Methylguanine Methyltransferase Expression Research Articles

DNAR-04. MICRORNA DEGRADATION ENHANCES GLIOBLASTOMA INTRA-TUMORAL HETEROGENEITY TO AUGMENT ACQUIRED TEMOZOLOMIDE RESISTANCE

Abstract INTRODUCTION O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form and dictates clonal evolution in response to chemotherapy. As such, MGMT is a major determinant of glioblastoma resistance to the standard-of-care chemotherapy temozolomide. miR-181d is a MGMT suppressing microRNA, whose expression inversely correlate with survival expectation after temozolomide treatment. Here, we characterize the expression of miR-181d and MGMT in response to temozolomide treatment. Method: Laboratory characterization, single cell analysis, and murine modeling RESULTS Treatment of glioblastoma cells with temozolomide (TMZ) induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1) dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation: 1) increased mean level of MGMT expression in a cell population and 2) enhanced cell-to-cell variability in MGMT expression to increase intra-tumoral heterogeneity in DNA repair capacity. This increased intra-tumoral heterogeneity enhanced glioblastoma resistance to temozolomide through a mechanism distinct from lowering the mean MGMT expression of the tumor population. Suppression of this heterogeneity by exogenous miR-181d over-expression enhanced the tumoricidal activity of temozolomide against glioblastomas. CONCLUSION We identified an interface between DNA damage response and miRNA degradation that mediate intra-tumoral heterogeneity and subsequent temozolomide resistance. This resistance mechanism is distinct from modulation of the mean MGMT expression of the population.

O6 -methylguanine methyltransferase promoter methylation status of glioblastoma cell line clonal population.

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

IDH1 mutation is prognostic for diffuse astrocytoma but not low-grade oligodendrogliomas in patients not treated with early radiotherapy.

Despite accumulating knowledge regarding molecular backgrounds, the optimal management strategy for low-grade gliomas remains controversial. One reason is the marked heterogeneity in the clinical course. To establish an accurate subclassification of low-grade gliomas, we retrospectively evaluated isocitrate dehydrogenase-1 (IDH1) mutation in clinical specimens of diffuse astrocytomas (DA) and oligodendroglial tumors separately. No patients were treated with early radiotherapy, and modified PCV chemotherapy was used for postoperative residual tumors or recurrence in oligodendroglial tumors. Immunohistochemical evaluation of IDH status, p53 status, O(6)-methylguanine methyltransferase expression, and the MIB-1 index were performed. The 1p and 19q status was analyzed with fluorescence in situ hybridization. Ninety-four patients were followed for a median period of 8.5years. For DAs, p53 was prognostic for progression- free survival (PFS) and IDH1 was significant for overall survival (OS) with multivariate analysis. In contrast, for oligodendroglial tumors, none of the parameters was significant for PFS or OS. Thus, the significance of IDH1 mutation is not clear in oligodendroglial tumors that are homogeneously indolent and chemosensitive. In contrast, DAs are heterogeneous tumors including some potentially malignant tumors that can be predicted by examining the IDH1 mutation status.

Abstract 5754: Temozolomide therapy for progressive metastatic paraganglioma/pheochromocytoma: SDHB mutation as a prognosis biomarker for efficacy

