Switching from FOLFIRI plus cetuximab to FOLFIRI plus bevacizumab based on early tumor shrinkage in RAS wild-type metastatic colorectal cancer: A phase II trial (HYBRID).

BACKGROUND: Long-term anti-EGFR antibody treatment increases the risk of severe dermatologic toxicities. This single-arm, phase II trial aimed to investigate the strategy of switching from cetuximab to bevacizumab in combination with FOLFIRI based on early tumor shrinkage (ETS) in patients with RAS wild-type metastatic colorectal cancer (mCRC). METHODS: Radiologic assessment was performed to evaluate ETS, defined as ≥20% reduction in the sum of the largest diameters of target lesions 8 weeks after the introduction of FOLFIRI plus cetuximab. ETS-negative patients switched to FOLFIRI plus bevacizumab, whereas ETS-positive patients continued FOLFIRI plus cetuximab for eight more weeks, with a switch to FOLFIRI plus bevacizumab thereafter. The primary endpoint was progression-free survival. RESULTS: This trial was prematurely terminated due to poor accrual after a total enrollment of 30 patients. In 29 eligible patients, 7 were ETS-negative and 22 were ETS-positive. Two ETS-negative patients and 17 ETS-positive patients switched to FOLFIRI plus bevacizumab 8 weeks and 16 weeks after initial FOLFIRI plus cetuximab, respectively. Median progression-free and overall survival durations were 13.4 and 34.7 months, respectively. Six (20%) patients experienced grade ≥3 paronychia, which improved to grade ≤2 by 18 weeks. Grade ≥3 acneiform rash, dry skin, and pruritus were not observed in any patients. CONCLUSIONS: Our novel treatment strategy delivered acceptable survival outcomes and reduced severe dermatologic toxicities.

A microdosing approach for characterizing formation and repair of carboplatin-DNA monoadducts and chemoresistance.

Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt-DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [(14)C]carboplatin-DNA monoadducts at the attomole level, which are the precursors to Pt-DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [(14)C]Carboplatin "microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and -proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin-DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo.

The relationship between proangiogenic gene expression levels in prostate cancer and their prognostic value for clinical outcomes.

BACKGROUND: Androgens stimulate the expression of vascular endothelial growth factor (VEGF) through activation of hypoxia inducible factor (HIF). These genes play a major role in cancer angiogenesis. This study assesses the relationship among expression levels for the androgen receptor (AR), HIF1a, VEGF-A, and VEGF-C genes in human prostate cancer tissue and their impact on prostate cancer outcomes. It also examines the impact of pre-operative androgen deprivation therapy (ADT) on the expression of these genes. METHODS: Radical prostatectomy specimens were obtained from 138 patients with D1 prostate cancer from the University of Southern California prostatectomy database; 30% received pre-operative and 23% received post-operative ADT. Gene expression levels were determined by quantitative real-time PCR. Specimens were stratified into three groups for each gene based on expression levels, and groups were compared for clinical outcomes (PSA and clinical recurrence, overall survival). RESULTS: There was a significant correlation in expression levels amongst all genes. Patients treated with pre-operative ADT had significantly lower HIF1a expression, mean 2.64 (CI 2.34-2.94) than patients not treated, mean 3.25 (CI 2.97-3.53, P = 0.006), adjusting for age, PSA, Gleason score, and stage. Higher VEGF-A expression was significantly associated with better overall survival (HR 0.49, P = 0.015). The risk of developing clinical recurrence was significantly lower with higher VEGF-C expression (HR 0.4, P = 0.014). CONCLUSIONS: Significant correlation was noted among AR, HIF1a, VEGF-A, and VEGF-C. This study shows that ADT is associated with lower HIF1a gene expression in human prostate cancer tissue and documents prognostic value for VEGF-A and VEGF-C expression levels.

Gene profiling and pathway analysis of neuroendocrine transdifferentiated prostate cancer cells.

