David Piccioni

Combined analysis of O6-methylguanine-DNA methyltransferase protein expression and promoter methylation provides optimized prognostication of glioblastoma outcome

BACKGROUND Promoter methylation of the DNA repair gene, O-6-methylguanine-DNA methyltransferase (MGMT), is associated with improved treatment outcome for newly diagnosed glioblastoma (GBM) treated with standard chemoradiation. To determine the prognostic significance of MGMT protein expression as assessed by immunohistochemistry (IHC) and its relationship with methylation, we analyzed MGMT expression and promoter methylation with survival in a retrospective patient cohort. METHODS We identified 418 patients with newly diagnosed GBM at University of California Los Angeles Kaiser Permanente Los Angeles, nearly all of whom received chemoradiation, and determined MGMT expression by IHC, and MGMT promoter methylation by methylation-specific PCR (MSP) and bisulfite sequencing (BiSEQ) of 24 neighboring CpG sites. RESULTS With use of the median percentage of cells staining by IHC as the threshold, patients with <30% staining had progression-free survival (PFS) of 10.9 months and overall survival (OS) of 20.5 months, compared with PFS of 7.8 months (P < .0001) and OS of 16.7 months (P < .0001) among patients with ≥30% staining. Inter- and intrareader correlation of IHC staining was high. Promoter methylation status by MSP was correlated with IHC staining. However, low IHC staining was frequently observed in the absence of promoter methylation. Increased methylation density determined by BiSEQ correlated with both decreased IHC staining and increased survival, providing a practical semiquantitative alternative to MSP. On the basis of multivariate analysis validated by bootstrap analysis, patients with tandem promoter methylation and low expression demonstrated improved OS and PFS, compared with the other combinations. CONCLUSIONS Optimal assessment of MGMT status as a prognostic biomarker for patients with newly diagnosed GBM treated with chemoradiation requires determination of both promoter methylation and IHC protein expression.

Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases

BackgroundPrimary and secondary brain cancers are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. Recent observations reveal that the microvascular endothelium of primary high-grade gliomas expresses prostate specific membrane antigen (PSMA). Breast cancers express PSMA and they frequently form secondary brain tumors. Hence we report here our pilot study addressing the feasibility of PSMA targeting in brain and metastatic breast tumors, by examining PSMA levels in all glioma grades (19 patients) and in breast cancer brain metastases (5 patients).MethodsTumor specimens were acquired from archival material and normal brain tissues from autopsies. Tissue were stained and probed for PSMA, and the expression levels imaged and quantified using automated hardware and software. PSMA staining intensities of glioma subtypes, breast tumors, and breast tumor brain metastases were compared statistically versus normals.ResultsNormal brain microvessels (4 autopsies) did not stain for PSMA, while a small proportion (<5%) of healthy neurons stained, and were surrounded by an intact blood brain barrier. Tumor microvessels of the highly angiogenic grade IV gliomas showed intense PSMA staining which varied between patients and was significantly higher (p < 0.05) than normal brain. Grade I gliomas showed moderate vessel staining, while grade II and III gliomas had no vessel staining, but a few (<2%) of the tumor cells stained. Both primary breast cancer tissues and the associated brain metastases exhibited vascular PSMA staining, although the intensity of staining was generally less for the metastatic lesions.ConclusionsOur results align with and extend previous data showing PSMA expression in blood vessels of gliomas and breast cancer brain metastases. These results provide a rationale for more comprehensive studies to explore PSMA targeted agents for treating secondary brain tumors with PSMA expressing vasculature. Moreover, given that PSMA participates in angiogenesis, cell signaling, tumor survival, and invasion, characterizing its expression may help guide later investigations of the poorly understood process of low grade glioma progression to glioblastoma.

Phase 1 trial of vocimagene amiretrorepvec and 5-fluorocytosine for recurrent high-grade glioma

