Danenberg Tumor Profile Method Research Articles

Thymidine phosphorylase affects clinical outcome following surgery and mRNA expression levels of four key enzymes for 5‑fluorouracil metabolism in patients with stage�I and II non‑small cell lung cancer

The expression levels of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil-based chemotherapy in patients with cancer. We herein investigated the differences in the mRNA levels of these enzymes in non-small-cell lung cancer (NSCLC) and evaluated their prognostic value for NSCLC treated by surgical resection. The intratumoral mRNA levels of TP, DPD, TS, and OPRT were quantified in 66 patients with pathological stage I and II NSCLC (adenocarcinoma or squamous cell carcinoma) following complete resection according to the Danenberg Tumor Profile method. The TP level was the only significant prognostic factor for disease-specific survival (DSS) following complete resection; the mean TP mRNA level differed significantly between the high and low mRNA expression groups. The DSS at 5 years was significantly higher in the low TP mRNA compared with that in the high TP mRNA expression group (83.4 vs. 58.6%, respectively; P=0.005). A Cox proportional hazards model revealed that pathological stage, sex, and TP expression were independent prognostic factors for DSS in patients with stage I and II NSCLC following complete resection. Thus, TP level may be used to monitor treatment efficacy and predict the outcome of NSCLC patients.

Prognostic value of biomarkers in metastatic colorectal cancer patients

BackgroundsThe prognostic value of biomarkers in metastatic colorectal cancer (mCRC) patients with liver metastases remains unclear. We assessed the difference of expression of biomarkers between primary tumors and liver metastases treated with chemotherapy in mCRC patients, as well as the prognostic value of these markers. MethodsForty-three mCRC patients with liver-limited disease from January 2007–November 2011 were analyzed. They all received resection of primary tumors followed by oxaliplatin-based chemotherapy. After chemotherapy, they all received hepatic resection. Forty-three paired primary and metastatic tumor specimens were collected to measure the messenger RNA expression of six biomarkers by the Danenberg tumor profile method (thymidylate synthase, dihydropyrimidine dehydrogenase [DPD], excision repair cross-complementing gene1, thymidine phosphorylase [TP], folylpolyglutamate synthase, and regenerating islet-derived family, member 4). ResultsThirty-six patients' messenger RNA was used for analysis. All markers showed similar expression between primary and metastatic sites. The low-expression group of Danenberg tumor profile and TP in the primary tumor showed significantly higher overall survival than the high-expression group (P < 0.001 and P = 0.033), but for DPD and TP in liver metastases, there were no significant differences of overall survival between the two groups. The ratios of marker expression in liver metastatic site to that in primary site of DPD and TP were significantly higher in chemo-responders than in non-chemo-responders (P = 0.034 and P = 0.022). ConclusionsBiomarkers' expressions in liver metastases were similar to those in the primary tumor. DPD and TP in the primary lesion may be a prognostic factor in chemotherapy-naïve mCRC patients with liver-limited disease, but those in liver tumor were not. Further validated analysis to our results would be warranted.

Expression level of dihydropyrimidine dehydrogenase is associated with clinical outcome in patients with T1G3 bladder cancer treated with Bacillus Calmette-Guerin