BACKGROUND: Neuroendocrine (NE) cells are present in both normal prostate and prostate cancer. In addition, NE differentiation can be induced by various factors, such as IL-6, in vitro and in vivo. However, the mechanism of this differentiation and the role of NE cells in prostate cancer are not well understood. In this study, we evaluated the gene expression and analyzed the pathways in prostate cancer cells exposed to various NE differentiation inducing factors in vitro. METHODS: Gene expression signatures between control LNCaP cells and each treatment induced NE cell line were compared using Affymetrix GeneChip with network and pathway analysis. RESULTS: All treatments were able to transdifferentiate LNCaP cells into NE phenotype as shown by morphology changes and NE marker measurements. Of the 54,675 oligonucleotide-based probe sets in microarray, 44,975 were mapped into the Ingenuity Pathway Analysis database and were filtered according to the t-test P value. At P < 0.002, the number of genes that were differentially expressed included 302 of the IL-6 treated cells, 201 of genistein, 233 of epinephrine, and 191 of the charcoal stripped serum ones. A pooled data approach also showed 346 differentially expressed genes at the same P value. Gene ontology analysis showed that cancer-related function had the highest significance. CONCLUSIONS: Despite some overlap, each NE transdifferentiation inducing treatment was associated with a changed expression of a unique set of genes, and such gene profiling may help to elucidate the molecular mechanisms involved in NE transdifferentiation of prostate cancer cells.

Messenger RNA expression of COX-2 and angiogenic factors in primary colorectal cancer and corresponding liver metastasis.

Several new drugs that are targeted towards various angiogenic factors have shown considerable potential for controlling tumor proliferation and metastases. Expression levels of the targeted genes in primary tumors and metastases should be understood to maximize the use of such drugs. The present study aimed to clarify associations between mRNA levels of cyclooxygenase 2 (COX-2) and angiogenic factors [vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8)] in primary colorectal cancer and in corresponding liver metastasis. We also compared these gene expressions of primary colorectal cancer between patients with and without liver metastasis. In 31 pairs of formalin-fixed and paraffin-embedded primary and metastatic liver tumors as well as 27 specimens of consecutive stage II patients without recurrence, mRNA was quantified by real-time reverse transcription-polymerase chain reaction following the laser capture microdissection. We found a significantly positive correlation in IL-8 between primary tumors and matched liver metastases (p=0.034, rs=0.39) and in VEGF (p=0.0083, rs=0.48), but not in COX-2, which was associated with both VEGF (p=0.044, rs=0.37) and IL-8 (p=0.0004, rs=0.64) in primary colorectal cancers. Multiple regression analysis revealed that COX-2 was independently associated with IL-8 (p<0.0001). There were no differences in mRNA levels between patients with and without liver metastasis. The mRNA levels of VEGF and IL-8 in liver metastasis can be predicted from those in primary colorectal cancer. COX-2 might exert angiogenic activity more through the IL-8, than the VEGF pathway. These angiogenic factors were sufficiently up-regulated before hematogenous metastasis. These preliminary data merit further validation studies.

Loss of heterozygosity at thymidylate synthase locus in Barrett's metaplasia, dysplasia, and carcinoma sequences.

BACKGROUND: Thymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue. Loss of heterozygosity (LOH) at the TS locus is known to occur in cancer patients, but there is no evidence that it is present in precancerous tissue. The aim of this study was to analyze the frequency and timing of LOH at the TS locus in Barrett-associated adenocarcinoma (BA) and its precursory lesions, such as intestinal metaplasia (IM) and dysplasia. METHODS: One hundred twenty-three samples (including 37 with gastroesophageal reflux disease (GERD), 29 with IM, 13 with dysplasia, and 44 with BA) were obtained from 100 patients. Biopsies were obtained from the lower esophageal mucosa/IM/dysplasia/BA, when available. Normal squamous tissue from the upper esophagus was taken as a control. All tissues were analyzed for the TS genotype and TS mRNA expression using the real-time reverse-transcription polymerase chain reaction (RT-PCR) method after laser-capture microdissection. RESULTS: Among the patients with informative heterozygous genotype in their control samples, no sample with LOH at the TS locus was observed in the lower esophageal mucosa in GERD patients (0/22 samples). However, 6 out of 21 samples (28.6%) had LOH in IM, 2 of 7 (28.6%) in dysplasia, and 10 of 25 (40.0%) in BA. No significant difference in TS mRNA expression levels was observed between TS genotypes. CONCLUSION: Our results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Both beta-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer.

