Cisplatin and carboplatin are widely used clinical chemotherapeutic agents, especially against testicular, ovarian, and head and neck cancers. O6-Benzylguanine (BG) has been shown to result in enhanced cytotoxicity, apoptosis, and DNA platination when used in conjunction with cisplatin and carboplatin in head and neck cancer cell lines. Microarray expression data indicated overexpression of 19 genes and underexpression of 22 genes specific to treatment with the combination of BG±cisplatin compared to cisplatin alone treatment in SQ20b head and neck cancer cells (p<0.05) using the Affymetrix HG-U133A GeneChip®. Among the overexpressed probe sets were genes involved in DNA damage and apoptosis, including GADD34, DDIT4, ATF4, and PTHLH. A similarly structured analog, 9-CH3-BG, does not enhance cisplatin-induced cytotoxicity or apoptosis nor is there enhanced expression of GADD34 in cisplatin or 9-CH3-BG±cisplatin-treated cells compared to control cells. Analysis of cells exposed to 9-CH3-BG±cisplatin allowed us to focus our array list on 32 probe sets specific to BG+cisplatin versus cisplatin, ruling out differentially expressed probe sets common to 9-CH3-BG+cisplatin versus cisplatin. Similarly, 14 probe sets were specific to BG±cisplatin versus BG, ruling out differentially expressed probe sets common to 9-CH3-BG±cisplatin versus 9-CH3-BG. Quantitative real-time PCR demonstrated a dose dependent increase in GADD34 expression in cells exposed to BG±cisplatin with levels approximately >2-fold for cells exposed to BG+cisplatin compared to cisplatin alone. Levels of GADD34 transcripts were determined with both cisplatin and BG+cisplatin at several different time points concomitant with and following drug treatment. At all timepoints, GADD34 transcript levels are approximately two-fold elevated in cells treated with BG+cisplatin compared to cisplatin alone. Furthermore, significant changes in GADD34 expression levels in SQ20b, SCC35, and SCC61 cells, with approximately three-fold, two-fold, and 3.5-fold increases in expression, respectively, upon treatment with BG±cisplatin compared with control. Elucidation of these molecular pathways will aid in our goal of synthesizing more powerful modulators to increase efficacy of platinum agents.

Pharmacological inhibitors of cyclin-dependent kinase (CDK)2 are currently in preclinical and clinical development. The purpose of our work was to evaluate a series of guanine derivatives for their ability to inhibit CDK2, affect cell cycle progression, and enhance the cytotoxic and apoptotic effects of cisplatin. A panel of guanine derivatives, including O(6)-benzylguanine (O(6)-BG), S(6)-benzyl-6-thioguanine (S(6)-BG), S(6)-[(cyclohexyl)methyl]-6-thioguanine (S(6)-CMG), O(6)-[(cyclohexyl)methyl]guanine (O(6)-CMG), O(6)-benzyl-9-methylguanine (9-CH(3)-BG), O(6)-[(cyclohexyl)methyl]-9-methyl-guanine (9-CH(3)-CMG), and 7-benzylguanine (N7-BG), exhibited varying degrees of CDK2 inhibition with O(6)-CMG being the most potent and 9-CH(3)-BG, 9-CH(3)-CMG, and N7-BG the least potent compounds. Treatment with S(6)-CMG and O(6)-CMG significantly decreased the percentage of cells in S phase. In SQ20b and SCC61 head and neck cancer cell lines, the most potent CDK2 inhibitor, O(6)-CMG, was also the most effective at enhancing cisplatin-induced cytotoxicity and apoptosis. Cisplatin-induced DNA platination increased in SQ20b cells pretreated with S(6)-BG, S(6)-CMG, and O(6)-CMG. Treatment with both O(6)-BG and trichostatin A, an indirect cell cycle inhibitor, demonstrated additive effects on cisplatin-induced cytotoxicity. In summary, we have identified a group of guanine derivatives that were effective modulators of cisplatin-induced cytotoxicity and apoptosis.

