Yasushi Tsuji

Mechanism of Synergistic Cytotoxic Effect between Tumor Necrosis Factor and Hyperthermia

We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L‐M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L‐M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect.

Endogenous Tumor Necrosis Factor Inhibits the Cytotoxicity of Exogenous Tumor Necrosis Factor and Adriamycin in Pancreatic Carcinoma Cells

Pancreatic carcinoma is one of the most devastating neoplasms with regard to its resistance to conventional therapy. In a previous report, we found that endogenous tumor necrosis factor (enTNF) exerts an intracellular protective effect against exogenous TNF- and Adriamycin (ADM)-induced cytotoxicity by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD). We also know that glutathione S-transferase pi (GST-pi) and glutathione (GSH) also scavenge OFR. It remains unclear to what extent enTNF and MnSOD induced by enTNF regulate the sensitivity to ADM and exogenous TNF among different carcinoma cells. In this study, we examined the relationship between ADM and exogenous TNF sensitivity and en-TNF expression and MnSOD activity in four pancreatic carcinoma lines. We determined whether ADM and exogenous TNF sensitivity could be predicted by measuring enTNF expression and MnSOD activity in the carcinoma cells. The sensitivity to TNF and ADM varied with the cell lines, and TNF sensitivity correlated well with Adriamycin sensitivity. Moreover, enTNF expression and Mn-SOD activity correlated positively with resistance to ADM and exogenous TNF. When MIAPaCa-2 cells, which had the lowest enTNF expression and the highest sensitivity to exogenous TNF and ADM, were transfected with the nonsecretory-type human TNF gene (pTNF delta pro) to increase enTNF synthesis, their intracellular MnSOD activity and exogenous TNF and ADM resistance were increased. These findings suggest that MnSOD plays a critical role in scavenging OFR induced by ADM and exogenous TNF. enTNF is the most important factor that regulates the production of MnSOD. Therefore, it is plausible that inhibition of enTNF expression or MnSOD activity in pancreatic carcinoma would improve the efficacy of therapies for pancreatic carcinoma.

A phase I/II study of S-1 plus cisplatin in patients with advanced gastric cancer: 2-week S-1 administration regimen.

BackgroundThe combination of a new oral dihydropyrimidine dehydrogenase-inhibitory fluoropyrimidine (S-1) and cisplatin (CDDP) is one of the most active chemotherapy regimens for gastric cancer. However, the optimum schedule for this combination has not yet been determined. This study was conducted to establish the maximum tolerated dose (MTD) and the recommended dose of CDDP when combined with 2-week S-1 administration, and to observe the safety and efficacy of the regimen as treatment for patients with advanced gastric cancer.MethodsS-1 was administered orally at a dose of 80 mg/m2 per day for 2 weeks, followed by a 2-week rest. CDDP was administered intravenously on day 8 of each course; the initial dose of CDDP was 60 mg/m2 and it was increased in 10-mg/m2 increments. Treatment was repeated every 4 weeks unless disease progression was observed.ResultsEleven patients were enrolled. The main toxicities were leucopenia, neutropenia, nausea, and anorexia. These toxicities were not severe, and were reversible and manageable. The MTD for CDDP was established as 80 mg/m2, as 2 of 5 (40%) patients developed dose-limiting toxicity (DLT) at this level. Therefore, the recommended dose of CDDP was determined to be 70 mg/m2. All 11 patients were evaluable for a response: 8 achieved a partial response and 1 had stable disease. The overall response rate was 73%.ConclusionThis regimen is considered to be generally well-tolerated and has substantial antitumor activity.

Recombinant Human Tumor Necrosis Factor Causes Regression in Patients with Advanced Malignancies

Fifteen patients with advanced solid tumors of various types were treated by the intratumoral administration of recombinant human tumor necrosis factor (rH-TNF). The treatment appeared to benefit the 4 cases of superficial tumors: there were 1 complete response, 1 partial response and 2 minor responses. In all 11 patients with deep-seated tumors, including 6 cases of pancreatic cancer, 4 of liver cell cancer and 1 of metastatic liver tumor, no tumor regression was observed, but progression stopped in all these tumors. Seven of the 11 with deep-seated tumors showed a decrease in tumor markers and/or the development of tumor necrosis. Fever, hypotension and fatigue were the main clinical side effects. No significant changes were found in hematologic, renal or liver parameters. These results suggest that administration of rH-TNF to the tumor site has the potential for controlling local tumor growth.

