Intratumoral expression profiling of genes involved in angiogenesis in colorectal cancer patients treated with chemotherapy plus the VEGFR inhibitor PTK787/ZK 222584 (vatalanib).

The phase III CONFIRM clinical trials demonstrated that metastatic colorectal cancer patients with elevated serum lactate dehydrogenase (LDH) had improved outcome when the vascular endothelial growth factor receptor (VEGFR) inhibitor PTK/ZK (Vatalanib) was added to FOLFOX4 chemotherapy. We investigated the hypothesis that high intratumoral expression of genes regulated by hypoxia-inducible factor-1 alpha (HIF1α), namely LDHA, glucose transporter-1 (GLUT-1), VEGFA, VEGFR1, and VEGFR2, were predictive of outcome in CONFIRM-1. Tumor tissue was isolated by laser-capture microdissection from 85 CONFIRM-1 tumor specimens; FOLFOX4/placebo n=42, FOLFOX4/PTK/ZK n=43. Gene expression was analyzed using quantitative RT-PCR. In univariate analyses, elevated mRNA expression of LDHA, GLUT-1, and VEGFR1 were associated with response to FOLFOX4/PTK/ZK. In univariate and multivariate analyses, elevated LDHA and VEGFR1 mRNA levels were associated with improved progression-free survival in FOLFOX4/PTK/ZK patients. Furthermore, increased HIF1α and VEGFR2 mRNA levels were associated with decreased survival in FOLFOX/placebo patients but not in patients who received FOLFOX4/PTK/ZK. These are the first data suggesting intratumoral mRNA expression of genes involved in angiogenesis/HIF pathway may predict outcome to VEGFR-inhibitors. Biomarkers that assist in directing VEGFR-inhibitors toward patients with an increased likelihood of benefit will improve the cost-effectiveness of these promising agents.

Pharmacogenetic profiling of CD133 is associated with response rate (RR) and progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC), treated with bevacizumab-based chemotherapy.

Recent studies suggest CD133, a surface protein widely used for isolation of colon cancer stem cells, to be associated with tumor angiogenesis and recurrence. We hypothesized that gene expression levels and germline variations in CD133 will predict clinical outcome in patients with metastatic colorectal cancer (mCRC), treated in first-line setting with 5-fluorouracil, oxaliplatin and bevacizumab (BV), and we investigated whether there is a correlation with gene expression levels of CD133, vascular endothelial growth factor (VEGF) and its receptors. We evaluated intra-tumoral gene expression levels by quantitative real-time (RT) PCR from 54 patients and three germline variants of the CD133 gene by PCR-restriction-fragment length polymorphism from 91 patients with genomic DNA. High gene expression levels of CD133 (>7.76) conferred a significantly greater tumor response (RR=86%) than patients with low expression levels (7.76, RR=38%, adjusted P=0.003), independent of VEGF or its receptor gene expression levels. Gene expression levels of CD133 were significantly associated with VEGF and its receptors messenger RNA levels (VEGFR-1 (P<0.01), -2 and -3, P<0.05). Combined analyses of two polymorphisms showed a significant association with progression-free survival (PFS) (18.5 months vs 9.8 months, P=0.004) in a multivariate analysis as an independent prognostic factor for PFS (adjusted P=0.002). These results suggest that CD133 is a predictive marker for standard first-line BV-based treatment in mCRC.

TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials.

To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

Dynamic changes in RAS gene status in circulating tumour DNA: a phase II trial of first-line FOLFOXIRI plus bevacizumab for RAS-mutant metastatic colorectal cancer (JACCRO CC-11).