CONTEXT:TGFBR1*6A is a common polymorphism of the type I transforming growth factor beta receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown.OBJECTIVES:To determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3-amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells.DESIGN, SETTING, AND PATIENTS:Tumor and germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004. In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A.MAIN OUTCOME MEASURES:TGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-beta-dependent cell proliferation.RESULTS:TGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-beta growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells.CONCLUSIONS:TGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-beta. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-beta signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

Purpose We conducted a two-phase clinical trial in patients with progressive malignant glioma (MG). The first phase of this trial was designed to determine the dose of O6-BG effective in producing complete depletion of tumor AGT activity for 48 hours. The second phase of the trial was designed to define the maximum tolerated dose (MTD) of a single dose of temozolomide when combined with O6-BG. In addition, plasma concentrations of O6-BG and O6-benzyl-8-oxoguanine were evaluated after O6-BG. Patients and Methods For our first phase of the clinical trial, patients were scheduled to undergo craniotomy for AGT determination after receiving a 1-hour O6-BG infusion at 120 mg/m2 followed by a continuous infusion at an initial dose of 30 mg/m2/d for 48 hours. The dose of the continuous infusion of O6-BG escalated until tumor AGT was depleted. Once the O6-BG dose was established a separate group of patients was enrolled in the second phase of clinical trial, in which temozolomide, administered as a single dose at the end of the 1-hour O6-BG infusion, was escalated until the MTD was determined. Results The O6-BG dose found to be effective in depleting tumor AGT activity at 48 hours was an IV bolus of 120 mg/m2 over 1 hour followed by a continuous infusion of 30 mg/m2/d for 48 hours. On enrolling 38 patients in six dose levels of temozolomide, the MTD was established at 472 mg/m2 with dose-limiting toxicities limited to myelosuppression. Conclusion This study provides the foundation for a phase II trial of O6-BG plus temozolomide in temozolomide-resistant MG.

Background Our aim was to identify candidate genes and genetic variants involved in cellular susceptibility to chemotherapeutic agents without a priori assumptions about the genes. To date, research has focused on known candidate genes involved in pharmacokinetic and pharmacodynamic pathways for specific chemotherapy. Methods Three-generation CEPH pedigrees were used to evaluate the genetic contribution to cellular growth inhibition by exposing the cells to increasing concentrations of cisplatin for 48 hours or carboplatin for 72 hours. Results The heritability of cisplatin- and carboplatin-induced cytotoxicity was found to be between 0.38–0.47 (p<0.0001) and 0.36–1.0 (p<0.02), respectively. The most significant findings from linkage analysis for cisplatin were on chromosome 1 at 44 cM in variance components analysis and chromosome 12 at 147 cM in nonparametric linkage analysis. Candidate genes within a 1-lod confidence interval surrounding the peaks on chromosome 1 (188 genes) and 12 (106 genes) included CASP9, SFN, STMN1, UBC, POLE and ZNF84. Using expression array, we compared gene expression differences at baseline and changes over time following treatment with cisplatin. Genes common to linkage analysis and expression array included ZNF84, RERE, NPPB, and SFRS8. Conclusions These data show the power of using large, extensively genotyped pedigrees with microarray analysis for evaluating the genetic contribution to sensitivity of cell growth inhibition by anticancer agents. Clinical Pharmacology & Therapeutics (2005) 77, P4–P4; doi: 10.1016/j.clpt.2004.11.018