Abstract Malignant pheochromocytoma and paraganglioma (PPGLs) are rare diseases with a heterogeneous behaviour. Nowadays systemic therapies, including 131I-MIBG therapy and the cyclophosphamide-dacarbazine-vincristine chemotherapy regimen (CVD), constitute the most popular options in case of aggressive tumors. We have investigated the antitumor effect of temozolomide (TMZ), in patients with metastatic PPGLs. All patients were assessed for TMZ efficacy, with CT scan and PET scan performed every 3 months. Safety and identification for prognosis factors of response were secondary endpoints. Fifteen consecutive patients with progressive (n=14) or symptomatic (n=1) metastatic PPGLs began TMZ therapy at our institution between November 2007 and January 2011. There were 12 men (80%); median age was 43 years, with a functioning tumor in 73% of the cases. Germline mutations were screened: 10 patients harbored SDH B mutation and 5 patients had no mutation among SDH A, SDH C, SDH D, NF1, RET or VHL genes. Primary was located in the adrenal gland for 53% of the patients, thorax for 40 % and neck for 7%. TMZ was given at a dose of 150 mg/m2/d for five days of a 28-days cycle for the first cycle and then at 200 mg/m2 for five days each subsequent cycle. Seven patients received TMZ as first line therapy, 2 patients as second line and 6 patients as third line. Median number of cycle administered was 7, mean dose intensity was 171 mg/m2 (95% CI 160.7-181) for each treatment cycle. TMZ was well tolerated: the most frequent all grade toxicities included asthenia, nausea and anemia. Grade 3 toxicities were lymphopenia (n=2) and hypertension (n=1). According to RECIST 1.1 criteria, 11 patients were evaluable: 4 patients experienced a partial response, 6 a stable disease, 1 patient a progressive disease. With a median follow up of 29 months (range 6.4-37.9), median overall survival was not reached and median progression free survival (PFS) was 9.6 months. Partial response was observed in 4 out of 10 SDHB mutated patients but no sporadic case. Patients with SDH B mutations or with no mutation had a median PFS of 16.7 months or 2.6 months, respectively (log-rank, p=0.013); Patients with SDH B mutations or with no mutation had a 12-month PFS of 60% or 0%, respectively. Expression of Methyl Guanine Methyl Transferase (MGMT) by immunohistochemistry (IHC) was analyzed in 4 patients, including 3 responders and, was found negative. MGMT IHC is ongoing in the remaining patients This study demonstrates for the first time the antitumor efficacy of TMZ in a large series of metastatic PPGLs as well as the potential role for SDH B mutation as a prognosis biomarker of response of response. Loss of MGMT expression in SDH B mutated patients could explain these results. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5754. doi:1538-7445.AM2012-5754

Pre-Screening for Low Methyl Guanine Methyltransferase (MGMT) Expression Results in High Response Rates to Temozolomide (TMZ) Therapy in AML and MDS Patients.

Abstract 422 Introduction:Many patients with acute myeloid leukemia (AML) and high-risk myelodysplasia (MDS) are not suitable candidates for standard induction therapy, and newer treatment approaches are needed. TMZ anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Expression of MethylGuanine DNA MethylTransferase (MGMT) restores normal guanine, decreasing DNA alkylation and thus anti-tumor activity of TMZ. Brandwein et al. (Leukemia 2007;21:821–4) previously demonstrated that response to TMZ in AML patients could be correlated with low MGMT expression. Thus MGMT expression prescreening could be used to identify patients more likely to respond to TMZ and provide an alternative for patients who are not candidates for standard induction therapy. Methods:We conducted a prospective multicenter study of patients with previously untreated AML and high risk MDS who were not candidates for standard induction therapy. Patient selection was based on MGMT expression as determined by Western blot in bone marrow or peripheral blood. Screening was performed by comparing the expression ratio between MGMT and b-actin protein levels. Patients with a ratio <0.25 were eligible to receive TMZ therapy 200 mg/m2/day × 7 days. Patients achieving a complete response (CR) could receive up to 12 monthly cycles of temozolomide 200 mg/m2/day × 5 days. Partial responders received a modified schedule of temozolomide 100 mg/m2/day × 21 days. Results:MGMT expression was tested in 188 patients; median age was 75 years old (range 46–94 years);62% were male. Primary diagnoses were AML (86%) and MDS (14%). Low or weak MGMT expression was detected in 46% of patient that were therefore potential candidates for TMZ therapy. Responses were evaluated in 42 patients, and were divided into 4 categories: Complete (CR/CRp), Morphologic Leukemia-Free State (MLFS), Partial (PR) Minimal (MR). Response was observed in 74% of cases including 29% CR, 10% MLFS, 19% PR and 17% MR. Prognostic factors associated with a higher response rate to temozolomide included MDS presentation, ECOG PS 0 and favorable/standard cytogenetic risk group. Induction and post-remission cycles were well-tolerated and most patients could be successfully treated on an outpatient basis.Patient who reached CR/CRp had a significantly longer median survival (>18 months) compared to those who reached MLFS, PR and MR (5.6 months). Median duration of therapy for patients reaching CR/CRp was 255 days and a majority (80%) became transfusion independent. Grade 3 and 4 toxicities were rare with the most common being fatigue (17%) and diarrhea (6%). Discussion:Pre-selection of patients based on MGMT expression level was associated with a higher response rate to TMZ as compared to previously reported unselected patients. Complete response was associated with increased survival and treatment had a favorable toxicity profile even in frail elderly patients. Targeted therapy with TMZ is feasible in patients with low MGMT expression and represents a new option for AML and MDS patients who are not candidate for standard induction therapy. Disclosures:Brandwein:Merck: Research Funding. Off Label Use: Temozolomide therapy for AML and MDS. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Galarneau:Merck: Employment. Pouliot:Merck: Employment.