Clinical benefit of high-sensitivity KRAS mutation testing in metastatic colorectal cancer treated with anti-EGFR antibody therapy.

Objective: We compared high-sensitivity KRAS mutation testing with direct sequencing for predicting the efficacy of antiepidermal growth factor receptor antibodies in patients with metastatic colorectal cancer (mCRC). Methods: We analyzed the KRAS status in 61 tumors from cetuximab-treated mCRC patients by both direct sequencing and a high-sensitivity method: 2-step PCR restriction fragmentation length polymorphism (RFLP). Therapeutic effects in each mutational status were evaluated. Results: The incidences of KRAS mutations determined by direct sequencing and 2-step PCR RFLP were 34.4 and 52.5%, respectively (p = 0.02). Patients were categorized into 3 groups [W/W, wild-type by both methods (n = 29); W/M, wild-type by direct sequencing, detected mutation by 2-step PCR RFLP (n = 11); M/M, mutant-type by both methods (n = 21)]. The response rate for cetuximab in the W/M group (0%) was the same as that in the M/M group, and was significantly lower than in the W/W group (41.4%) (p < 0.001). Progression-free survival in the W/M group (11.0 weeks) was similar to that in the M/M group (8.0 weeks), and was significantly shorter than in the W/W group (18.0 weeks) (p < 0.002). Conclusion: High-sensitivity KRAS mutation testing is useful for selecting true responders to cetuximab.

Successful treatment of a case of hepatocellular carcinoma with tumor necrosis factor and local hyperthermia

SummaryA case of unresectable hepatocellular carcinoma which responded favorably to combined therapy with tumor necrosis factor (TNF) and local hyperthermia is reported. A 58-year-old man was admitted to our hospital in June 1988 for treatment of hepatocellular carcinoma affecting S4 and S8. After three sessions of transcatheter arterial embolization (TAE) therapy, the serum α1 fetoprotein level decreased, and a reduction in the size of the lesions was also noted. Thereafter, the patient received local hyperthermia once a week (60 minutes of irradiation from a Thermotron-RF8 at l,100W), but the α1-fetoprotein level increased again in February 1989. On examination, enlargement of the S8 lesion and a new nodule in S7 were recognized. Since TAE was contraindicated due to liver dysfunction, human recombinant TNF (1 × 106U) was given by intravenous infusion together with local hyperthermia once a week. Eight sessions of the combined therapy reduced the serum α1-fetoprotein level markedly (7,512.0 to 2,782.0pg/ml) and after eighteen sessions, 58.1% regression of tumor size (partial response) on computed tomography scans was observed. This anecdotal case supports previous experimental evidence suggesting that TNF plus hyperthermia may be effective for treating unresectable hepatocellular carcinoma.

First-line sunitinib plus FOLFIRI in Japanese patients with unresectable/metastatic colorectal cancer: A phase II study

This phase II, open‐label, single‐arm study investigated sunitinib + FOLFIRI in Japanese patients with treatment‐naïve unresectable/metastatic colorectal cancer. Patients received i.v. FOLFIRI (levo‐leucovorin 200 mg/m2 + irinotecan 180 mg/m2, followed by 5‐fluorouracil 400 mg/m2 bolus then 2400 mg/m2 46‐h infusion) every 2 weeks, and oral sunitinib 37.5 mg/day on Schedule 4/2 (4 weeks on, 2 weeks off), until disease progression or treatment withdrawal. Progression‐free survival (PFS) was the primary endpoint, with a target median of 10.8 months (35% improvement over FOLFIRI alone). Seventy‐one patients started a median of 3 (range 1–11) sunitinib cycles (median relative dose intensity, <60%). The median PFS was 6.7 months (95% confidence interval, 4.7–9.2) by independent review, 7.2 months (95% confidence interval, 5.4–9.5) by investigator assessment. Objective response rate (complete responses + partial responses) was 36.6% (independent review) and 42.3% (investigator assessment). Clinical benefit rate (complete responses + partial responses + stable disease) was 83.1% (independent review) and 88.7% (investigator assessment). Common all‐causality, any‐grade, adverse events were: neutropenia and leukopenia (both 97.2%); thrombocytopenia (84.5%); diarrhea and nausea (both 78.9%); decreased appetite (74.6%); and fatigue (66.2%). Neutropenia (96%) was the most frequent grade 3/4 adverse event. This study was closed early due to findings from a concurrent phase III study of sunitinib + FOLFIRI in non‐Japanese patients with metastatic colorectal cancer. In conclusion, the median PFS for sunitinib + FOLFIRI in Japanese patients was shorter than the 10.8 month target, indicating that sunitinib did not add to the antitumor activity of FOLFIRI. This study was registered with www.ClinicalTrials.gov (NCT00668863). (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02320.x, 2012)