Temozolomide is a DNA-methylating agent used in the treatment of malignant gliomas. In this study, we have examined if inhibition of poly(ADP-ribose) polymerase (PARP) could increase the cytotoxicity of temozolomide, particularly in cells deficient in DNA mismatch repair. Athymic mice, transplanted with mismatch repair–proficient [D-245 MG] or deficient [D-245 MG (PR)] xenografts, were treated with a combination of temozolomide and the PARP inhibitor, INO-1001. For the tumors deficient in mismatch repair, the most effective dose of INO-1001 was found to be 150 mg/kg, given i.p. thrice at 4-hour intervals with the first injection in combination with 262.5 mg/kg temozolomide (0.75 LD10). This dose of temozolomide by itself induced no partial regressions and a 4-day growth delay. In two separate experiments, the combination therapy increased the growth delay by 21.6 and 9.7 days with partial regressions observed in four of eight and three of nine mice, respectively. The addition of INO-1001 had a more modest, yet statistically significant, increase in tumor growth delay in the mismatch repair–proficient xenografts. In these experiments, mice were treated with a lower amount of temozolomide (88 mg/kg), which resulted in growth delays of 43.1 and 39.2 days. When the temozolomide treatment was in combination with 200 mg/kg INO-1001, there was an increase in growth delay to 48.9 and 45.7 days, respectively. These results suggest that inhibition of PARP may increase the efficacy of temozolomide in the treatment of malignant gliomas, particularly in tumors deficient in DNA mismatch repair.

The epidermal growth factor receptor (EGFR) plays a prominent role in cell growth and development. Its regulation in humans is complex and incompletely understood. In this study, 12 new polymorphisms were discovered in the 5'-regulatory region of EGFR gene and 2 common single nucleotide polymorphisms (-216G/T and -191C/A) were found in the essential promoter area, one of which is located in a Sp1 recognition site (-216). Transient transfection in human cancer and primary cell lines showed significantly different promoter activity between the two most common haplotypes (-216G-191C and -216T-191C). The replacement of G by T at position -216 increases the promoter activity by 30%. A transient transfection assay in the Sp1-deficient cell line (Schneider cell line 2) showed a strong dependence of EGFR promoter activity on Sp1 and confirmed the effect of the aforementioned polymorphisms. Electrophoretic mobility shift assay also showed a significantly higher binding efficiency of nuclear protein or pure Sp1 protein to the T allele compared with the G allele. We then investigated the allelic imbalance of EGFR transcription in fibroblast cell lines with heterozygous genotype at -216G/T but C/C homozygous genotype at -191C/A. The expression of mRNA carrying T-C haplotype was significantly stronger compared with that of G-C haplotype (P < 0.02). Thus, we successfully showed that a common polymorphism in the EGFR promoter was associated with altered promoter activity and gene expression both in vitro and in vivo. Our findings have implications for cancer etiology and therapy and may also be relevant to the inherited susceptibility of other common diseases.

2010 Background: CPT-11 is oxidized to inactivated metabolites (including APC) by CYP3A enzymes and activated to SN-38 by carboxylesterase-2 (CES-2). SN-38 is inactivated to it glucuronide (SN-38G) by UGT1A1 and UGT1A9. Other enzymes and ABC transporters are involved in pathways of CPT-11 disposition. We had previously measured the plasma PK of patients treated with CPT-11 and analyzed the correlation with UGT1A1 variants. This study aims to generate hypotheses regarding the phenotypic effects of variation in other drug metabolizing and transporter genes. Methods: CPT-11, SN-38, and SN-38G AUCs were measured after a 350 mg/m2 IV dose in 64 adults with refractory solid tumors as part of a pharmacogenetic study. Genotyping of common variants (q>0.10) was performed for the following genes (number of variants in parenthesis): CES-2 (n=2), ABCC1 (n=7), ABCC2 (n=6), and ABCB1 (n=8). CYP3A4*1B, CYP3A5*3, UGT1A9 -11810T/9T and HNF1 79A>C were also typed. Results: The synonymous 3972T>C variant in ABCC2 was correlated with CPT-11 AUC (p=0.02), APC AUC (p<0.0001) and APC/CPT-11 AUC ratios (p<0.0001, ANOVA). For SN-38 and SN-38G AUC, the correlation with 3972T>C was analyzed in the subset of patients with 6/6 and 6/7 UGT1A1 genotype (n=54) to minimize the confounding effect of 7/7. SN-38G and SN-38G/SN-38 AUC ratios were correlated with 3972T>C (p≤0.001, ANOVA). No significant correlation was observed between SN-38 AUC and 3972T>C (p=0.9). Other gene variants showed either no or borderline statistical significance in the ANOVA test. The TT 3972 genotype was associated with higher AUC of CPT-11 (p=0.02), APC and SN-38G (both p<0.0001, t test) compared to CT+CC patients. The frequencies of CC, CT, and TT genotypes were 0.44, 0.44, and 0.13, respectively. Conclusions: This study identifies 3972T>C as a variant potentially affecting ABCC2 activity and suggests that ABCC2 may have a significant impact on CPT-11 clearance. These results also suggest that APC may be a substrate for ABCC2. The phenotypic effect of 3972T>C was previously unknown and further studies will test its biological function and clinical relevance. No significant financial relationships to disclose.