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.

Identification of AML and MDS Patients Who Could Potentially Benefit From Temozolomide Therapy: Proportion of Patients with Low Methyl Guanine Methyltransferase (MGMT) Expression Level

AbstractAbstract 3985 Introduction:Temozolomide is an imidazotetrazine derivative of dacarbazine that functions as an alkylating agent. Temozolomide anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Resistance to temozolomide may largely be conferred by O6-methylguanine DNA methyltransferase (MGMT) over-expression. The level of MGMT expression will determine the ability of a cell to withstand damage from an alkylating chemotherapeutic agent. Brandwein et al. (Leukemia 2007;21:821-4) previously showed that the complete response rate to temozolomide therapy in patients with acute myeloid leukemia (AML) was higher when MGMT expression was low. Methods:In this prospective multicenter study we evaluated MGMT expression by Western blot in bone marrow or peripheral blood from older patients with previously untreated AML and high risk myelodysplastic syndrome (MDS). Expression was characterized by comparison with b-actin and results were expressed as an MGMT:b-actin ratio: Negative: < 0.2; Weak: 0.2 – 0.25; and Strong:> 0.25 MGMT promoter methylation was also evaluated using methylation-specific PCR. Both protein expression and promoter methylation were tested in a central lab. Results:Data were obtained for 152 patients; median age was 76 years old (range 46–94 years). A majority (63%) were male. Primary diagnosis was AML (84%) and MDS (16%). MGMT expression level could be determined in 128 of the 152 patients tested (84%). Expression ratios were: Negative (41%), Weak (11%), and Strong (48%). MGMT promoter methylation could be detected in 16% of cases in patients with either Negative or Weak expression ratio and in 2% of patients with Strong expression ratio. A majority of patients with promoter methylation (82%) had low MGMT expression. Discussion:One-half of patients with AML/MDS had either a Negative or Weak MGMT expression level, and would therefore be potential candidates for temozolomide therapy. These low levels could be explained by promoter methylation in a minority of cases. Thus, low level of MGMT expression in AML/MDS may be due to direct alteration of the MGMT gene or modulation of expression at the post-transcriptional level. Disclosures:Brandwein:Merck: Honoraria, Research Funding. Off Label Use: temozolomide for acute myeloid leukemia. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Greaves:Schering-Plough/Merck: Employment. Pouliot:Schering-Plough/Merck: Employment.

Temozolomide Treatment for Aggressive Pituitary Tumors: Correlation of Clinical Outcome with O6-Methylguanine Methyltransferase (MGMT) Promoter Methylation and Expression

The typically indolent behavior of pituitary tumors is juxtaposed with high rates of tumor cell invasion into adjacent dural structures, and occasional aggressive behavior. Although clinically significant invasion and malignant transformation remain uncommon, there are limited treatment options available for the management of these aggressive tumors. Recently, case reports have described efficacy of temozolomide for the treatment of aggressive pituitary tumors. Seven patients with aggressive pituitary tumors have been treated with temozolomide. We compared O(6)-methylguanine methyltransferase (MGMT) promoter methylation and MGMT expression in 14 surgical specimens from these seven patients and correlated these molecular features with the clinical response to temozolomide. Significant tumor regression was seen in two patients (29%), a 20% reduction in tumor volume with subsequent stable tumor size was noted in one patient, arrest of tumor growth occurred in three patients, and progressive metastatic disease developed during treatment in one patient. The DNA promoter site for MGMT was unmethylated in all 14 adequate specimens, and variable MGMT expression was seen in all 14 cases. There was no correlation between MGMT expression and clinical outcomes. We conclude that medical therapy with temozolomide can be helpful in the management of life-threatening pituitary tumors that have failed to respond to conventional treatments. The optimal duration of treatment in patients with stabilization or reduction of tumor size has not been established, and long-term follow up studies are needed.

MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib

Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. In this study, we used the O(6)-methylguanine methyltransferase (MGMT)-negative GBM cell line U87MG stably transfected with MGMT (U87/MGMT) to assess whether MGMT expression affects the response to sunitinib. We showed that the addition of sunitinib to standard therapy (temozolomide [TMZ] and radiation therapy [RT]) significantly improved the response of MGMT-positive but not of MGMT-negative cells. Gene expression profiling revealed alterations in the angiogenic profile, as well as differential expression of several receptor tyrosine kinases targeted by sunitinib. MGMT-positive cells displayed higher levels of vascular endothelial growth factor receptor 1 (VEGFR-1) compared with U87/EV cells, whereas they displayed decreased levels of VEGFR-2. Depleting MGMT using O(6)-benzylguanine suggested that the expression of these receptors was directly related to the MGMT status. Also, we showed that MGMT expression was associated with a dramatic increase in the soluble VEGFR-1/VEGFA ratio, thereby suggesting a decrease in bioactive VEGFA and a shift towards an antiangiogenic profile. The reduced angiogenic potential of MGMT-positive cells is supported by: (i) the decreased ability of their secreted factors to induce endothelial tube formation in vitro and (ii) their low tumorigenicity in vivo compared with the MGMT-negative cells. Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and tumorigenicity of GBM cells and suggests the combination of sunitinib and standard therapy as an alternative strategy for GBM patients with MGMT-positive tumors.

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

AbstractCorrection of murine models of β-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human γ-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with β-thalassemic HSCs transduced with a γ-globin/MGMT vector initially had subtherapeutic levels of red cells expressing γ-globin. To enrich γ-globin–expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of γ-globin–expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of γ-globin–expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for β-thalassemia.

Correlation of O6-Methylguanine Methyltransferase (MGMT) Promoter Methylation With Clinical Outcomes in Glioblastoma and Clinical Strategies to Modulate MGMT Activity

Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) remains a significant barrier to the successful treatment of patients with malignant glioma. The relative expression of MGMT in the tumor may determine response to alkylating agents, and epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression in gliomas. MGMT promoter methylation is correlated with improved progression-free and overall survival in patients treated with alkylating agents. Strategies to overcome MGMT-mediated chemoresistance are being actively investigated. These include treatment with nontoxic pseudosubstrate inhibitors of MGMT, such as O(6)-benzylguanine, or RNA interference-mediated gene silencing of MGMT. However, systemic application of MGMT inhibitors is limited by an increase in hematologic toxicity. Another strategy is to deplete MGMT activity in tumor tissue using a dose-dense temozolomide schedule. These alternative schedules are well tolerated; however, it remains unclear whether they are more effective than the standard dosing regimen or whether they effectively deplete MGMT activity in tumor tissue. Of note, not all patients with glioblastoma having MGMT promoter methylation respond to alkylating agents, and even those who respond will inevitably experience relapse. Herein we review the data supporting MGMT as a major mechanism of chemotherapy resistance in malignant gliomas and describe ongoing studies that are testing resistance-modulating strategies.

Phase I analysis of BCNU-impregnated biodegradable polymer wafers followed by systemic interferon alfa-2b in adults with recurrent glioblastoma multiforme