BACKGROUND: Few prospective studies have used liquid biopsy testing in RAS-mutant metastatic colorectal cancer (mCRC), and its clinical significance remains unknown. Therefore, this study aimed to carry out a biomarker analysis by liquid biopsy using updated data of the phase II trial of FOLFOXIRI plus bevacizumab as first-line chemotherapy for RAS-mutant mCRC. MATERIALS AND METHODS: A total of 64 patients who received modified FOLFOXIRI regimen (irinotecan 150 mg/m2, oxaliplatin 85 mg/m2, levofolinate 200 mg/m2, and fluorouracil 2400 mg/m2) plus bevacizumab biweekly were enrolled. The primary endpoint was the objective response rate (ORR). Plasma samples were collected at pre-treatment, 8 weeks after treatment, and progression in participants included in the biomarker study. The levels of circulating tumour DNA (ctDNA) and specific KRAS and NRAS variants were evaluated using real-time PCR assays. RESULTS: There were 62 patients (median age: 62.5 years, 92% performance status 0, 27% right side) who were assessable for efficacy and 51 for biomarker analysis. ORR was 75.8% (95% confidence interval 65.1% to 86.5%). The median progression-free survival was 12.1 months, and the median overall survival (OS) was 30.2 months. In 78% of patients, RAS mutations disappeared in the ctDNA at 8 weeks after treatment; these patients tended to have better outcomes than those with RAS mutations. Interestingly, RAS mutations remained undetectable during progression in 62% of patients. Survival analysis indicated that the median OS from progression was significantly longer in patients with RAS mutation clearance than in those with RAS mutation in the ctDNA at disease progression (15.1 versus 7.3 months, hazard ratio: 0.21, P = 0.0046). CONCLUSIONS: Our biomarker study demonstrated no RAS mutations in ctDNA at disease progression in 62% of patients with RAS-mutant mCRC. Both OS and post-progression survival were better in patients with clearance of RAS mutations in ctDNA after triplet-based chemotherapy.

Southwest Oncology Group study S0413: a phase II trial of lapatinib (GW572016) as first-line therapy in patients with advanced or metastatic gastric cancer.

BACKGROUND: Lapatinib (GW572016) is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2/ErbB2), which are reported as overexpressed in 15%-45% of gastric cancers, making them potential targets. PATIENTS AND METHODS: The primary objective of this study was to assess response rate. Secondary objectives included overall survival (OS), toxicity, and the relationship of EGFR, ErbB2, and markers of angiogenesis with clinical outcome. Lapatinib was administered to chemonaive metastatic gastric cancer patients at a dose of 1500 mg orally daily for 28 days. RESULTS: The study enrolled 47 patients from February 2005 until May 2006. Four patients (9%) had a confirmed partial response (PR), 1 (2%) had an unconfirmed PR, and 10 (23%) had stable disease. Median (95% confidence interval) time to treatment failure was 1.9 (1.6-3.1) months and OS was 4.8 (3.2-7.4) months. Significant adverse events: one grade 4 cardiac ischemia/infarction, one grade 4 fatigue, and one grade 4 emesis. One treatment-related death was due to central nervous system ischemia. An exploratory analysis of markers revealed gene expression of HER2, interleukin (IL)-8 and genomic polymorphisms IL-8, and vascular endothelial growth factor correlated with OS. CONCLUSIONS: Lapatinib is well tolerated, with modest single-agent activity in advanced/metastatic gastric cancer patients. Potential molecular correlatives were identified which warrant further validation.

Pharmacogenetic profiling of Aurora kinase B is associated with overall survival in metastatic colorectal cancer.

Aurora kinases are conserved eukaryotic serine-threonine kinases, which serve as key regulators of mammalian mitosis. Several studies revealed a distinct correlation between inaccurate chromosome segregation, leading to chromosomal number instability, cancer progression and poor outcome. The aim of this study was to investigate the correlation of Aurora kinases A (AURKA) and B (AURKB) with overall survival (OS) by quantifying gene expression analysis and evaluation of single-nucleotide polymorphisms (SNPs) in human colorectal cancer samples and assessing the associations with clinicopathological features. We evaluated intratumoral gene expression levels and SNPs of AURKA and -B from 41 patients with metastatic colorectal cancer (mCRC). Patients with a high expression level of AURKB (>1.28) lived significantly shorter (n=11, median OS=6.4 months, 95% confidence interval (CI): 3.0-14.5 months) compared with patients with a low expression level (≤ 1.28) (n=30, median OS=18.4 months, 95% CI: 14.7-27.8 months, P=0.026, Wald's test). Patients harboring any G-allele in AURKB 885A>G showed a significantly decreased OS (P=0.05, log-rank test). We did not find any associations with clinicopathological variables and AURKA gene expression levels. Our results suggest a potential role for AURKB inhibition in patients with mCRC; thereby supporting its potential role as a target in mCRC.

The gene expression profile of cardia intestinal metaplasia is similar to that of Barrett's esophagus, not gastric intestinal metaplasia.