BACKGROUND: Beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been frequently considered as constitutive house keeping genes for RT-PCR and used to normalize changes in specific gene expressions. However, these expressions have been shown to be affected by the sample type and experimental conditions. We investigated which housekeeping gene is useful to study gene expression of paraffin embedded human tissue samples of prostate cancer. METHODS: Fifteen pairs of cancer and corresponding normal tissue were obtained from patients with prostate cancer. We evaluated gene expression of beta-actin, GAPDH, androgen receptor (AR), and heat-shock 70-kd protein 5 (HSPA5) using laser captured microdissection and quantitative RT-PCR. AR and HSPA5 gene expression were normalized to each of these reference genes using the 2(-DeltaDeltaCt) method of relative quantification. The quantity 2(Ct(normal)-Ct(cancer)) divided by ratio of cDNA(cancer)/cDNA (normal) was used for comparing differences between cancer and normal tissue in GAPDH and beta-actin expression. RESULTS: Ct value of beta-actin was significantly correlated with that of GAPDH (r = 0.443, P = 0.014). AR and HSPA5 gene expression levels using beta-actin for normalization were significantly correlated with these gene expression levels using GAPDH (AR; r = 0.689, P = 0.004, HSPA5; r = 0.879, P < 0.001). Both reference genes were expressed more highly in cancer tissue than in normal tissue, with that of GAPDH being significantly different between cancer tissue and normal tissue (P = 0.029). CONCLUSIONS: The good correlation between gene expression values obtained when using beta-actin and GAPDH as reference genes suggests that either gene is a valid denominator for gene expression studies in prostate cancer.

Messenger RNA expression of vascular endothelial growth factor and its receptors in primary colorectal cancer and corresponding liver metastasis.

BACKGROUND: Antiangiogenic therapies have been developed recently in combination with traditional chemotherapy for patients with metastatic colorectal cancer. The aim of this study was to clarify the association between messenger RNA (mRNA) levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFR) in primary colorectal cancer and those in corresponding liver metastasis. METHODS: Thirty-one paired formalin-fixed, paraffin-embedded tumor tissues of colorectal cancer and liver metastasis were dissected by laser capture microdissection. After the mRNA was isolated, a quantitative fluorescent dye real-time reverse transcription-polymerase chain reaction system was used for gene expression measurement. RESULTS: There was a positive correlation between VEGF mRNA levels in primary colorectal cancer and those in matched liver metastasis (P = .0083, r (s) = 0.48). However, there was no association between mRNA levels of VEGFRs in primary tumor and those in liver metastasis. The mRNA levels of VEGF were associated with those of VEGFR-1 (P = .0026, r (s) = 0.39) but not with those of VEGFR-2. The mRNA levels of VEGF were higher than that of either of the VEGFRs (P < .0001). CONCLUSIONS: We can predict VEGF mRNA levels, but not those of VEGFRs, in liver metastasis by measuring those in primary colorectal cancer. The mRNA expression of VEGFRs may be more dependent on the surrounding environment than that of VEGF. These results should be useful for the formulation of antiangiogenic therapies for metastatic colorectal cancer. Further studies will be necessary to validate these preliminary data.

Messenger RNA expression of TS and ERCC1 in colorectal cancer and matched liver metastasis.