Carmustine (BCNU)-impregnated biodegradable polymer wafers have been shown to prolong survival in patients with recurrent malignant glioma. Interferon alfa-2b (IFNalpha(2b)) has demonstrated antitumor activity against a number of cancers, but its use in glioma has been limited. The use of these agents in combination is appealing because the mode of antitumor activity likely differs. This is a report of a phase I dose-finding study for IFNalpha(2b) in combination with surgery and BCNU wafers in patients with recurrent glioblastoma. Patients with progressive malignant glioma that was confirmed intraoperatively to be glioblastoma were treated with surgical resection and implantation of 8 BCNU wafers. A week later, IFNalpha(2b) was initiated three times a week at a dose of 3 MU/m(2), which was escalated in increments of 3 MU/m(2). The treatment cycle encompassed 8 weeks. Toxicity was monitored by clinical and laboratory testing. Correlative studies of methylguanine methyltransferase (MGMT) expression and gene expression array analysis were carried out. Ten patients were enrolled, and 9 patients had evaluable data. Dose-limiting toxicity in the form of fatigue occurred at 9 MU/m(2). Two complete imaging responses were observed at the 3 MU/m(2) dose. MGMT expression and gene expression arrays did not correlate with toxicity or response. Multimodal therapy with surgery, BCNU wafers, and IFNalpha(2b) appears to be a feasible and safe treatment strategy. The maximum tolerated dose of IFNalpha(2b) was determined to be 6 MU/m(2). Analysis of MGMT expression and gene expression was feasible.

Phase II Study of Extended-Dose Temozolomide in Patients With Melanoma

We conducted a phase II trial of extended-dose temozolomide (TMZ) in patients with melanoma to test the hypothesis that the approximately 30% response rate observed in patients treated with extended-dose TMZ with antiangiogenic agents was caused by TMZ alone. We hypothesized that expression of methylguanine methyltransferase (MGMT) in the tumor would correlate with drug resistance to TMZ. Patients with stage IV or unresectable stage III melanoma were treated with TMZ 75 mg/m(2)/d for 6 weeks followed by a 2-week rest period. Cycles were repeated until progression. Patients were stratified by M1c disease or not. The primary end point was objective response proportion. MGMT expression was assessed by methylation-specific pyrosequencing of the promoter and by immunohistochemistry. Forty-nine patients (25 with M1c disease) were assessable. Three patients (12.5%) in each cohort experienced partial responses; there were no complete responses. Ten patients (21%) had stable disease lasting more than 24 weeks. Median time to progression was 3.3 months. Median survival was 10.1 months; survival was similar in the two cohorts. The estimated 18-month survival was 27%. There was no correlation between response and either immunohistochemistry staining for MGMT or for MGMT promoter methylation. Seventy-five percent of patients developed CD4(+) lymphopenia after three cycles. Extended-dose TMZ therapy did not result in a 30% responses rate, which has been observed using extended-dose TMZ with antiangiogenic agents. Response did not correlate with MGMT expression or promoter methylation as a continuous variable, suggesting that other resistance mechanisms are important.

Molecular Analysis of Colorectal Cancer Tumors from Patients with Mismatch Repair–Proficient Hereditary Nonpolyposis Colorectal Cancer Suggests Novel Carcinogenic Pathways

A subset of colorectal cancers (CRC) arises in families that, despite fulfilling clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC), do not show evidence of a mismatch repair (MMR) deficiency. The main objective of this study was to characterize these tumors at the molecular level. After comprehensive germ line mutation scanning, microsatellite analysis, and MMR protein expressions, we selected a well-defined cohort of 57 colorectal tumors with no evidence of MMR defects. In this group of tumors, we analyzed KRAS, BRAF, and APC somatic mutations, as well as methylguanine methyltransferase (MGMT) and beta-catenin expression. We correlated these alterations with clinicopathologic data and explored the relationship between KRAS G > A transitions and lack of MGMT expression. The mutation profile at the RAS/RAF/MAPK pathway mimics sporadic microsatellite-stable CRCs. We found an average age of diagnosis 10 years older in KRAS-mutated patients (P = 0.001). In addition, we show that KRAS G > A transitions are actively selected by tumors, regardless of MGMT status. Similarities with HNPCC high-microsatellite instability tumors are observed when APC data are analyzed. The APC mutation rate was low and small insertions/deletions accounted for 70% of the alterations. In addition, we found a low frequency of beta-catenin nuclear staining. Finally, we did not find evidence of tumors arising in individuals from the same family sharing molecular features. We show evidence that CRC tumors arising in HNPCC families without MMR alterations have distinctive molecular features. Overall, our work shows that systematic analysis of somatic alterations in a well-defined subset of CRCs is a good approach to provide new insights into the mechanisms of colorectal carcinogenesis.