The etiology and significance of cardia intestinal metaplasia (CIM) is disputed. CIM may represent a form of Barrett's esophagus due to reflux or could reflect generalized gastric intestinal metaplasia due to Helicobacter pylori. The aim of this study was to utilize gene expression data to compare CIM to Barrett's and gastric intestinal metaplasia. Endoscopic biopsies were classified by endoscopic and histologic criteria as CIM (n= 33), Barrett's (n= 25), or gastric intestinal metaplasia of the antrum or body (n= 18). The squamocolumnar and gastroesophageal junctions were aligned in CIM patients and patients with diffuse gastric intestinal metaplasia were excluded. H. pylori was tested for in the biopsies of all patients. After laser-capture microdissection, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of a panel of nine genes that has been shown to differentiate Barrett's from other foregut mucosa. Cluster analysis with linear discriminant analysis of the expression data was used to classify each sample into groups based solely on similarity of gene expression. Cluster analysis was performed for three groups (CIM vs. Barrett's vs. gastric intestinal metaplasia) and two groups (CIM + Barrett's vs. gastric intestinal metaplasia). There was no difference in H. pylori infection among groups (P= 0.66). Clustering into three groups resulted in frequent misclassification between CIM and Barrett's while misclassification of gastric intestinal metaplasia was uncommon. The CIM and Barrett's groups were then combined for two group clustering and linear discriminant analysis correctly predicted 95% of CIM and Barrett's samples and 83% of gastric intestinal metaplasia samples based on gene expression alone. In conclusion, the gene expression profiles of CIM and Barrett's esophagus were similar in 95% of biopsies and differed significantly from that of gastric intestinal metaplasia. The indistinguishable gene expression profile of CIM and BE suggests that they may share a common etiology in the majority of patients with a similar biology, and calls into question the perception that CIM is an innocuous process.

Molecular profiling including epidermal growth factor receptor and p21 expression in high-risk breast cancer patients as indicators of outcome.

BACKGROUND: Patients with high-risk primary breast cancer remain at high risk for relapse. More precise prognostic and predictive tools are needed to improve treatment of such patients. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tumors from 239 high-risk breast cancer patients were examined for expression of human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), estrogen receptor, progesterone receptor, Ki-67, p16, p21, p27, and p53 by immunohistochemistry. Gene expression of EGFR, HER2, glutathione S-transferase-Pi (GSTP1), excision repair cross complementation1 (ERCC1), p21, beta-tubulin-3, multidurg resistance (MDR1), cyclooxygenase2 (COX2), and cyclin-E was measured by RT-PCR. RESULTS: Eighty percent of patients presented with locally advanced, or > or =10 axillary nodal metastasis, and 20% with inflammatory breast cancer. The median age was 46 years (26-62 years) and the median number of involved axillary lymph nodes was 12 (0-42). At a median follow-up of 86 months, relapse-free survival (RFS) and overall survival for the entire group were 50% (95% CI 43% to 57%) and 62% (95% CI 56% to 69%). Multivariate Cox stepwise analysis resulted in a simple model for RFS consisting only of p21 expression, EGFR expression assessed by RT-PCR, and number of axillary nodal metastases. CONCLUSION: A prognostic model on the basis of the expression of a limited number of proteins and genes may help to guide target-specific therapies in patients with high-risk breast cancer.

Adenocarcinoma developing in de novo Barrett's mucosa in the remnant esophagus after esophagectomy: clinical and molecular assessment.

For many patients after subtotal esophagectomy and gastric pull-up, reflux of gastric contents to the esophageal stump is the leading clinical problem. Besides symptoms such as heartburn and regurgitation, de novo formation of columnar mucosa in the esophageal remnant is a well-known and frequent phenomenon. In this context, the remnant supra-anastomotic esophagus serves as an in vivo model for the study of Barrett's carcinogenesis. We present a retrospective case analysis of a patient who developed de novo Barrett's metaplasia followed by de novo invasive carcinoma 28 months after gastric pull-up by assessing clinical and molecular parameters.

Predictors of response and survival for neoadjuvant treated patients with esophageal adenocarcinoma.