BackgroundRecently, it has been shown that 5-fluorouracil (5-FU) with a strong dihydropyrimidine dehydrogenase (DPD) inhibitor elicits a significant response in bladder cancer with a high level of DPD. However, only a few studies investigated the association between the level of the enzyme that regulates the metabolism of 5-FU and prognosis in bladder cancer. Furthermore, to our knowledge, there has also been no such report in T1G3 bladder tumors treated with BCG. Therefore, we evaluated enzymes that regulate the metabolism of 5-FU in T1G3 tumors treated with BCG immunotherapy using the Danenberg tumor profile (DTP) method, a highly accurate measurement of RNA from paraffin-embedded specimens.MethodsThis study included 28 patients with T1G3 bladder cancer, each of whom underwent complete transurethral tumor resection and BCG intravesical instillation at our institution. The median follow-up period was 39 months (range, 3 to 159 months). The DTP method was used to analyze the mRNA expression of 3 enzymes related to 5-FU: DPD, orotate phosphoribosyltransferase (OPRT), and thymidylate synthase (TS).ResultsAmong the 28 patients, 13 developed recurrences (46.4%) and 5 experienced disease progression (17.9%). An elevated DPD mRNA level was significantly associated with recurrence (p = 0.048) and progression (p = 0.045). However, TS and OPRT mRNA levels were not significantly associated with any other clinical features or outcomes. Furthermore, the high DPD group had a significantly lower recurrence-free survival rate than the low DPD group (p = 0.047). Among patients with low DPD, the 2- and 5-year recurrence-free survival rates were 88.9% and 74.1%, respectively; while among patients with high DPD, the corresponding rates were 61.3% and 36.8%, respectively. TS and OPRT were not significantly associated with recurrence-free survival rates.ConclusionDPD is significantly associated with recurrence and progression among T1G3 bladder cancer patients treated with BCG.

Evaluation of 5-fluorouracil metabolic enzymes as predictors of response to adjuvant chemotherapy outcomes in patients with stage II/III colorectal cancer: a decision-curve analysis.

The effectiveness of 5-fluorouracil (5-FU)-based adjuvant chemotherapy is reported in patients with colorectal cancer (CRC), but the usefulness of 5-FU metabolic enzymes as predictive biomarkers of the efficacy of this chemotherapy remains unclear. This study aims to verify whether 5-FU metabolic enzymes are predictive biomarkers in the clinical setting of adjuvant chemotherapy for stage II/III CRC. In total, 179 patients with stage II/III CRC who were treated at our institute between 2000 and 2010 were enrolled. Messenger RNA (mRNA) expression of major 5-FU metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase (TP), orotate phosphoribosyl transferase, and β-actin (control) was evaluated using the Danenberg Tumor Profile method. mRNA expression and other clinicopathological data were investigated with regard to CRC relapse. A total of 78 patients underwent surgery alone, while 101 underwent adjuvant chemotherapy (5-FU plus leucovorin [LV] or tegafur plus uracil /LV) following surgery. Relapse-free survival was longer and risk of recurrence was lower in association with high TP mRNA expression than in association with low TP mRNA expression in the adjuvant chemotherapy group (hazard ratio 0.66; 95 % confidence interval 0.47-0.92; p = 0.016), but not in the surgery alone group. mRNA expression of no other enzymes was associated with relapse in both groups. In decision-curve analyses, the predictive efficiency of TP mRNA expression plus clinicopathological factors was slightly better than that of clinicopathological factors only. TP mRNA expression in tumors predicted the effects of adjuvant chemotherapy for stage II/III CRC, although the beneficial effects were marginal.

Analysis of the mRNA expression of chemotherapy-related genes in colorectal carcinoma using the danenberg tumor profile method.

The establishment of individualized chemotherapy for colorectal carcinoma based on the expression of genes involved in chemotherapeutic sensitivity or prognosis is necessary. To achieve this, the expression profiles of genes within tumors and their relationship to clinicopathological factors must be elucidated. Here, we selected 10 genes (TS, DPD, TP, FPGS, GGH, DHFR, ERCC1, TOPO-1, VEGF, and EGFR), examined differences in their mRNA expression between the upper and lower thirds of tumors by laser-captured microdissection and real-time RT-PCR (the Danenberg tumor profile), and analyzed the relationships between their expression profiles and clinicopathological factors. Interestingly, the mRNA expression of DPD, TP, and VEGF was significantly higher in the lower third than in the upper third of tumors (P = 0.044, 0.023, and 0.013, resp.). Furthermore, increased ERCC1 mRNA expression in the lower third of tumors correlated with recurrence (P = 0.049), and VEGF mRNA expression was significantly higher in cases with recurrence than in cases without recurrence, both in the upper and lower thirds of tumors (P = 0.018 and 0.036, resp.). These results implied that heterogeneity in DPD, TP, and VEGF expression may exist in colorectal carcinoma and that ERCC-1 and VEGF may be markers predicting recurrence after curative operation.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Biopsy for custom-made treatment: mRNA expression level of cancer-critical genes from gastric/colorectal cancer tissues obtained by endoscopic biopsy.