Prediction of response to temozolomide (TMZ)-based therapy by loss of MGMT expression in patients with advanced neuroendocrine tumors (NET)

4505 Background: TMZ, an oral alkylating agent, has recently been shown to be active in a subset of patients with NETs. In other tumor types, methyl-guanine methyl transferase (MGMT) expression is associated with TMZ resistance through its ability to remove methyl/alkyl groups from the O6 -position of guanine, thereby preventing TMZ-induced DNA damage. Methods: To better define NET pts who benefit from TMZ, we performed a retrospective analysis of 76 NET pts treated with TMZ-based regimens at 3 institutions, identified using IRB-approved observational and clinical trial databases. Immunohistochemical MGMT expression was measured in a subset of pts with available paraffin-embedded archival tissue specimens in a blinded fashion, utilizing mouse monoclonal antibodies to MGMT. Nuclear MGMT expression was scored as either present or absent in tumor cells and correlated with tumor type and treatment outcome. Results: Of 76 pts, 30 received TMZ/thalidomide and 46 received TMZ/bevacizumab. Both regimens incorporate oral TMZ, administered at a dose of 150 mg/m2 qd for 7d, every other week. 63 pts were enrolled on phase II studies, and 13 pts were treated outside a formal study protocol. Pt characteristics were: M:F=42:34; median age 57.5 (20–75); tumor type carcinoid (38), pancreatic NET (35), paraganglioma/pheochromocytoma (3). PR or CR (as measured by RECIST) was observed in 11/35 (31%) pancreatic NETs and 0/38 carcinoid pts (P&lt;0.001), as well as 1/3 pts with paraganglioma/pheo. In 21 pts with available specimens, complete absence of MGMT expression was observed in 5/8 pancreatic NET and 0/13 carcinoid tumors (P&lt;0.01). In this pt subset, absence of MGMT expression was associated with response to TMZ ( Table 1 ). Conclusions: Treatment with TMZ is associated with radiologic response in approximately one third of pts with pancreatic NET, but not in pts with carcinoid tumors. Absence of MGMT expression appears to define pts with pancreatic NET who achieve significant benefit from TMZ. [Table: see text] No significant financial relationships to disclose.

Phase II trial of extended-dosing temozolomide in patients with melanoma

8531 Background: To test the hypothesis that the ∼30% response rate we observed in metastatic melanoma (mel) patients (pts) treated with temozolomide (TMZ) using an extended dosing schedule with either thalidomide or interferon was largely due to TMZ alone, we conducted a phase II trial of TMZ alone. We also hypothesized that mel expression of methylguanine methyltransferase (MGMT) would correlate with drug resistance. Methods: Stage IV mel pts without brain metastases or prior chemotherapy were stratified into two cohorts based on whether or not they had stage M1c disease. Each cycle consisted of TMZ 75 mg/m2/day for 6 weeks followed by 2 weeks with no treatment. Treatment cycles were repeated as long as pts did not progress. The primary endpoint was the proportion of complete or partial responses by RECIST criteria. The trial was powered to exclude the null hypothesis that the response rate in each cohort was &lt;30%. MGMT promoter methylation status was assessed by methylation-specific pyrosequencing. In pts with insufficient tumor material for pyrosequencing, MGMT expression was measured by immunohistochemistry using the monoclonal antibody MT3.1. Results: To date we have accrued 48 pts, 23 in the M1c cohort. 7 pts are still receiving treatment and are not yet evaluable for response. There have been 4 partial responses observed, 2 in each cohort. The median number of cycles was 2 (range 1–6). 9/41 pts (22%) were stable for =6 months and received =3 cycles. 56% of pts developed CD4+ lymphopenia (&lt;500/μl) during treatment; 37% of pts developed CD4+ lymphopenia &lt;200/μl and received PCP prophylaxis. Opportunistic infections were not observed. Conclusions: The overall response rate is 9.8% (95% CI 0.8%-19%). The estimated median survival is 11 months for the stage III/M1a/b cohort and 9.7 months for the M1c cohort. To date, the response proportion to TMZ alone appears to be &lt;30%. There would need to be 3 more responses in a cohort to reject the null hypothesis for that cohort. MGMT analyses are ongoing and will be used to determine if promoter methylation status correlates with objective response rate or time to progression. The study was supported by Schering-Plough. No significant financial relationships to disclose.