Mainly patients with advanced esophageal adenocarcinoma who respond to neoadjuvant chemotherapy show a significant survival benefit after resection. Therefore, prediction of response before treatment is desirable. The aim of this study was to assess genetic predictors of response and survival for patients with esophageal adenocarcinoma prior to neoadjuvant therapy. Thirty-two patients with advanced esophageal adenocarcinoma who underwent neoadjuvant therapy with resection of their tumor were analyzed for thymidylate synthase (TS), excision repair cross complementing (ERCC1) and Gluthatione S-transferase (GSTP-1) mRNA levels prior to the treatment. These results were analyzed in regards of response and survival. In total, 18 patients responded to this protocol. Seventeen of those did show a gene expression level at or below the respective median of at least one gene. This had a profound impact on survival, demonstrating an increase in survival for patients who have TS, ERCC1, or GSTP-1 mRNA level at or below the median. These results demonstrate a potential predictive value of a gene expression profile available prior to therapy. These data have to be confirmed by a larger prospective trial.

Plasma DNA: a molecular marker of surgical insult and postoperative recovery in esophageal cancer.

AIM: To assess plasma DNA changes intraoperatively, to relate plasma DNA to the magnitude of the surgical insult and to monitor the changes during the postoperative recovery period. MATERIAL AND METHOD: Prospective study of 35 patients with esophageal cancer who had esophagectomy of different magnitudes: 19 esophagectomy without thoracotomy and 16 esophagectomy with thoracotomy. The plasma DNA was measured prior to surgery, throughout the course of the operation on four different intervals, and on postoperative days 1, 3, 5, and 7. RESULTS: A significant difference was seen in the median plasma DNA intraoperatively between the two groups: esophagectomy without thoracotomy, 507 ng/ml/min (range 211-2,708), esophagectomy with thoracotomy, median 1,098 ng/ml/min (range 295-22,284; p = 0.014). Postoperative complications were identified in 6 patients who demonstrated a significant elevation in plasma DNA on postoperative days 5 and 7. CONCLUSION: Plasma DNA increases during surgery as a result of cell damage and the rise correlates with the magnitude of surgery. The descent of plasma DNA postoperatively correlates with surgical recovery. Elevation of the plasma DNA during the postoperative period correlates with postoperative complications. Plasma DNA is an objective molecular marker of surgical insult and can be used to monitor postoperative recovery after esophagectomy.

Molecular factors of 5-fluorouracil metabolism in colorectal cancer: analysis of primary tumor and lymph node metastasis.

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) are predictive markers for tumor response to 5-fluorouracil-based therapies. To determine whether gene expression values measured in primary cancer tissue would be useful for prediction of response of lymph node metastases, the expressions of these genes were quantitatively analyzed in 35 pairs of primary colorectal cancer (CRC) and corresponding lymph node metastases using real-time PCR. DPD and TP mRNA levels were significantly lower in the primary colorectal tumor and lymph node metastases compared with the normal adjacent stroma tissue (p<0.01), whereas TS mRNA levels were significantly higher in the primary tumor and lymph node metastases than in the normal adjacent tissue (p<0.001). Median gene expression levels of TP and TS did not differ significantly between primary colorectal tumor and corresponding lymph node metastasis but median DPD gene expression levels in the lymph node metastases were significantly higher compared to matched primary colorectal tumors (p=0.015). There was a significant correlation for DPD, TP and TS gene expression levels between primary colorectal tumor specimens and the matched lymph node metastasis. These results suggest that biopsies of the tumor of origin may be valid for determining predictive markers for chemotherapy response in patients with metastatic CRC.

MethyLight: a high-throughput assay to measure DNA methylation.

Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.

Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma.

BACKGROUND: The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. METHODS: We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P: value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. RESULTS: Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P =.016). CONCLUSION: The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.

Effect of 5-fluorouracil substitution on the self-splicing activity of Tetrahymena ribosomal RNA.