33 Background: In this study, we measured the mRNA expression level of cancer-critical genes from the gastric/colorectal cancer tissues obtained through endoscopic biopsy before treatments and compared its consistency with the sample tissues surgically resected from identical cases. Methods: The study was made on 13 gastric and 19 colorectal cancer cases with patients’ consent. We picked identical cases and measured mRNA expression levels from the tissues endoscopically taken before treatment and the surgically resected ones. For the measurement, DNP (DanenbergTumorProfile) method was used. Examination items are as follows: TS, DPD, TP, FPGS, GGH, DHFR, ERCC1, Topo-I, EGFR, and VEGF. Results: Upon comparing the consistency between endoscopically sampled biopsy tissues and surgically taken tissues from identical cases, it was found that eight out of ten items showed strong correlations in colorectal cancer cases. The results are as follows: FPGS(r=0.91, p&lt;0.001); GGH(r=0.87, p&lt;0.001); EGFR(r=0.86, p&lt;0.001); Topo I (r=0.81, p&lt;0.001); TS(r=0.79, p&lt;0.001); DHFR(r=0.70, p&lt;0.01); VEGF(r=0.67, p&lt;0.01); and TP(r=0.62, p&lt;0.05). In case of gastric cancers, strong correlations were found in three out of ten items with the following results: EGFR (r=0.98, p&lt;0.001); TS (r=0.91, p&lt;0.001; and DPD (r=0.74, p&lt;0.05). Conclusions: Today’s progresses of preoperative chemotherapy and radiotherapy and developments of endoscopic and surgical treatments allow diverse options for treatments. In such circumstances, the significance of knowing the expression of cancer-critical genes between individuals before treatment in conducting custom-made treatment is profound. There are two issues in applying the expression of cancer-critical genes found through endoscopic biopsy: one is whether enough cancer cells can be obtained through biopsy, and the other is whether the sampled cancer cells reflect the characteristics of primary focus. While there remain issues to be addressed, certain results were achieved in this study. Currently, we are working on accumulating cases to compare them and find out whether the results can be applied to custom-made treatments.

Role of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in gallbladder carcinoma

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are important enzymes in the metabolism of 5-fluorouracil, which have been examined as possible predictive markers. We conducted this study to clarify the role of TS and DPD expressions in gallbladder carcinoma (GBC). The subjects were 28 patients who underwent surgical resection of GBC. We examined intratumoral TS and DPD mRNA expressions, using the Danenberg tumor profile method. The expression levels were classified into two groups, based on median values. Clinicopathological variables, including prognosis, were then compared between the high and low expression groups. There was a significant difference in the incidence of lymph node metastasis between the high and low TS expression groups. The incidence of advanced clinical stage was higher in the low TS expression group than in the high TS expression group. However, no clear correlation was observed between the DPD mRNA expression and any clinicopathological variable. There was no significant difference in the postoperative survival rates between the groups, in accordance with the expression of TS or DPD genes. Low TS mRNA was correlated with a high incidence of lymph node metastasis and advanced clinical stage. Therefore, TS gene expression may help identify patients at increased risk of the progression of GBC.

Role of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA in intrahepatic cholangiocarcinoma

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are important enzymes in the metabolism of 5-fluorouracil and possible predictive markers. We conducted this study to clarify if TS and DPD gene expressions are a prognostic indicator for intrahepatic cholangiocarcinoma (IHCC). The subjects of this study were 21 patients with IHCC who had undergone surgical resection. Intratumoral TS and DPD mRNA expressions were examined using the Danenberg tumor profile method and classified into two groups according to the median value of each. We then compared the clinicopathological variables, including prognosis, between the high and low expression groups. Low DPD expression was correlated with macroscopic type, namely, mass-forming + infiltrative (P = 0.08). Postoperative survival rates in the low DPD expression group were significantly lower than those in the high DPD expression group. Multivariate analysis revealed macroscopic type to be an independent prognostic factor, whereas TS mRNA expression did not correlate with any clinicopathological variables, including prognosis. Low DPD mRNA expression was related to macroscopic type and associated with poor prognosis. DPD mRNA expression in tumor cells is suggested to be an important regulator of malignant behavior in IHCC.