5-fluorouracil (5-FU) and oxaliplatin play important roles in chemotherapy for patients with colorectal cancer. The expression levels of thymidylate synthase (TS) and excision repair cross-complementing factor 1 (ERCC1) have been reported to be prognostic markers for patients with 5-FU/oxaliplatin chemotherapy. The aim of this study was to clarify the association between messenger RNA (mRNA) levels of TS and ERCC1 in primary colorectal cancer and those in corresponding liver metastasis. Formalin-fixed paraffin-embedded tumor specimens of 31 patients with resection for both colorectal cancer and liver metastasis were dissected by laser capture microdissection. After RNA extraction, TS and ERCC1 mRNA levels in both primary tumor and corresponding liver metastasis were measured by real-time reverse transcription-polymerase chain reaction. Both TS and ERCC1 mRNA levels in primary tumors were significantly associated with those in synchronous liver metastases (TS, rs=0.875, p=0.0024; ERCC1, rs=0.835, p=0.0038). TS mRNA levels in primary tumors were also associated with those in metachronous liver metastases (rs=0.659, p=0.0065), but not in ERCC1 (rs=0.319, p=0.19). In both genes, mRNA levels in metachronous liver metastases were higher than those in primary tumors (TS, p=0.0084; ERCC1, p=0.037). However, there was no difference in the TS and ERCC1 mRNA levels between primary tumors and synchronous liver metastasis. The measurement of TS and ERCC1 mRNA levels in primary colorectal cancer can predict those in synchronous liver metastases, but not in metachronous ones.

Thymidylate synthase polymorphisms and mRNA expression are independent chemotherapy predictive markers in esophageal adenocarcinoma patients.

Thymidylate synthase (TS) is known to have polymorphisms in the 5' and 3' untranslated region (UTR). These polymorphisms have been reported to be associated with high TS expression and chemoresistance to 5-FU. The aim of this study was to examine the prognostic roles of the 5'-UTR and 3'-UTR TS polymorphisms in esophageal adenocarcinoma patients, as well as their relation with TS mRNA expression. Eighty-three patients with esophageal adenocarcinoma were assessed. Thirty-four had received 5-FU containing chemotherapy and 49 were treated with surgery alone. Surgically resected tumor tissues were analyzed for TS genotype and TS mRNA expression using a quantitative real-time RT-PCR method. No survival difference was seen between the patients with 3RG allele (3RG group) and non-3RG group among surgery-alone patients. However, among patients with a history of 5-FU-based chemotherapy, the non-3RG group showed significantly better overall survival compared to the 3RG group (p=0.02). Moreover, whereas chemotherapy produced a significant increase in survival for the non-3RG group patients, those in the 3RG group obtained no survival benefit from chemotherapy. When patients were classified by low or high TS mRNA expression levels, low TS expressers obtained survival benefit from chemotherapy while high TS expressers did not, although there was no difference of median TS mRNA levels between 3RG and non-3RG group. The 3'-UTR polymorphism was not associated with overall survival. These results suggest that the status of the TS 5'-UTR polymorphism and TS mRNA expression are independent predictive markers for survival benefit from 5-FU-based therapy.

Thymidylate synthase, dihydropyrimidine dehydrogenase, ERCC1, and thymidine phosphorylase gene expression in primary and metastatic gastrointestinal adenocarcinoma tissue in patients treated on a phase I trial of oxaliplatin and capecitabine.

