Hisataka Fujiwara

Evaluation of the ratio of lymph node metastasis as a prognostic factor in patients with gastric cancer

Background. Lymph node metastasis in patients with gastric cancer is one of the important prognostic factors. However, there is no consensus concerning the best classification for lymph node metastasis as a prognostic factor. So, to evaluate the ratio of the number of metastatic lymph nodes to the total number of dissected lymph nodes (the ratio of LN meta) as a prognostic factor, we compared the ratio of LN meta with lymph node status according to the Japan Classification of Gastric Carcinoma and the total number of metastatic lymph nodes with multivariate analysis. Methods. Between 1991 and 1997, a total of 360 patients with primary gastric cancer who underwent gastrectomy with D2 or more extended lymph node dissection were included in this study. Ten kinds of prognostic factors and three types of different classifications for lymph node metastasis were analyzed by multivariate analysis using the Cox regression. Results. The average number of dissected lymph nodes and metastatic lymph nodes were 55.0 (range, 11–184) and 2.6 (range, 0–86), respectively. There were significant differences of the 5-year cumulative survival rates among each group of the ratio of LN meta (0%, 1%–9%, 10%–24%, and more than 25%). Age, tumor size, curability, and the ratio of LN meta were selected as independent prognostic factors by forward stepwise selection. The ratio of LN meta showed the highest hazard ratio by Cox regression. Conclusion. The ratio of LN meta appears to be an important prognostic factor and the best classification factor for lymph node metastasis.

Quantitative Measurement of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase mRNA Level in Gastric Cancer by Real-time RT-PCR

We used real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) to assay expression of the mRNA of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in gastric cancer tissue with the objective of establishing a system to measure TS and DPD in ultra‐low‐volume samples. Nude mouse xenografts of 5 human gastric cancer cell lines and 85 clinical samples were used as the specimens in this study. Sensitivity to 5‐fluorouracil (5‐FU) was determined on the basis of the relative tumor proliferation rate in mice and the results of ATP assay using serum‐free cultures of the clinical samples. mRNA expression was measured in tumor tissue by real‐tune RT‐ PCR using the ABI PRISM 7700 system. The values for expression of the mRNA for TS and DPD were corrected according to the level of glyceraldehyde‐3‐phosphate dehydrogenase mRNA expression. The xenografts yielded correlations between TS and DPD mRNA expression and the activity of the enzymes (TS: rs=0.700, DPD: rs=0.900), and an inverse correlation was noted between the mRNA levels and sensitivity to 5‐FU (TS: rs=‐0.900, DPD: rs=‐0.800). The clinical samples showed an inverse correlation between 5‐FU sensitivity and mRNA expression (TS: rs=‐0.518, DPD: rs=‐0.564). Sensitivity to 5‐FU was noted only in cases in which TS mRNA expression and DPD mRNA expression were both low. Real‐time RT‐PCR can provide a highly sensitive assessment of TS and DPD mRNA expression in gastric cancer, and it was useful for predicting 5‐FU sensitivity.

Superior antitumour activity of S-1 in tumours with a high dihydropyrimidine dehydrogenase activity

To elucidate the mechanism of the enhanced antitumour activity of S-1 (1 M tegafur, 0.4 M 5-chloro-2, 4-dihydroxypyridine, and 1 M potassium oxonate) in terms of the phosphorylation and degradation pathways of 5-fluorouracil (5-FU) metabolism, we investigated tumoral thymidylate synthase (TS) content, dihydropyrimidine dehydrogenase (DPD) activity, the TS inhibition rate (TS-IR), and 5-FU incorporated into RNA (F-RNA) in four human gastric cancer xenografts (MKN-28, MKN-74, GCIY and GT3TKB) and compared the results obtained with S-1 with those obtained with 5-FU and UFT (1 M tegafur, 4 M uracil). 5-FU was administered intraperitoneally (i.p.) to mice at a dose of 50 mg/kg, three times, on days 0, 4 and 8. S-1 and UFT were administered orally at doses of 10 and 24 mg/kg, respectively, once a day, for 9 consecutive days. Antitumour activity was evaluated as the maximum inhibition of tumour growth in each animal. S-1 showed a better antitumour activity than 5-FU and UFT in tumours with a high DPD activity (GCIY and GT3TKB). There were inverse correlations between the antitumour activity and both TS content and DPD activity in the 5-FU and UFT groups. However, no such correlations were observed in the S-1 group. In GCIY and GT3TKB xenografts, TS-IR was significantly higher in the S-1 group than in the 5-FU or UFT groups. In GT3TKB xenografts, the F-RNA level was significantly higher in the S-1 group than in the 5-FU or UFT groups. The superior cytotoxicity of S-1 appears to be attributable to both an increased inhibition of DNA synthesis and an enhanced blockade of RNA function against tumours with a high DPD activity.

