Yasunori Yoshimoto

Genomic and Molecular Profiling Predicts Response to Temozolomide in Melanoma

Purpose: Despite objective response rates of only ∼13%, temozolomide remains one of the most effective single chemotherapy agents against metastatic melanoma, second only to dacarbazine, the current standard of care for systemic treatment of melanoma. The goal of this study was to identify molecular and/or genetic markers that correlate with, and could be used to predict, response to temozolomide-based treatment regimens and that reflect the intrinsic properties of a patients tumor. Experimental Design: Using a panel of 26 human melanoma-derived cell lines, we determined in vitro temozolomide sensitivity, O6-methylguanine-DNA methyltransferase (MGMT) activity, MGMT protein expression and promoter methylation status, and mismatch repair proficiency, as well as the expression profile of 38,000 genes using an oligonucleotide-based microarray platform. Results: The results showed a broad spectrum of temozolomide sensitivity across the panel of cell lines, with IC50 values ranging from 100 μmol/L to 1 mmol/L. There was a significant correlation between measured temozolomide sensitivity and a gene expression signature–derived prediction of temozolomide sensitivity (P < 0.005). Notably, MGMT alone showed a significant correlation with temozolomide sensitivity (MGMT activity, P < 0.0001; MGMT expression, P ≤ 0.0001). The promoter methylation status of the MGMT gene, however, was not consistent with MGMT gene expression or temozolomide sensitivity. Conclusions: These results show that melanoma resistance to temozolomide is conferred predominantly by MGMT activity and suggest that MGMT expression could potentially be a useful tool for predicting the response of melanoma patients to temozolomide therapy.

Sorafenib, a Multikinase Inhibitor, Enhances the Response of Melanoma to Regional Chemotherapy

Melanoma responds poorly to standard chemotherapy due to its intrinsic chemoresistance. Multiple genetic and molecular defects, including an activating mutation in the BRaf kinase gene, are associated with melanoma, and the resulting alterations in signal transduction pathways regulating proliferation and apoptosis are thought to contribute to its chemoresistance. Sorafenib, a multikinase inhibitor that targets BRaf kinase, is Food and Drug Administration approved for use in advanced renal cell and hepatocellular carcinomas. Although sorafenib has shown little promise as a single agent in melanoma patients, recent clinical trials suggest that, when combined with chemotherapy, it may have more benefit. We evaluated the ability of sorafenib to augment the cytotoxic effects of melphalan, a regional chemotherapeutic agent, and temozolomide, used in systemic and regional treatment of melanoma, on a panel of 24 human melanoma-derived cell lines and in an animal model of melanoma. Marked differences in response to 10 μmol/L sorafenib alone were observed in vitro across cell lines. Response to sorafenib significantly correlated with extracellular signal-regulated kinase (ERK) downregulation and loss of Mcl-1 expression (P < 0.05). Experiments with the mitogen-activated protein kinase/ERK kinase inhibitor U0126 suggest a unique role for ERK downregulation in the observed effects. Sorafenib in combination with melphalan or temozolomide led to significantly improved responses in vitro (P < 0.05). In the animal model of melanoma, sorafenib in combination with regional melphalan or regional temozolomide was more effective than either treatment alone in slowing tumor growth. These results show that sorafenib in combination with chemotherapy provides a novel approach to enhance chemotherapeutic efficacy in the regional treatment of in-transit melanoma. Mol Cancer Ther; 9(7); 2090–101. ©2010 AACR.

Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine.

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials. [Mol Cancer Ther 2006;5(3):732–8]

Optimizing a Novel Regional Chemotherapeutic Agent against Melanoma: Hyperthermia-Induced Enhancement of Temozolomide Cytotoxicity

Purpose: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. Experimental Design: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5°C), or hyperthermic (43°C) conditions. Results: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. Conclusion: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide.

Defining regional infusion treatment strategies for extremity melanoma: Comparative analysis of melphalan and temozolomide as regional chemotherapeutic agents