BACKGROUND: Over-expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in tumor tissue is associated with insensitivity to 5-fluorouracil (5-FU). Over-expression of ERCC1 correlates with insensitivity to oxaliplatin (OX) therapy, while high thymidine phosphorylase (TP) levels predict for increased sensitivity to capecitabine (Xel). METHODS: Biopsies of metastatic tumor were taken before OX (130 mg/m2 day 1) given with Xel (1200-3000 mg/m2 in two divided doses days 1-5 and 8-12) every 3-weeks. Micro-dissected metastatic and primary tumors were analyzed for relative gene expression by real-time quantitative polymerase chain reaction. The clinical protocol prospectively identified the molecular targets of interest that would be tested. Endpoints for the molecular analyses were correlation of median, first and third quartiles for relative gene expression of each target with response, time to treatment failure (TTF), and survival. RESULTS: Among 91 patients participating in this trial; 97% had colorectal cancer. The median number of prior chemotherapy regimens was 2, and most had prior 5-FU and irinotecan. In paired samples, median mRNA levels were significantly higher in metastatic versus primary tumor (-fold): TS (1.9), DPD (3.8), ERCC1 (2.1) and TP (1.6). A strong positive correlation was noted between DPD and TP mRNA levels in both primary (r = 0.693, p < 0.0005) and metastatic tissue (r = 0.697, p < 0.00001). There was an association between TS gene expression and responsive and stable disease: patients whose intratumoral TS mRNA levels were above the median value had significantly greater risk of early disease progression (43% vs 17%), but this did not translate into a significant difference in TTF. ERCC1 gene expression above the third quartile was associated with a shorter TTF (median 85 vs 162 days, p = 0.046). Patients whose TS mRNA levels in metastatic tumor tissue were below the median had a longer overall survival (median 417 vs 294 days, p = 0.042). CONCLUSION: Target gene expression in primary tumor was significantly lower than that in paired metastatic tissue. High ERCC1 mRNA levels in metastatic tumor was associated with a shorter TTF. Lower expression of TS mRNA correlated with a lower chance of early PD with XelOX therapy and improved overall survival.

Thymidylate synthase, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase mRNA and protein expression levels in solid tumors in large scale population analysis.

It has been reported that the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil (5-FU)-based therapy in cancer patients. We investigated the differences in the mRNA and protein expression of these enzymes in various tumor tissues. A total of 17,613 specimens of head and neck, gastric, colorectal, breast, lung and pancreatic cancer were collected from multiple facilities in Japan, and the mRNA and protein expression levels of the above enzymes were examined in 4,830 and 12,783 of these specimens, respectively. The mRNA levels were analyzed using RT-PCR in laser-captured microdissected formalin-fixed paraffin-embedded specimens, while the protein levels were analyzed by enzyme-linked immunosorbent assays. The median values of the relative TS, DPD and OPRT mRNA levels were 2.06, 0.803 and 1.17, respectively, while the median protein levels were 22.1, 134.8 and 3.81 ng enzyme/mg protein, respectively. The carcinomas were classified into two sets of four groups each using the overall median levels of TS and DPD or TS and OPRT as cutoff values. Approximately 60% of the gastric cancers exhibited elevated mRNA and protein expression levels of DPD, while >65% of the colorectal cancers showed low levels of DPD expression. Overall, 75% of the head and neck cancers exhibited high expression levels of DPD. Among the lung and pancreatic cancers, 50-74% showed low TS/high DPD expression. In conclusion, the mRNA expression and protein levels of TS, DPD and OPRT differed according to the type of cancer. The results of this large-scale population analysis are expected to be useful as reference data for predicting the relationship between the respective enzyme levels and the efficacy of 5-FU-based chemotherapy.

Prognostic value of the androgen receptor and its coactivators in patients with D1 prostate cancer.

BACKGROUND: Prostate cancer treated with androgen ablation eventually becomes resistant. Because the androgen receptor (AR) signaling axis affects disease progression, AR coactivator molecules could provide clinical prognostic value. This study investigates the association between AR coactivator molecules and clinical outcome measures in patients with prostate cancer. PATIENTS AND METHODS: Expression levels of AR and its coactivators, SRC1, TIF2, and Her2/neu were determined by quantitative RT-PCR in 148 prostatectomy specimens. AR protein expression was determined by immunohistochemistry. The prognostic value of these expression levels on clinical outcomes was examined. RESULTS: Increased gene and protein AR expression was not correlated with any of the clinical outcome measures. A non-monotonic correlation was observed between SRC1 and overall survival, as well as Her2/neu and time to prostate-specific PSA recurrence. CONCLUSION: Although no statistically significant relationships were found, the weak association between some clinical outcomes and two AR coactivators may help improve the current predictive nomogram for patients with prostate cancer.