A retrospective study on TS mRNA expression and prediction of the effects of adjuvant oral 5-fluorouracil in breast cancer

Nucleic acid-metabolizing enzymes, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have attracted attention as candidates for response determinants of 5-fluorouracil (5-FU). Whether the expression levels of these enzymes can be adopted as valuable parameters for 5-FU sensitivity in breast cancer has yet to be elucidated. In the present study, intratumoral mRNA expression of TS, DPD, TP and OPRT were determined in formalin-fixed paraffin-embedded surgical specimens collected from 217 breast cancer patients, using the Danenberg Tumor Profile method, which combines microdissection and real-time-polymerase chain reaction. The significance of these enzymes as prognostic and 5-FU efficacy-predicting factors was evaluated. Our data showed that a low DPD expression is related to a high nuclear grade and other factors including hormone receptor-negativity. Low expression levels of TP were found in hormone receptor-negative tumors. TS and OPRT expression were not related to various clinicopathological factors, but patients with a high TS mRNA expression showed a significantly poorer prognosis in cases where 5-FU was not administered. The efficacy of 5-FU was more significant when administered for more than 6 months in the group with a high TS mRNA expression. These data suggest that TS mRNA expression in breast cancer tissue is an ideal predictor of outcomes for patients with no administration of 5-FU, and of the efficacy of 5-FU.

Role of tumor angiogenesis in gallbladder carcinoma: with special reference to thymidine phosphorylase

Angiogenesis plays an important role in tumor growth and metastasis. Thymidine phosphorylase (TP), as well as vascular endothelial growth factor (VEGF), is known to be an angiogenic factor. The aim of this study was to clarify whether TP regulates angiogenesis and to determine the clinicopathological role of TP expression in gallbladder cancer. Thirty-seven patients with gallbladder cancer who underwent curative surgical resection were included in this study, and we evaluated results in 21 of these patients in whom TP gene expression could be measured. Intratumoral TP gene expression was evaluated using the Danenberg tumor profile method. TP gene expression was classified into two groups according to median values: high and low. Tissue TP expression was classified into two groups (positive and negative) by a pathologist. Clinicopathological variables were compared between groups with high and low TP gene expression (TP high and TP low groups). No correlation was observed between TP gene expression (high and low groups) and any clinicopathological variables except survival rate. However, the survival rate was significantly lower in the TP high group (P<0.05). TP gene expression was associated with tissue TP expression evaluated by immunohistochemical staining (P<0.05). In addition, tumor microvessel density was significantly higher in the TP high group (P<0.05). Expression of the TP gene in gallbladder carcinoma is associated with angiogenesis and may be a new independent prognostic parameter.

Thymidylate Synthase, Thymidine Phosphorylase and Orotate Phosphoribosyl Transferase Levels as Predictive Factors of Chemotherapy in Oral Squamous Cell Carcinoma

We conducted a clinicopathologic study on protein and mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) using biopsy tissue specimens before treatment. The mRNA levels have been measured in tumor cells microdissected from paraffin-embedded specimens (Danenberg Tumor Profile method: DTP method). We studied the mRNA and protein expression as effect predictive factors in chemotherapy. The subjects consisted of 20 cases of untreated oral squamous cell carcinoma who had undergone chemotherapy with TS-1 (16 males and 4 females, tongue in 8 cases, upper gingiva in 3 cases, lower gingiva in 3 cases, buccal mucosa in 5 cases and floor of the mouth in 1 case). TS gene expressions of the responders were lower than those for the nonresponders. Furthermore, regarding males who were less than 70 years of age, stage I and II, well differentiated type and tongue, TS mRNA expression of the responders were lower than that for the nonresponders. The mRNA expression of OPRT for the male responders was lower than that for the nonresponders. No remarkable difference was observed by immunohistochemistry. In this study, the measurement of the TS levels using the DTP method may potentially act as a predictive factor of antitumor effectiveness.