Five different human melanoma xenografts were used in a xenograft model of extremity melanoma to evaluate the variability of tumor response to regionally administered melphalan or temozolomide and to determine if various components of pertinent drug resistance pathways for melphalan [glutathione S-transferase (GST)/glutathione] and temozolomide [O6-alkylguanine DNA alkyltranferase (AGT)/mismatch repair (MMR)] could be predictive of tumor response. Xenograft-bearing rats underwent regional isolated limb infusion with either melphalan (90 mg/kg) or temozolomide (2,000 mg/kg). The levels of AGT activity, GST activity, glutathione level, and GST/AGT expression were examined in this group of xenografts and found to be quite heterogeneous. No correlation was identified between melphalan sensitivity and the GST/glutathione cellular detoxification pathway. In contrast, a strong correlation between the levels of AGT activity and percentage increase in tumor volume on day 30 (r = 0.88) was noted for tumors treated with temozolomide. Regional therapy with temozolomide was more effective when compared with melphalan for the xenograft with the lowest AGT activity, whereas melphalan was more effective than temozolomide in another xenograft that had the highest AGT activity. In three other xenografts, there was no significant difference in response between the two chemotherapy agents. This study shows that AGT activity may be useful in predicting the utility of temozolomide-based regional therapy for advanced extremity melanoma tumors. Our observations also point out the limited ability of analysis of the GST/glutathione pathway to predict response to chemotherapies like melphalan whose resistance is primarily mediated through a complex mechanism of detoxification. [Mol Cancer Ther 2007;6(5):1492–500]

Gene Expression Signatures as a Guide to Treatment Strategies for In-Transit Metastatic Melanoma

In-transit metastatic melanoma, which typically presents as multifocal lesions, provides a unique setting to evaluate the utility of gene signatures for defining optimal regional therapeutic strategies and assessing the efficacy of treatment. The goal of this study was to determine whether a single multifocal lesion is representative of residual tumor burden in terms of gene expression signatures predictive of response to therapy. Using microarray-based gene expression profiling, we examined 55 in-transit melanoma lesions across 29 patients with multifocal disease. Principal component analysis, unsupervised hierarchical clustering, one-way ANOVA, binary regression analysis, and gene signatures predictive of oncogenic pathway activation were used to compare patterns of gene expression across all multifocal lesions from a patient. Patterns of gene expression were highly similar (P < 0.006; average r = 0.979) across pretreatment lesions from a single patient compared with the significantly different patterns observed across patients (P < 0.05). The findings presented in this study show that individual melanoma tumor nodules in patients with multifocal disease harbor similar patterns of gene expression and a single lesion can be used to predict response to chemotherapy, evaluate the activation status of oncogenic signaling pathways, and characterize other aspects of the biology of an individual patients disease. These results will facilitate the use of gene expression profiling in melanoma regional therapy clinical trials to not only select optimal regional chemotherapeutic agents but to also allow for a more rational identification of candidates for specific targeted therapies and evaluation of their therapeutic efficacy. Mol Cancer Ther; 9(4); 779–90. ©2010 AACR.

Lymph node metastases identified with mediastinoscopy in a patient with superficial carcinoma of the esophagus.

Superficial esophageal cancers limited to the lamina propria are not associated with lymph node metastases. Mediastinoscopic transhiatal esophagectomy was planned in a patient with widespread superficial cancer of the midthoracic esophagus. Sampling of the upper mediastinal lymph nodes revealed metastases. The operation was converted to a transthoracic esophagectomy with radical lymphadenectomy. Histopathologic examination of the resection specimen showed three metastatic lymph nodes, despite local invasion limited to the lamina propria. This is the first report of a patient with superficial esophageal cancer and lymph node metastases.

Treatment of cholecystoduodenal fistula by laparoscopy.

We describe a patient with cholecystoduodenal fistula treated by a laparoscopic approach. Use of a flexible videoscope, flexible retractor, and endoscopic transecting stapler allows for laparoscopic treatment of cholecystoenteric fistulae.

Enlarged extrahepatic portal vein aneurysm in a non-cirrhotic patient: a therapeutic dilemma.

The authors report a case of a 75-year-old woman who was seen about 5 years ago and found to have asymptomatic gallstone and extrahepatic portal vein aneurysm (PVA) adjacent to the gallbladder. Further investigations revealed no evidence of cirrhosis. A follow-up ultrasound revealed that the PVA increased in diameter from 20 to 25 mm over 5 years and surgical intervention was recommended. Cholecystectomy and omental wrapping around PVA were performed. Postoperative follow-up investigations for 3 years revealed no increases in PVA diameter. Although PVA is clinically rare, the authors believe that their case report presents an overview of related literature, discusses indications for surgery in PVA and describes their surgical method for managing PVA.

Wire-guided transoral esophagogastrostomy for carcinoma of the cervical esophagus.

The double stapling technique has been used to improve the ease and safety of colorectal anastomosis. When applied in esophageal surgery, a higher esophagogastric anastomosis is feasible. Laryngeal preservation, which improves the quality of life dramatically, also can be achieved in patients with carcinoma of the cervical esophagus. Reports of this technique are limited because of the difficulties in introducing the stapling device transorally into the neck. We describe here a special device to facilitate a safe transoral anastomosis.