Toca 511 and Toca FC show promising results in treating recurrent high-grade glioma, and a specific molecular signature correlates with treatment-related survival. Tag-team attack on glioma Toca FC (extended-release 5-fluorocytosine) and Toca 511 (vocimagene amiretrorepvec) are an investigational therapeutic combination for glioma, consisting of two parts: a prodrug that is inactive on its own and a modified virus that infects the tumor and delivers an enzyme, which then activates the drug and allows it to kill the glioma cells. Cloughesy et al. tested this therapy in 45 human patients with recurrent or progressive high-grade glioma and discovered that the treatment was well tolerated and improved survival compared to an external control group. In addition, the authors identified a gene signature that correlated with response to the treatment, which may help identify the patients most likely to benefit from this approach. Toca 511 (vocimagene amiretrorepvec) is an investigational nonlytic, retroviral replicating vector (RRV) that delivers a yeast cytosine deaminase, which converts subsequently administered courses of the investigational prodrug Toca FC (extended-release 5-fluorocytosine) into the antimetabolite 5-fluorouracil. Forty-five subjects with recurrent or progressive high-grade glioma were treated. The end points of this phase 1, open-label, ascending dose, multicenter trial included safety, efficacy, and molecular profiling; survival was compared to a matching subgroup from an external control. Overall survival for recurrent high-grade glioma was 13.6 months (95% confidence interval, 10.8 to 20.0) and was statistically improved relative to an external control (hazard ratio, 0.45; P = 0.003). Tumor samples from subjects surviving more than 52 weeks after Toca 511 delivery disproportionately displayed a survival-related mRNA expression signature, identifying a potential molecular signature that may correlate with treatment-related survival rather than being prognostic. Toca 511 and Toca FC show excellent tolerability, with RRV persisting in the tumor and RRV control systemically. The favorable assessment of Toca 511 and Toca FC supports confirmation in a randomized phase 2/3 trial (NCT02414165).

Use of Liquid Biopsies in Clinical Oncology: Pilot Experience in 168 Patients

Purpose: There is a growing interest in using circulating tumor DNA (ctDNA) testing in patients with cancer. Experimental Design: A total of 168 patients with diverse cancers were analyzed. Patients had digital next-generation sequencing (54 cancer-related gene panel including amplifications in ERBB2, EGFR, and MET) performed on their plasma. Type of genomic alterations, potential actionability, concordance with tissue testing, and patient outcome were examined. Results: Fifty-eight percent of patients (98/168) had ≥1 ctDNA alteration(s). Of the 98 patients with alterations, 71.4% had ≥ 1 alteration potentially actionable by an FDA-approved drug. The median time interval between the tissue biopsy and the blood draw was 2.7 months for patients with ≥ 1 alteration in common compared with 14.4 months (P = 0.006) for the patients in whom no common alterations were identified in the tissue and plasma. Overall concordance rates for tissue and ctDNA were 70.3% for TP53 and EGFR, 88.1% for PIK3CA, and 93.1% for ERBB2 alterations. There was a significant correlation between the cases with ≥ 1 alteration with ctDNA ≥ 5% and shorter survival (median = 4.03 months vs. not reached at median follow-up of 6.1 months; P < 0.001). Finally, 5 of the 12 evaluable patients (42%) matched to a treatment targeting an alteration(s) detected in their ctDNA test achieved stable disease ≥ 6 months/partial remission compared with 2 of 28 patients (7.1%) for the unmatched patients, P = 0.02. Conclusions: Our initial study demonstrates that ctDNA tests provide information complementary to that in tissue biopsies and may be useful in determining prognosis and treatment. Clin Cancer Res; 22(22); 5497–505. ©2016 AACR.

Deferred use of bevacizumab for recurrent glioblastoma is not associated with diminished efficacy

BACKGROUND The optimal timing to initiate bevacizumab (BV) therapy for recurrent glioblastoma (GBM) is currently unclear. To address this issue, we examined progression-free survival (PFS) and survival time (ST) in a large retrospective cohort of GBM patients treated with BV at different recurrences. METHODS We identified 468 primary GBM patients who underwent biopsy or surgery followed by radiation therapy and temozolomide (RT/TMZ), and then received BV. PFS and ST were compared between patients stratified by the recurrence that BV was initiated (upfront, first recurrence, second recurrence, or 3+ recurrences). We also examined the effect on PFS and ST of the addition of chemotherapy to BV. In a larger cohort of GBM patients, we determined overall treatment continuation rates at each recurrence and identified variables predictive of inability to continue treatment. RESULTS BV PFS was similar for all 3 recurrence groups (median, 4.1 months). There were no differences in BV ST (median, 9.8 months). The addition of chemotherapy to BV improved PFS but not ST. Analysis of treatment continuation rates indicated that the number of patients unable to undergo further treatments is modest, and that patients unable to tolerate BV delay can be identified by age ≥60 years and low extent of resection. CONCLUSIONS Deferred use of bevacizumab is not associated with diminished efficacy. Analysis of treatment continuation rates identified patients who may be unable to delay BV therapy. Our findings suggest that there is a fixed survival after BV initiation and that delayed BV treatment is preferable for most patients.

