v N2; P(interaction) = .014). Favorable DFS was observed for dMMR versus proficient MMR proximal tumors (HR, 0.71; 95% CI, 0.53 to 0.94; P = .018) but not dMMR distal tumors (HR, 1.71; 95% CI, 0.99 to 2.95; P = .056), adjusting for mutations and covariates. Any survival benefit of dMMR was lost in N2 tumors. Mutations in BRAF(V600E) (HR, 1.37; 95% CI, 1.08 to 1.70; P = .009) or KRAS (HR, 1.44; 95% CI, 1.21 to 1.70; P < .001) were independently associated with worse DFS. The observed MMR by tumor site interaction was validated in an independent cohort of stage III colon cancers (P(interaction) = .037). CONCLUSION The prognostic impact of MMR depended on tumor site, and this interaction was validated in an independent cohort. Among dMMR cancers, proximal tumors had favorable outcome, whereas distal or N2 tumors had poor outcome. BRAF or KRAS mutations were independently associated with adverse outcome.