Serum lactate dehydrogenase levels and glycolysis significantly correlate with tumor VEGFA and VEGFR expression in metastatic CRC patients.

OBJECTIVES: In an attempt to elucidate the relationship between biomarkers of tumor hypoxia, glycolysis and angiogenesis, we tested the hypothesis that intratumoral gene expression of the hypoxia response (hypoxia inducible factor [HIF1 alpha and 2 alpha]), glycolysis (lactate dehydrogenase A [LDHA]), glucose metabolism (glucose transporter-1 [Glut-1]) and genes involved in angiogenesis (i.e., VEGFA, VEGFR1-3, and neuropilin [NRP]1) are upregulated in metastatic colorectal cancer (mCRC) patients with high serum lactate dehydrogenase (LDH). PATIENTS AND METHODS: 78 formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from 36 patients with mCRC. Tumor gene expression was correlated with serum LDH levels from the same group of patients. FFPE tissues were dissected using laser-captured microdissection and analyzed for gene expression using a quantitative real-time RT-PCR method. RESULTS: Intratumoral gene expression of VEGFA and VEGFR1 showed a statistically significant correlation with serum LDH levels (p = 0.006, r = 0.45 and p = 0.004, r = 0.50, respectively). Intratumoral expression of LDHA gene showed a significant correlation with Glut-1, VEGF, HIF1 alpha, HIF2 alpha and VEGFR1 (p = 0.007, r = 0.44; p < 0.001, r = 0.57; p = 0.013, r = 0.41; p = 0.044, r = 0.34; p = 0.026, r = 0.40). Serum LDH levels also correlated with microvessel density analyzed by immunohistochemical analysis. CONCLUSION: The results demonstrated a significant correlation between the intratumoral gene expression of LDHA, HIF1 alpha, HIF2 alpha, Glut-1, NRP1, VEGFA and VEGFR1. Patients with high serum LDH have increased intratumoral gene expression of VEGFA and VEGFR1. The results also support the hypothesis that serum LDH levels may serve as a surrogate marker for activation of the HIF-related genes in the tumor.

DPD is a molecular determinant of capecitabine efficacy in colorectal cancer.

Capecitabine is a fluoropyrimidine-based drug that offers physicians a more convenient treatment for advanced colorectal cancer (CRC), with manageable toxicity and antitumor activity comparable to that of continuous-infusion therapies with 5-fluorouracil (5-FU). However, there are no validated and established predictive factors for clinical outcome of capecitabine efficacy in CRC. The gene expressions of the pyrimidine metabolism enzymes dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and thymidylate synthase (TS) have previously been shown to be response determinants of fluoropyrimidine-based drugs in various tumors. Therefore, we investigated whether intratumoral mRNA expression levels of these genes are also associated with the clinical outcome of patients with metastatic CRC treated with first-line capecitabine. Thirty-seven patients with metastatic CRC were enrolled in this study and treated with single agent capecitabine. The intratumoral mRNA levels of DPD, TP and TS were assessed from paraffin-embedded tissue samples using laser-capture-microdissection methods and quantitative real-time PCR. There were 20 women and 17 men with a median age of 61 years (range 49-74). The median progression-free survival was 6.7 months (95% CI, 4.8-11.6 months), with a median follow-up of 14.4 months (range 1.3-18.7 months). Complete response was observed in 1 (3%), partial response in 6 (20%), stable disease in 14 (47%) and progressive disease in 9 (30%) patients (response was inevaluable in 7 patients). Higher gene expression levels of DPD were associated with resistance to capecitabine (P=0.032; Kruskal-Wallis test). Patients with a lower mRNA amount of DPD (

Reduction of interleukin 8 gene expression in reflux esophagitis and Barrett's esophagus with antireflux surgery.