Methylguanine Methyltransferase Testing in Glioblastoma: When and How?

Controversy remains on the optimal treatment schedule of the alkylating agent temozolomide (TMZ) in malignant glioma and the value of methylguanine methyltransferase (MGMT) determination. This repair enzyme is key in reverting lethal DNA damage induced by alkylating agent chemotherapy, and in the absence of this enzyme, cells are more susceptible to TMZ. In this issue of the Journal of Clinical Oncology, Chinot et al report on a (too) small phase II study investigating a week-on and week-off dosedense TMZ administration regimen as neoadjuvant chemotherapy before definitive radiation in 29 eligible patients with unresectable glioblastoma. The aim of this alternative dose-dense administration schedule is not only to increase the dose-intensity by more than two-fold, but to overcome drug resistance by depleting the tumor cells of MGMT. In peripheral blood mononuclear cells, it has been shown that repeated and continuous exposure to TMZ will exhaust the cells of MGMT, but whether this also applies to tumor cells remains to be demonstrated. The results of this study were disappointing. Median survival was only 6 months (95% CI, 1 to 11), with only 28% of patients alive at 1 year. Even when considering that this is a poor prognosis group of patients, these results appear inferior to standard therapy, including concomitant chemoradiotherapy, where a median survival of 9 months was reported for patients with unresectable tumors. Chinot et al attempted to determine MGMT expression in 25 patients by immunohistochemistry, choosing an arbitrary cutoff based on the median percentage of cells staining positively. They found that 6 of 11 patients with low MGMT expression, but only one of the 14 patients with high MGMT expression responded. The study emphasizes again the importance of MGMT to predict a response to the alkylating agent therapy. The predictive value of MGMT for benefit from alkylating agent chemotherapy, whether this be a nitrosourea or temozolomide, has now been repeatedly shown. Methylation of the MGMT gene promoter has been found to be a predictive marker for benefit from TMZ treatment in patients treated with temozolomide chemotherapy. Analysis of a representative subgroup of patients in a randomized trial showed that the addition of TMZ only benefited patients with a methylated (and thus silenced) MGMT gene promoter, while overall survival and progression-free survival was comparable for TMZ-treated patients with a functional MGMT gene to patients having been treated with initial radiotherapy alone. Based on this data, one may want to conclude that patients without MGMT promoter methylation should not be treated with alkylating agent chemotherapy. In these patients, TMZ has marginal activity at best, and alternative strategies may offer better chances. However, outside clinical trials, alternative strategies are currently not available. Nevertheless, the treatment, although generally well tolerated, carries its risks for toxicity. Also, in countries without reimbursement for chemotherapy, some patients may choose to forego the expense for temozolomide in the absence of MGMT promoter methylation. Furthermore, this analysis was performed retrospectively, and although very provocative and suggestive, requires prospective validation. In an ongoing Intergroup trial by the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer (RTOG 0525/EORTC 26052-22053), patients with newly diagnosed glioblastoma are stratified by MGMT methylation status before random assignment to a TMZ administration schedule (standard daily dose for 5 days or a dose-dense treatment for 3 of 4 weeks). To be eligible for this trial, patients must have undergone at least partial tumor resection, and tumor material must be available for centralized MGMT promoter methylation determination. Thus, not only efficient trial management is necessary, but also the goodwill and collaboration of the neurosurgeons and pathologists at many sites. Repeatedly, investigators advocate performing MGMT determination using the simpler immunohistochemistry staining, rather than a methylation-specific polymerase chain reaction assay (MSP) of the function of the MGMT gene. Although some associations may be found by immunohistochemical MGMT expression, this method has many shortcomings. Heterogeneous MGMT expression within tumors, and high levels of MGMT protein present in infiltrating lymphocytes, microglia, and blood vessels limit the reproducibility of the method. More important, MGMT is an inducible enzyme that may be upregulated after chemotherapy or radiotherapy, thus the expression of the protein at diagnosis might not reflect the functional protein during therapy. The MSP assay requires small quantities of DNA that can be extracted from paraffin-embedded tissue samples or, better yet, from cryopreserved tissue samples. In glioma, the presence of a methylated MGMT allele can be attributed solely to the neoplastic cells, irrespective of contamination of nontumor tissue. In our opinion, determination JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 12 APRIL 2

