In a large cohort of archival neuroendocrine tumor specimens, we found that MGMT deficiency, as measured by immunohistochemistry, was more common in pancreatic neuroendocrine tumors than in carcinoid tumors. Consistent with this difference, we found that 34% of patients with pancreatic neuroendocrine tumors treated with temozolomide-based regimens experienced a Response Evaluation Criteria in Solid Tumors-defined radiologic tumor regression, whereas responses in carcinoid tumor patients were rare. MGMT deficiency was directly associated with treatment response to temozolomide in the subgroup of 21 treated patients who also had available tumor tissue.
Like temozolomide, streptozocin and dacarbazine induce methylation at the O6
position of guanine (27
). This common cytotoxic mechanism suggests that the mechanisms of drug resistance for these agents may also be similar and that the ability of MGMT to repair treatment-induced formation of O6
methylguanine may contribute to drug resistance to all three drugs. Our observations that temozolomide-based therapy is more effective in pancreatic neuroendocrine tumors than in carcinoid tumors in fact mirror earlier results with the alkylating agents streptozocin and dacarbazine.
In an initial randomized trial, the combination of streptozocin and doxorubicin was associated with a combined biochemical and radiologic response rate of 69% in patients with pancreatic neuroendocrine tumors (5
). In a retrospective analysis of 84 pancreatic neuroendocrine tumor patients treated with streptozocin, 5-fluorouracil, and doxorubicin, using more formal radiologic response criteria, the overall response rate was 39% (4
). Dacarbazine was associated with an overall response rate of 33% in patients with pancreatic neuroendocrine tumors in a phase II study (6
). The response rate of 34% observed with temozolomide in the current study is similar to that observed in these prior studies.
Response rates associated with these alkylating agents in carcinoid tumors are lower. In a recent trial, 249 patients with advanced carcinoid tumors were randomized to receive either streptozocin/5-fluorouracil or 5-fluorouracil/doxorubicin (7
). The response rates associated with these regimens were 16% and 15.9%, respectively. The reported response rates associated with single-agent dacarbazine in carcinoid tumors are 8% to 16% (2
). Only a single carcinoid tumor patient (2%) responded to temozolomide-based therapy in our series.
Our results are similar to those of a smaller study of temozolomide monotherapy in 36 patients with neuroendocrine tumors (13
). As in our study, 4 of 5 responding patients in the monotherapy study had low MGMT expression; responses were uncommon in patients with high MGMT expression. In contrast to our observations, however, temozolomide monotherapy was associated with an overall response rate of 31% (4 of 13) in patients with bronchial carcinoid tumors. We identified only 8 bronchial carcinoid tumor patients in our series, limiting our ability to more formally evaluate the efficacy of temozolomide in this subpopulation. Interestingly, the single carcinoid patient who responded to temozolomide in our study had a bronchial carcinoid tumor. Although several tumors classified as MGMT “intact” showed heterogeneous staining in our study, we observed a markedly heterogeneous pattern of MGMT expression in three atypical bronchial carcinoid tumors, providing a possible explanation for the sensitivity of some carcinoid tumors to temozolomide.
Streptozocin-based therapy has been associated with improved OS in patients with pancreatic neuroendocrine tumors (5
). We observed trends toward improved PFS and OS among temozolomide-treated patients with pancreatic neuroendocrine tumors when compared with treated patients with carcinoid tumors in our study. Survival comparisons in our cohort are limited by both the retrospective nature of our analysis and potential differences in the treated subpopulations. Nevertheless, given the often similar natural history of patients with these malignancies, our observations raise the possibility that the higher observed rate of treatment response may also translate into improved survival in patients with pancreatic neuroendocrine tumors. Although we also observed a trend toward improved PFS and OS in patients with MGMT-deficient compared with MGMT-intact tumors, we cannot rule out the possibility that MGMT status had an independent effect on survival. Prospective, randomized studies will be necessary to confirm these associations.
There remains considerable controversy regarding the optimal method of MGMT analysis in clinical studies. Direct analysis of MGMT enzymatic activity generally requires use of carefully preserved frozen tissue or cell lysates and is not readily applicable to analysis of archival tumor samples from large clinical studies (31
). Epigenetic silencing of the MGMT
gene by CpG island promoter methylation is a common mechanism of MGMT
gene regulation, and promoter methylation status, assessed by methylation-specific PCR, has been widely used as a surrogate marker of MGMT activity in clinical specimens (34
). In patients with glioblastoma, MGMT
promoter methylation has been associated with improved survival and benefit from temozolomide in most, although not all, studies (20
). Direct measurement of MGMT protein expression using immunohistochemistry, as was done in our study, is the technically easiest and perhaps the most commonly used technique to measure MGMT status in tumor samples. As with MGMT
promoter methylation, low levels of immunohistochemical MGMT expression have been associated with improved response to temozolomide in glioblastoma in many studies, although correlations have not always been consistent (19
Our observation that MGMT deficiency is more common in pancreatic neuroendocrine than in carcinoid tumors would suggest that MGMT promoter methylation status may also be more prevalent in pancreatic neuroendocrine tumors. However, previously reported studies of CpG island methylation in neuroendocrine tumors have found either no significant difference in MGMT promoter methylation rates between carcinoid and pancreatic neuroendocrine tumors or higher rates of promoter methylation in carcinoid tumors compared with pancreatic neuroendocrine tumors (39
). A poor correlation between MGMT
promoter methylation and immunohistochemical expression of MGMT has been reported in several studies directly comparing these two methods (41
). One study evaluating 31 glioblastoma samples found evidence of MGMT promoter methylation in 61% of samples but low level immunohistochemical MGMT expression (<20% nuclear staining) in only 31% (41
). In a second study, substantial numbers of MGMT-positive cells were detected in the majority (73%) of tumor specimens carrying a methylated promoter (42
). Tumor heterogeneity, as well as the presence of endothelial cells and other nonneoplastic components expressing MGMT in tumor samples, may have contributed to the discordant results observed in these studies.
We sought to minimize these limitations in our study by prospectively using a strict definition for MGMT deficiency, in which specimens were only considered deficient if they showed complete absence of detectable MGMT in tumor cells by immunohistochemistry. We further specifically identified nonneoplastic components of the tumors using these elements as positive internal controls. Nevertheless, technical limitations and interobserver variability remain a concern in the interpretation of MGMT immunohistochemical assays. We also cannot rule out the possibility that mechanisms other than MGMT expression affect neuroendocrine tumor sensitivity to temozolomide. Parallel DNA repair mechanisms, including the base excision repair system, may affect temozolomide sensitivity, resulting in an imperfect correlation between MGMT expression and treatment response (44
In summary, MGMT deficiency, as measured immunohistochemically, appears to be more common in pancreatic neuroendocrine tumors than in carcinoid tumors. Consistent with this finding, in a retrospective analysis, we observed a 34% response rate to temozolomide-based therapy in pancreatic neuroendocrine tumors compared with 2% in carcinoid tumors. MGMT deficiency was directly associated with temozolomide response in the patient subgroup with available tumor tissue and treatment data. Our findings suggest that MGMT status could be used as a predictive marker to identify neuroendocrine tumor patients who are likely to respond to treatment with alkylating agents. Standardization of techniques to assess MGMT status in tumor tissue, together with prospective trials to confirm a correlation between MGMT status and treatment response in neuroendocrine tumor patients treated with alkylating agents, is warranted.