HYPOTHESIS: Chronic inflammation of esophageal mucosa secondary to refluxed gastric juice increases gene expression of interleukin 8 (IL-8). Antireflux surgery can reduce this overexpression. DESIGN: Prospective analysis of archival paraffin-embedded tissue. SETTING: Academic tertiary medical center. PATIENTS AND METHODS: One hundred eight patients with reflux symptoms were classified according to pH monitoring and endoscopic and histologic findings. Twenty patients did not have reflux or mucosal injury; 47 had reflux disease (16 esophagitis and 31 Barrett's esophagus), 20 had dysplasia, and 21 had adenocarcinoma. Microdissection was performed to exclude inflammatory cells and stromal tissue. After RNA isolation and reverse transcription, IL-8 messenger RNA expression was measured using quantitative real-time polymerase chain reaction. All patients with reflux disease had Nissen fundoplication with biopsies at matched levels within the esophagus preoperation and postoperation. RESULTS: Expression of IL-8 was increased in patients with reflux compared with those without reflux. Patients with the highest IL-8 expression were those with Barrett's dysplasia and adenocarcinoma (P<.001). In patients with reflux, Nissen fundoplication led to significantly decreased IL-8 expression compared with preoperative levels in esophagitis (P = .01) and Barrett's esophagus (P = .03). CONCLUSIONS: Interleukin 8 messenger RNA expression increases during the progression of reflux disease from normal squamous mucosa to esophageal adenocarcinoma. Elimination of reflux with Nissen fundoplication significantly reduces IL-8 expression in both squamous and Barrett's mucosa. These results demonstrate that effective antireflux surgery can modulate the gene expression of esophageal mucosa and may impact the natural history of reflux disease.

Plasma DNA as a molecular marker for completeness of resection and recurrent disease in patients with esophageal cancer.

OBJECTIVE: To identify a marker for completeness of resection and recurrent disease in patients with esophageal cancer. DESIGN: Case series. SETTING: Department of Surgery of the University of Southern California. PATIENTS: Forty-four healthy subjects and 45 patients with esophageal cancer prior to esophagectomy. Six patients were unresectable and 39 had a complete resection. MAIN OUTCOME MEASURES: Plasma DNA levels were measured using polymerase chain reaction. Twenty resected patients had follow-up plasma DNA levels measured. RESULTS: Preoperatively, plasma DNA levels exceeded the normal level in 38 (84%) of 45 patients. Preoperatively, 12 patients received neoadjuvant therapy and 11 had plasma DNA levels higher than normal. All 6 unresectable patients had DNA levels higher than normal. At initial follow-up, the plasma DNA levels remained higher than normal in 2 (10%) of 20 patients, and systemic disease was subsequently detected in each. Plasma DNA levels dropped lower than or remained normal in 18 (90%) of 20. In 14 of 18 patients, there was no evidence of recurrent disease at a median of 12 months (range, 3-20 months); in 4 patients, the plasma DNA level rose higher than normal on follow-up and all developed subsequent systemic disease on computed tomographic or positron emission tomographic scan. Six of the 20 patients developed systemic disease during the follow-up (2 had persistently elevated plasma DNA levels, and 4 developed elevated plasma DNA levels at subsequent follow-ups). In 4 of these 6 patients, elevated plasma DNA levels were detected prior to imaging evidence of disease. CONCLUSIONS: Plasma DNA levels are significantly elevated in patients with esophageal cancer and following complete resection should return to normal. Persistently elevated plasma DNA levels after resection or levels that rise on follow-up indicate residual or recurrent disease.

Predictive value of MSH2 gene expression in colorectal cancer treated with capecitabine.