Phase II trial of extended-dosing temozolomide in patients with melanoma

8042 Background: We previously reported a 30% response rate in metastatic melanoma (mel) patients (pts) treated with temozolomide (TMZ) using an extended dosing schedule of 6 weeks on/ 2 weeks off with either thalidomide or interferon. We hypothesized that these responses were largely due to TMZ alone and conducted a phase II trial using extended dosing TMZ alone. We also tested the hypothesis that a) mel expression of methylguanine methyltransferase (MGMT) would correlate with drug resistance, and b) TMZ would cause decreased CD4+ T cell function during the first cycle of therapy. Methods: A phase II trial stratified metastatic mel patients into two cohorts based on whether or not they had stage IV M1c. 25 patients/cohort are planned. No brain metastases or prior chemotherapy were allowed. Each cycle consisted of TMZ 75mg/m2/day for 6 weeks followed by 2 weeks with no treatment. Primary endpoint was the proportion of objective responses by RECIST criteria. The trial was powered to exclude the null hypothesis that the response rate in each cohort was ≤10%. MGMT promoter methylation status was assessed by methylation-specific PCR. MGMT tumor expression was measured by immunohistochemistry (IHC). CD4+ T cell function was assessed by measuring interferon-γ responses to CMV by intracellular cytokine FACS analysis (FastImmune, BD Biosciences) at pretreatment, week 4 and week 8 of cycle 1. Results: To date 19 evaluable pts, 8 in the M1c cohort, have progressed with no objective responses; 6 other patients are currently on study. 4/19 pts who have gone off study because of progression were stable for 6 months and received ≥3 cycles. To date, 14/15 tumors analyzed for MGMT promoter methylation were unmethylated (≤15%). Promoter methylation of 21% was seen in one tumor. Conclusions: The MGMT promoter of metastatic mel tumors are hypomethylated predicting expression of the enzyme. This is being assessed directly by IHC and will be correlated with promoter methylation and clinical response. The effect of TMZ on anti-CMV CD4+ T cell function is being assessed. Accrual is ongoing and will be completed in Spring 2006. The study was supported by Schering-Plough. No significant financial relationships to disclose.

Cisplatin represses transcriptional activity from the minimal promoter of the O6-methylguanine methyltransferase gene and increases sensitivity of human gallbladder cancer cells to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-2-chloroethyl)-3-nitrosourea

O(6)-methylguanine methyltransferase (MGMT) repairs O(6)-alkylguanine in cellular DNA introduced by the clinically used alkylating drug 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Thus, cancer cells with MGMT expression are resistant to ACNU treatment. Cisplatin has been reported to suppress MGMT expression; however, the molecular mechanism by which cisplatin reduces MGMT expression remains to be elucidated. Using gallbladder cancer cells (KMG-C) expressing MGMT, we analyzed whether a low dose of cisplatin suppresses MGMT expression, followed by an enhanced drug effect of ACNU in vitro and in vivo. We also investigated the promoter region critical for the transcriptional repression of MGMT gene by cisplatin using 5 deletion mutants in reporter promoter assays. In RT-PCR analysis, the expression of MGMT mRNA in KMG-C cells was dose- and time-dependently repressed. Drug sensitivity to ACNU was increased 2-fold by pretreatment with cisplatin, compared with only ACNU treatment, in MTT assays as well as tumor-bearing nude mice. Although the 5'-flanking region is deleted as far as -69 bp upstream of the transcription start site, cisplatin dose dependently inhibited luciferase activity. However, cisplatin did not cause such repression when 59 bp region from -69 to -10 bp was deleted. We confirmed that cisplatin enhanced sensitivity to ACNU in KMG-C cells expressing MGMT both in vitro and in vivo. Furthermore, a low dose of cisplatin repressed the transcription of the MGMT promoter. The 59 bp region in the MGMT promoter was crucial for repression by cisplatin. These results might form the basis of a chemotherapeutic strategy involving alkylating agents via prior cisplatin-induced biochemical modulation.