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Carboxylesterases are members of the serine esterase super family important in the metabolism of a wide variety of substrates, including xenobiotics and prodrugs. There are two known carboxylesterases expressed in human liver, small intestine and other tissues, carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2). The aim of this study was to identify polymorphisms in the CES2 gene and determine whether these polymorphisms affect expression levels of CES2 or rate of metabolism of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin). Microsome samples prepared from liver tissues of 78 normal individuals were used to determine the rate of hydrolysis of irinotecan and procaine (an anaesthetic hydrolysed by CES2 but not CES1). The rate of hydrolysis of irinotecan is highly variable among individuals, ranging from 2.7-138 pmol/mg protein/h (mean +/- SD 26.0 +/- 22.9). Fifteen single nucleotide polymorphisms (SNPs) were identified, one is in an exon, 9 are in introns, three are in the 3'-untranslated region (UTR), and two are in the 5'-flanking region. Eight of the 15 SNP loci have rare allele frequencies greater than 5%, of which three were greater than 20%. Genotyping of samples from the SNP Consortium demonstrated different distributions among African-Americans, Asian-Americans and European-Americans. We also analysed the haplotype structure and estimated linkage disequilibrium (LD). A SNP located in the 5'-UTR (5'-UTR-363) was found in LD with loci in intron 1 (Intron1 + 947, Intron1 + 1361, Intron1 + 1643). Haplotypes with homozygous rare alleles on these loci exhibit lower mRNA levels as determined by real time polymerase chain reaction (P < 0.01) and the incorporation of rare alleles in haplotypes correlate with reduced mRNA (P = 0.03). The 5'-UTR-363 SNP is located in one of the three promoters of CES2. However, we did not observe significant differences in CES2 activities (irinotecan and procaine hydrolysis) among individuals with different haplotypes.

Objectives To determine the activity of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) and MGMT promoter methylation status of pediatric rhabdomyosarcoma (RMS) and examine MGMT in RMS tumors from different prognostic groups. Methods Fifteen samples each of the alveolar (ARMS) and embryonal (ERMS) subtypes were obtained for analysis of MGMT activity and promoter methylation status. MGMT activity was assayed by measuring the removal of O6-[3H] methylguanine from [3H]-methylated substrate by a tumor extract containing the enzyme. Promoter methylation status was examined using methylation-specific polymerase chain reaction (PCR). Results MGMT activity was successfully assayed from 25 samples, 10 ERMS and 15 ARMS. All ERMS and 11 of the 15 ARMS samples displayed high activity levels. There was significant intertumor variability among both subtypes but no significant difference in mean activity between the two histologic groups. There were trends toward increased activity in ERMS tumors and tumors from anatomically unfavorable locations. Only one tumor was hypermethylated at the MGMT promoter region. Conclusions This analysis suggests that a low percentage of RMS samples are hypermethylated at the MGMT promoter and that most have significant MGMT activity, implying that clinical trials with MGMT-modulating agents may have a role in the treatment of these tumors. This analysis does not support MGMT activity as an explanation of the differential response to chemotherapy demonstrated by ARMS and ERMS, but does suggest that MGMT may be involved in RMS treatment failure regardless of subtype and in the poorer response shown by tumors from unfavorable locations.