PURPOSE: The objective of the present study was to evaluate the gene expression of the DNA mismatch repair gene MSH2 as a predictive marker in advanced colorectal cancer (CRC) treated with first-line capecitabine. PATIENTS AND METHODS: Microdissection of paraffin-embedded tumor tissue, RNA extraction, and quantitative polymerase chain reaction were performed on tumors obtained from 37 patients with advanced CRC. RESULTS: The median relative gene expression of MSH2 was 0.65 (quartiles 0.5-0.8) in nonresponders and 1.25 (quartiles 0.92-1.38) for responders (P = 0.038). High expression of MSH2 was associated with a hazard ratio of 0.5 (95% confidence interval, 0.23-1.11; P = 0.083) in survival analysis. CONCLUSION: The higher gene expression of MSH2 in responders and the trend for predicting overall survival indicates a predictive value of this marker in the treatment of advanced CRC with capecitabine.

Towards the molecular characterization of disease: comparison of molecular and histological analysis of esophageal epithelia.

Reliable quantification of gene expression offers the possibility of more accurate and prognostically relevant characterization of tissues than potentially subjective interpretations of histopathologists. We measured the expression of 18 selected genes and compared them to histological features in a spectrum of esophageal disease to evaluate the feasibility of molecular characterization of normal and pathologic esophageal epithelia. Esophageal tissue biopsies from 82 patients with foregut symptoms were laser capture microdissected, and the expression levels of 18 selected genes were measured by quantitative real-time polymerase chain reaction. Linear discriminant analysis, which uses combinations of genes to distinguish between histological groups, was performed to compare gene expression and the following five histological groups: (1) normal squamous epithelium (n = 35), (2) reflux esophagitis (n = 13), (3) non-dysplastic Barrett's (n = 33), (4) dysplastic Barrett's (n = 16), (5) adenocarcinoma (n = 31). A panel of seven genes had 90-94% predictive power to distinguish non-dysplastic and dysplastic Barrett's esophagus. Clustering analysis revealed structure in gene expression values even in the absence of histology. Expression levels in 17 genes differed significantly across histological groups. Classification based on gene expression agreed with histopathological assessment in the following percentage of cases: normal squamous epithelium = 53%, reflux esophagitis = 31%, non-dysplastic Barrett's = 76%, dysplastic Barrett's = 40%, and adenocarcinoma = 59%. Interestingly, predictive power improved markedly when inflammatory and dysplastic tissues were removed (77-94%). Gene expression classification agrees well with histopathological examination. When differences occur, it is unclear whether this effect is due to intraobserver variability in pathological diagnosis or to a genuine difference between gene expression and histopathology.

Vascular endothelial growth factor messenger RNA expression level is preserved in liver metastases compared with corresponding primary colorectal cancer.

PURPOSE: Increased vascular endothelial growth factor (VEGF) expression is associated with colorectal cancer liver metastases. It is reasonable to expect that measurement of VEGF in liver metastases would provide the best prediction of therapy benefit for VEGF-targeted drugs, such as bevacizumab (Avastin). In this study, we evaluated how VEGF mRNA level in primary colorectal cancer was related to that in corresponding liver metastases. Thirty-one pairs of primary colorectal cancer and corresponding liver metastases were analyzed. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tumor specimens were dissected by using laser-captured microdissection. RNA was extracted and cDNA was prepared by reverse transcription. Quantitation of VEGF and internal reference gene (beta-actin) was done using real-time PCR (Taqman PCR). RESULTS: There was no difference between median VEGF mRNA levels of primary colorectal cancer and liver metastases (median value 3.79 versus 3.97: P = 0.989). On an individual basis, there was a significant correlation in VEGF mRNA expression between primary colorectal cancer and corresponding liver metastases (r(s) = 0.6627, P < 0.0001). In addition, the VEGF mRNA levels of the patients who had two or more liver metastatic tumors were significantly higher than those of the patient who had solitary liver metastatic tumor in both primary cancer (5.02 versus 3.34: P = 0.0483) and liver metastases (4.38 versus 3.25: P = 0.0358). CONCLUSION: Good prediction of VEGF mRNA levels in liver metastases can be obtained by measuring those of primary colorectal cancer. The risk of multiple liver metastatic tumors might be predictable by measuring VEGF mRNA expression in primary colorectal cancer. Further study is required to confirm these preliminary results.