Enhanced Antitumor Effect of Recombinant Human Tumor Necrosis Factor in Combination with Recombinant Human Granulocyte Colony‐stimulating Factor in BALB/c Mice

The synergistic antitumor effect of tumor necrosis factor (TNF) and granulocyte colony‐stimulating factor (G‐CSF) was investigated. G‐CSF was administered subcutaneously to BALB/c mice inoculated with Meth‐A cells at a dose of 2.5 μg/day for 5 consecutive days. When TNF (1 × 103 U) was administered intravenously to mice which had been pretreated with G‐CSF, tumor growth showed a 74.1% inhibition 17 days after the tumor cell inoculation, compared to that of untreated mice. In this experiment, G‐CSF significantly (P<0.025) enhanced the antitumor effect of TNF. The in vitro cytotoxicity of TNF (10 U/ml) towards Meth‐A cells was increased about 5.2‐fold in the presence of neutrophils (E/T=50) as compared to the cytotoxicity obtained with TNF alone. A combination of TNF and G‐CSF (50 ng/ml) in the presence of neutrophils, resulted in a 2.1 times greater cytotoxicity against Meth‐A cells as compared to that obtained without G‐CSF. Significant augmenting effects of G‐CSF on superoxide (O2−) production by TNF‐stimulated neutrophils were observed. These observation suggest that the neutrophil plays an important role in the antitumor action of TNF on Meth‐A cells, and that the antitumor effect of TNF is enhanced by combination with G‐CSF.

Cyclic Platelet and Leukocyte Count Oscillation in Chronic Myelocytic Leukemia Regulated by the Negative Feedback of Transforming Growth Factor β

We report a case of chronic myelocytic leukemia (CML) with cyclic oscillation of platelet and leukocyte counts and attempt to elucidate the oscillatory mechanism from the standpoint of cytokine regulation of hematopoiesis. A 57-year-old woman with a diagnosis of CML exhibited platelet and white blood cell (WBC) count fluctuations of a cyclic nature. The average duration of the cycles was about 8 weeks. The patient suffered from headache, fatigue, and malaise at the peak of the cycle. The peak thrombopoietin concentration in peripheral blood coincided with a period of decrease in platelet numbers. The change in transforming growth factor β (TGF-β) level paralleled that of the platelet numbers. A progenitor cell assay revealed the suppression of trilineage colony formation in the presence of plasma from the blood cell peak point, and this suppression was completely blocked when the plasma was incubated with an anti-TGF-β antibody. From these findings, we concluded that the cyclic oscillation of the platelet, WBC, and reticulocyte counts had been induced by excess negative feedback to megakaryopoiesis by TGF-β.

Fatality Caused by Self-Bloodletting in a Patient with Factitious Anemia

Death by bloodletting among patients with factitious anemia has never been reported to our knowledge.We report the first known case. A 25-year-old woman with severe iron deficiency anemia confessed her habit of bloodletting at her first visit to our hospital, in March 1998.We prescribed oral iron and referred her to a psychiatrist.The diagnosis was borderline personality disorder. The psychiatrist began counseling the patient and prescribed a major tranquilizer. The patient’s method of bloodletting was to insert an 18-gauge needle without syringe into her vein after inducing congestion in her arm. This method was considered to involve risk of death, because once the patient fell into a faint caused by blood loss, the bloodletting could not be stopped. Although we attempted to persuade the patient to stop bloodletting by this method, she died after self-bloodletting in September 1999. It is not known whether the death was intentional suicide or an accident.