Prediction of sensitivity to fluoropyrimidines by metabolic and target enzyme activities in gastric cancer

BackgroundThis study was designed to investigate the role of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) in tumor progression and sensitivity to 5-fluorouracil (5-FU).MethodsA total of 275 tumor samples from 275 patients with gastric cancer were utilized in this study. TS activity was determined in 130 samples by 5-fluorodeoxyuridine monophosphate binding assay. DPD activity was measured in 140 samples by radioenzymatic assay, and TP protein level was determined in 157 samples by an enzyme-linked immunosorbent assay (ELISA) system. These parameters were compared with several clinicopathologic factors and sensitivity to 5-FU determined by in-vitro ATP assay. The antitumor activities of 5-FU, uracil plus tegafur (UFT), and 1 M tegafur — 0.4 M 5-chloro-2,4-dihydroxypyridine — 1 M potassium oxonate (S-1 [TS-1®]) were also compared, using three human gastric cancer xenografts in nude mice.ResultsThere was no correlation between either TS or TP and sensitivity to 5-FU. However, a weak inverse correlation was found between DPD activity and sensitivity to 5-FU. High DPD activity in tumor resulted in poor prognosis, especially in patients who received 5-FU-based adjuvant chemotherapy. Although TP was significantly correlated with depth of tumor invasion and with lymphatic and venous invasions, TP alone had no impact on survival. On the other hand, TS, as well as peritoneal, hepatic, and lymph node metastases, was selected as an independent prognostic factor in gastric cancer. In the animal model, there was no significant difference in antitumor activities among the drugs in a tumor with low DPD activity. However, S-1 showed superior antitumor activity to 5-FU or UFT in tumors with high DPD activity.ConclusionDPD is considered to be a most important predictive factor of 5-FU sensitivity. The use of DPD inhibitory fluoropyrimidines is strongly recommended for tumors with high DPD activity.

Ghrelin and leptin levels in cachectic patients with cancer of the digestive organs.

BackgroundCancer cachexia, a catabolic state characterized by weight loss, occurs frequently in patients with terminal-stage neoplastic diseases. Gastrointestinal hormones and cytokines may be associated with anorexia and wasting in cancer cachexia.MethodsThis study aimed to examine the mechanism of anorexia in cachectic patients through a prospective investigation of plasma cytokines, ghrelin, and leptin in 16 cachectic patients with cancer of the digestive organs and 10 healthy volunteers.ResultsTumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1 receptor antagonist (IL-1Ra), and ghrelin levels were significantly higher in cachectic cancer patients than in the healthy volunteers, whereas leptin was significantly lower in the cachectic cancer patients. Plasma leptin levels and cytokine levels (TNF-α and IL-6) correlated significantly with body mass index (BMI), but plasma ghrelin levels did not correlate with BMI or with the grade of symptoms.ConclusionNeither weight loss nor the grade of symptoms seemed to be directly associated with the increase in ghrelin levels. Hence, it is considered that the increase in ghrelin levels cannot simply be explained by an increase in ghrelin secretion, suggesting that other mechanisms, such as the decreased inactivation of ghrelin, may also play a role. Further studies are needed to clarify the mechanisms of the increase in ghrelin levels. Additionally, the changes in plasma cytokines (TNF-α and IL-6) and leptin in cachectic cancer patients suggest that these molecules may be useful markers for the evaluation of cancer cachexia.

Experimental Evaluation of Bursting Pressure in Lymphatic Vessels with Ultrasonically Activated Shears

To evaluate the ability of ultrasonically activated shears (USAS) to occlude lymphatic vessels, we investigated the bursting pressure of lymphatic vessels occluded with USAS and compared with pressures in an artery and a vein. The inguinal lymphatic vessels, testicular arteries, and testicular veins were removed from male pigs. The vessels were occluded by USAS at power level 2. The bursting pressures of the harvested vessels were measured ex vivo. The bursting pressure of the artery (average, 1154 mmHg; range, 1000-1341 mmHg) was significantly greater than that in the vein (average, 747 mmHg; range, 560-973 mmHg; p = 0.0024) or the lymphatic vessel (average, 610 mmHg; range, 491-996 mmHg; p = 0.0003). There was no significant difference of bursting pressure between the vein and lymphatic vessel (p = 0.2226). Although the bursting pressures of the vein and lymphatic vessel were significantly lower than that of the artery, they were much higher than that found in physiologic conditions. USAS is considered to be a powerful tool for lymph node dissection for malignant diseases in open surgery as well as laparoscopic surgery.

Molecular marker identification for relapse prediction in 5-FU-based adjuvant chemotherapy in gastric and colorectal cancers.

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.