Recent studies from several laboratories have suggested that the anticancer drug-5-fluorouracil (FUra) promotes abnormal splicing of precursor RNA molecules. In order to determine the effects of FUra on the chemistry of RNA splicing, we studied the splicing reaction of FUra-containing Tetrahymena rRNA [(FUra) RNA], a Group I self-splicing system having one intron [intervening sequence (IVS)] and two exons. When subjected to splicing conditions, the (FUra) precursor RNA gave all of the normal splicing products, ligated exons, IVS, circulation IVS (C-IVS), and the hydrolyzed circle (L-19 IVS) as well as other hydrolysis side products. No abnormal products indicative of missplicing were observed at pH 7.5. However, the presence of FUra in the RNA decreased the rates and extents of formation of all of the product species. At pH 7.5, the rate of ligated exon formation of (FUra) RNA was inhibited 3-fold and the maximum yield of ligated exons was 50% of normal. Substitution with FUra inhibited the rate of formation of C-IVS about 2-fold, while the extent of formation of this product was decreased by more than 3-fold compared to uracil-containing RNA [(Ura) RNA]. The circularization of (Ura) IVS remained constant to pH 9 and then increased, while that of (FUra) IVS declined abruptly after pH 7.3, indicating that ionization of the FUra residues of (FUra) RNA abolishes its catalytic activity. A temperature dependence experiment showed that the circularization activity of (FUra) IVS was lost at a temperature 15 degrees C lower than that of the (Ura) IVS. The labile phosphodiester bond of the (FUra) C-IVS was more stable to hydrolysis than was that of the (Ura) C-IVS at all pH values. The data suggest that a major effect of FUra substitution is to destabilize the active conformation of RNA because of weaker base pairing between FUra and adenine owing to partial ionization of the FUra residues.

Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes.

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Inhibition of cyclin D1 expression has been recently shown (M. Kornmann, et al., J. Clin. Invest, 101: 344-352, 1998) to suppress pancreatic cancer cell growth and increase cytotoxic actions of cisplatinum. The aim of the present study was to determine whether inhibition of cyclin D1 expression also modulates the effects of other antineoplastic drugs and whether it is associated with alterations in the level of expression of drug resistance genes. The suppression of cyclin D1 expression after the stable transfection of a cyclin D1 antisense construct in PANC-1 and COLO-357 human pancreatic cancer cells resulted in a significant increase in sensitivity to the fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine and to mitoxantrone. All of the antisense-expressing dones exhibited a decrease in thymidylate synthase and an increase in thymidine phosphorylase mRNA expression as determined by reverse transcription-PCR analysis and decreased levels of MDR-1 and MRP mRNA as determined by Northern blotting. These findings demonstrate that the inhibition of cyclin D1, in addition to suppressing the growth of pancreatic cancer cells, enhances their responsiveness to multiple chemotherapeutic agents and suggest that this effect may be due to the altered expression of several chemoresistance genes.

CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.

The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.

Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors.

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). We have correlated TS protein and gene expression with the response in patients with colorectal (n = 9) and gastric cancer (n = 12) treated with infusional 5-FU plus leucovorin (LV) or infusional 5-FU/LV and cisplatin, respectively. TS protein expression was analyzed by Western blot using TS106 monoclonal antibody and densitometry scanning. TS gene expression was measured by PCR analysis using beta-actin as an internal standard and expressed as a TS:beta-actin mRNA ratio. A close linear relationship was noted between TS protein expression and TS gene expression (r2 = 0.60) for the 21 tumor samples analyzed. TS immunohistochemical staining on 15 of the 21 samples revealed that the TS staining intensity correlated closely with TS protein and mRNA expression. In two biopsy samples, TS protein levels and TS gene expression did not correlate; however, one of these exhibited a focal TS staining pattern. Both the TS protein level and TS gene expression were significantly associated with response to 5-FU-based therapy. Patients with responsive disease had a mean TS protein level of 0.17 +/- 0.03 arbitrary units (range, 0.05 to 0.38), whereas in patients whose tumors did not respond, the mean TS protein level was significantly higher 0.60 +/- 0.09 (range, 0.06 to 1.01; P < 0.01). A similar pattern was noted with TS gene expression. In patients with responsive disease, the mean TS:beta-actin gene ratio was 1.36 +/- 0.3 (range, 0.5-3.3 x 10(-3). In contrast, biopsies from patients with unresponsive disease had a mean TS:beta-actin gene ratio of 15.4 +/- 2.6 x 10(-3) (range, 2.7-35.9; P < 0.01). TS protein and TS mRNA expression are highly correlated, and each predict for response to 5-FU/LV-based chemotherapy in patients with colorectal and gastric cancer.

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.

NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity.

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0-3447 nmol/min/mg protein), as measured by the reduction of dichlorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 +/- 144 nmol/min/mg protein, mean +/- SD) and are sensitive to MMC, showed high NQO1 expression relative to beta-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 +/- 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 +/- 6 and 36 +/- 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.