O(6)-Benzylguanine (O(6)-BG), a potent inactivator of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), is presently in clinical trials combined with alkylating agents that modify the O(6) position of DNA guanine residues, i.e., 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Previous work demonstrated that O(6)-BG also enhances the cytotoxicity of cyclophosphamide, ifosfamide, and nitrogen mustards in Chinese hamster ovary cells. We have extended this study to include other clinically relevant agents that form interstrand and intrastrand cross-links including cisplatin and carboplatin. Pretreatment of a series of head and neck tumor cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon tumor cells with O(6)-BG (100 micro M for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED(50) of cisplatin and a concomitant increase in the percentage of cells undergoing apoptosis. The enhancement was independent of AGT activity. Similar enhancement was observed with carboplatin, but no enhancement was seen in AGT-deficient cell lines with radiation or temozolomide, demonstrating the dependence of the effect on bifunctional, cross-linking agents. Furthermore, levels of platinum on DNA after treatment with cisplatin increased 1.4-fold in SQ20b cells and 4.5-fold in JSQ3 cells immediately after treatment with O(6)-BG plus cisplatin and remained elevated for 48 h. Consistent with greater cytotoxicity and apoptosis is the approximately 2-fold higher amount of DNA damage when cells are treated with O(6)-BG plus cisplatin compared with cisplatin alone. Modulation of cisplatin therapy with O(6)-BG might improve the prognosis of patients with head and neck, ovarian, testicular, or lung cancer who are treated with this drug.

PURPOSE:O6-benzylguanine (BG) provides a means to effectively inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and increase the chemotherapeutic effectiveness of chloroethylating and methylating agents in preclinical and clinical studies. Two different doses of BG have been reported as the optimal biochemical modulatory dose for patients (i.e., 100 and 120 mg/m2). The objective of our study was to compare these doses by measuring AGT in surgically removed specimens after treatment with BG. EXPERIMENTAL DESIGN:BG was administered to patients as an i.v. infusion 16 +/- 4 h before surgical resection of their systemic tumor. AGT activity was measured in the tumor using a methylated DNA substrate. The target end point was defined as > or =11 of 13 patients with undetectable tumor AGT levels (<10 fmol/mg protein). RESULTS:Of the 28 patients enrolled, 25 of whom were analyzed for AGT activity, the most common primary sites of cancer included the colon (n = 11), bladder (n = 3), rectum (n = 4), and stomach (n = 3). Positive (DaOY cells) and negative (Chinese hamster ovary cells) control cell lines were included in each assay. Seven of the 12 patients treated with 100 mg/m2 BG had AGT activity of >10 fmol/mg protein (15-147 fmol/mg protein). Only 2 of the 13 patients treated with 120 mg/m2 BG had AGT activity of >10 fmol/mg protein (11 and 12 fmol/mg protein). CONCLUSIONS:From our surgically removed tissue data, a dose of 120 mg/m2 BG is recommended to deplete systemic tumors of AGT activity.

Much effort has been directed at identifying molecular markers in human tumors that predict response and/or survival after treatment with chemotherapeutic agents. Molecular predictors help to identify patients most likely to benefit from a particular therapy, with the ultimate goal of selecting optimal treatment for each patient. Gene microarray analysis has already identified genes that may be useful for predicting the clinical behavior of certain tumors (1,2). In malignant lymphoma, microarray analysis has clearly identified subgroups of tumors within the category of diffuse large B-cell lymphoma (B-DLCL) that are histologically indistinguishable but differ considerably in outcome after treatment with standard therapies (3). The specific genes responsible for the different outcomes have not yet been identified. In this issue of the Journal, Esteller et al. (4) describe a new predictive marker of survival in patients with B-cell lym