Intraoperative Analgesic Regimen and Cholecystectomy: A Closer Look

Dear Editor, We appreciate the interest shown by Drs. Kendall MC and Castro-Alves LJ in our recent article “Comparison of SingleIncision Laparoscopic Cholecystectomy versus Needlescopic Cholecystectomy: A Single institutional Randomized Clinical Trial” published in Digestive Surgery [1]. They have raised three questions regarding the intraoperative analgesic regimen, type I error setting in statistical analyses, and patient-centered outcomes. First, with regard to the standardized intraoperative analgesic regimen, all the enrolled patients underwent continuous intravenous infusion of remifentanil during surgery and 50 mg of flubiprofen axetil was administrated at the end of surgery. We did not employ the ultrasound-guided nerve block [2] due to the long occupation of the operation room, and we did not employ any postoperative opioid administrations because overall analgesic use occurred only once during the hospital stay in the needlescopic cholecystectomy group. However, as we mentioned in the article, infiltration of the umbilical site with lidocaine or ropivacaine may reduce the postoperative wound pain in the single-incision laparoscopic cholecystectomy group owing to a larger incision compared to needlescopic cholecystectomy [3]. With regard to the query raised about the type I error setting in statistical analyses, we wish to state that type I error is called α error. We determined an α level of 0.05 ahead of time; hence, type I error was considered in all calculations and statistical analyses. Finally, patient-centered outcomes usually evaluate the maintenance or improvement of the quality of life [4]. As they say, less pain or less incision is not necessarily connected to patients’ quality of life and personal satisfaction; hence, further studies about patient-centered outcomes are warranted to clarify the true meaning of minimally invasive surgery.

Comparison of Single-Incision Laparoscopic Cholecystectomy versus Needlescopic Cholecystectomy: A Single Institutional Randomized Clinical Trial

Background: Both single-incision laparoscopic cholecystectomy (SILC) and needlescopic cholecystectomy (NSC) are superior to conventional laparoscopic cholecystectomy in terms of cosmetic outcome and incisional pain. We conducted a prospective, randomized clinical trial to evaluate the surgical outcome, postoperative pain, and cosmetic outcome for SILC and NSC procedures. Methods: In this trial, 105 patients were enrolled (52 in the SILC group; 53 in the NSC group). A visual analogue scale (VAS) was used to evaluate the cosmetic outcome and incisional pain for patients. Logistic regression analyses were used to evaluate the operative difficulty that was present for both procedures. Results: There were no significant differences in patient characteristics or surgical outcomes, including operative time and blood loss. The mean VAS scores for cosmetic satisfaction were similar in both groups. There were significant differences in the mean VAS scores for incisional pain on postoperative day 1 (p = 0.009), and analgesics were required within 12 h of surgery (p = 0.007). Obesity (body mass index ≥25 kg/m2) was the only significant influential factor for operating time over 100 min (p = 0.031). Conclusion: NSC is superior to SILC in terms of short-term incisional pain. Experienced laparoscopic surgeons can perform both SILC and NSC without an increase in operative time.

Abstract 880: Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach.

Introduction: The relapse rate of gastrointestinal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy is approximately 40%. Therefore, it is important to identify prediction markers of the chemotherapeutic efficacy. Recently, we identified NF-κB as a candidate relapse prediction biomarker from a cell-based screening followed by an immunohistochemical retrospective study (Ishida, et al PLoSONE, 2012). To evaluate the biological significance of NF-κB in the context of 5-FU-based chemotherapy, we analyzed the biological response of NF-κB stimulated by 5-FU. Materials and Methods: Human gastric cancer cell lines (MKN45, MKN74, KE39, GSS, KatoIII) were used. Induction and localization of NF-κB in response to 5-FU by western blot and immunohistochemistry were observed. Transcriptional products induced by 5-FU were investigated using a DNA microarray. Knockdown of p65 and p53 genes was performed to examine their transcriptional machinery. Both DNA binding domain mutation and codon72 polymorphism in p53 gene were also investigated. Pro/Pro variant from codon72 affects the binding efficacy for NF-κB subunit p65. Results: By applying 5-FU, both total p65 and phosphorylated-p65 levels were increased in the nucleus. By DNA microarray analysis, 10 transcript products induced by 5-FU were identified. Interestingly, 5 of 10 transcripts have been known as that of p53 downstream. Theoretical prediction of promoter binding sites of p53 and NF-κB revealed that p53 binding sequences were located closer to the transcription start site than those of NF-κB. However, the density of the consensus sequences of NF-κB was higher than that of p53. Interestingly, there were many binding sequences of NF-κB in TP53 promoter, but only a few of those of p53 in p65 promoter. Knockdown of p65 gene also decreased p53 protein as well as its downstream gene expression, such as p21. In contrast, NF-κB was not affected by TP53 knockdown. MKN45 and GSS, whose codon72 variant is Pro/Pro are relatively resistant to 5-FU, but Arg/Arg variant of KE39 and MKN74 is >10times sensitive to 5-FU. KatoIII with a large deletion of TP53 is resistant to 5-FU. These facts may suggest that NF-κB and p53 binding is relevant to 5-FU sensitivity. Conclusion: Present results suggest that: (i) NF-κB plays a more important role than p53 in response to 5-FU; and (ii) NF-κB may play a compensatory function in p53 mutant cells. From a viewpoint of predicting 5-FU based chemotherapeutic efficacy, NF-κB is considered to be a 5-FU-chemosensitivity prediction marker. Citation Format: Fumitaka Endo, Satoshi Nishizuka, Kazushige Ishida, Kohei Kume, Taishi Ide, Hirokatsu Katagiri, Kaoru Ishida, Takeshi Iwaya, Hisataka Fujiwara, Keisuke Koeda, Go Wakabayashi. Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 880. doi:10.1158/1538-7445.AM2013-880