Gene Expression of 5-Fluorouracil Metabolic Enzymes in Head and Neck Squamous Cell Carcinomas

To measure the gene expression of the metabolic enzymes of 5-fluorouracil (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase) to assess the efficacy of 5-fluorouracil in patients with head and neck cancer. Tissue specimens of 64 patients with head and neck cancer (41 men and 23 women with a mean age of 62.6 years) who had been surgically treated from April 1997 to March 2004 were included in this study. The mRNA expressions of thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase genes were examined from the surgically resected tissues using the Danenberg tumor profile method. The mRNA levels of the 4 enzymes were compared between tumor tissue and adjacent normal tissue. We have employed the DTP method for the preparation of specimens. There are few institutions in which cancer section specimens are frozen and stored. The DTP method employed in this study is quite simple and easy because it utilizes paraffin block specimens, which are commonly created and maintained at institutions. Measurement by assays, including radioenzymatic assays, provides information regarding the state of the entire body from which the tissue specimen was taken. Therefore, the influence from interstitial cells and immunocytes should be considered. The DTP method allows for the selection of the exact cells needed for specimen preparation through the laser capture microdissection method. By pinpointing the cancer tissues and analyzing them, the DTP method can substantially minimize the influence from non-cancerous cells and yield reliable data. n/a All specimens showed that thymidylate synthase, thymidine phosphorylase, and orotate phosphoribosyltransferase levels were significantly higher in the primary tumors than in the adjacent normal tissues, while the levels of dihydropyrimidine dehydrogenase were significantly lower in the primary tumors than in the adjacent normal tissues. Prediction of the efficacy of 5-fluorouracil therapy in the treatment of head and neck cancer from the measurement of mRNA levels of its metabolic enzymes appears to be feasible and possibly beneficial.

Evaluation of the expression of pyrimidine metabolic enzyme genes in colorectal cancer cells using the Danenberg tumor profile method

目的: 近年, Danenbergらによりホルマリン固定パラフィン包埋切片から特定の細胞を採取し, ピリミジン代謝酵素のmRNA発現量を測定するDanenberg Tumor Profile法が開発された. 今回この方法を用いて腫瘍細胞, 正常細胞におけるピリミジン代謝酵素のmRNA発現量の計測, 検討を行った. 対象および方法: 大腸癌33症例の原発腫瘍組織および近傍の正常組織のパラフィン包埋切片から腫瘍および正常細胞のみを採取し, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphprylase (TP) のmRNA量をreal time reverse transcript-polymerase chain reaction法にて定量した. 結果: 各酵素のmRNAの発現量について, DPDは正常細胞に比べ腫瘍細胞で有意に低く (p<0.001), 一方OPRTは腫瘍細胞で有意に高かった (p<0.001). 各酵素のmRNA発現量について, 腫瘍細胞, 正常細胞間で比較したところ, いずれの酵素についても両細胞間で有意な相関は認めなられかった. 一方, 酵素間での発現量を比較したところ, DPDとTP間およびTSとOPRT間に, 腫瘍細胞と正常細胞の両者で有意な正の相関を認めた. 結論: 5-fluorouracil (5-FU) の分解を担うDPDのmRNAが腫瘍細胞で有意に低く, 5-FUの活性化のために重要なOPRTのmRNAが腫瘍細胞で有意に高いことは, 5-FUが腫瘍に選択的に作用することを示唆している. 今後Danenberg Tumor Profile法を用いて, 腫瘍組織において腫瘍細胞と間質細胞を区別してピリミジン代謝酵素を測定することは, 5-FUの効果予測にとって臨床的に意義あることと考えられる.