On the Road to Precision Cancer Medicine: Analysis of Genomic Biomarker Actionability in 439 Patients

Despite the increased use of molecular diagnostics, the extent to which patients who have these tests harbor potentially actionable aberrations is unclear. We retrospectively reviewed 439 patients with diverse cancers, for whom next-generation sequencing (mostly 236-gene panel) had been performed. Data pertaining to the molecular alterations identified, as well as associated treatment suggestions (on- or off-label, or experimental), were extracted from molecular diagnostic reports. Most patients (420/439; 96%) had at least one molecular alteration: 1,813 alterations (in 207 distinct genes) were identified [the majority being mutations (62%) or amplifications (29%)]. The three most common gene abnormalities were TP53 (44%), KRAS (16%), and PIK3CA (12%). The median number of alterations per patient was 3 (range, 0–16). Nineteen patients (4%) had no alterations; 48 patients (11%) had only one alteration; and 372 patients had two or more abnormalities (85%). The median number of potentially actionable anomalies per patient was 2 (range, 0–8). Most patients (393/439; 90%) had at least one potentially actionable alteration, and in all these cases the aberration could at least be targeted by an experimental drug in a clinical trial. A total of 307 patients (70%) had an alteration that was actionable with an approved drug, but in only 89 patients (20%) was the drug approved for their disease (on-label). Next-generation sequencing identified theoretically actionable aberrations in 90% of our patients. Many of the drugs are, however, experimental or would require off-label use. Strategies to address drug access for patients harboring potentially actionable mutations are needed. Mol Cancer Ther; 14(6); 1488–94. ©2015 AACR.

Identification of Retinol Binding Protein 1 Promoter Hypermethylation in Isocitrate Dehydrogenase 1 and 2 Mutant Gliomas

BACKGROUND Mutations in isocitrate dehydrogenase 1 (IDH1) and associated CpG island hypermethylation represent early events in the development of low-grade gliomas and secondary glioblastomas. To identify candidate tumor suppressor genes whose promoter methylation may contribute to gliomagenesis, we compared methylation profiles of IDH1 mutant (MUT) and IDH1 wild-type (WT) tumors using massively parallel reduced representation bisulfite sequencing. METHODS Reduced representation bisulfite sequencing was performed on ten pathologically matched WT and MUT glioma samples and compared with data from a methylation-sensitive restriction enzyme technique and data from The Cancer Genome Atlas (TCGA). Methylation in the gene retinol-binding protein 1 (RBP1) was identified in IDH1 mutant tumors and further analyzed with primer-based bisulfite sequencing. Correlation between IDH1/IDH2 mutation status and RBP1 methylation was evaluated with Spearman correlation. Survival data were collected retrospectively and analyzed with Kaplan-Meier and Cox proportional hazards analysis. All statistical tests were two-sided. RESULTS Methylome analysis identified coordinated CpG island hypermethylation in IDH1 MUT gliomas, consistent with previous reports. RBP1, important in retinoic acid metabolism, was found to be hypermethylated in 76 of 79 IDH1 MUT, 3 of 3 IDH2 MUT, and 0 of 116 IDH1/IDH2 WT tumors. IDH1/IDH2 mutation was highly correlated with RBP1 hypermethylation (n = 198; Spearman R = 0.94, 95% confidence interval = 0.92 to 0.95, P < .001). The Cancer Genome Atlas showed IDH1 MUT tumors (n = 23) to be RBP1-hypermethylated with decreased RBP1 expression compared with WT tumors (n = 124). Among patients with primary glioblastoma, patients with RBP1-unmethylated tumors (n = 102) had decreased median overall survival compared with patients with RBP1-methylated tumors (n = 22) (20.3 months vs 36.8 months, respectively; hazard ratio of death = 2.48, 95% confidence interval = 1.30 to 4.75, P = .006). CONCLUSION RBP1 promoter hypermethylation is found in nearly all IDH1 and IDH2 mutant gliomas and is associated with improved patient survival. Because RBP1 is involved in retinoic acid synthesis, our results suggest that dysregulation of retinoic acid metabolism may contribute to glioma formation along the IDH1/IDH2-mutant pathway.

Detection of 2-hydroxyglutaric acid in vivo by proton magnetic resonance spectroscopy in U87 glioma cells overexpressing isocitrate dehydrogenase-1 mutation

The arginine 132 (R132) mutation of isocitrate dehydrogenase -1 (IDH1(R132)) results in production of 2-hydroxyglutarate (2-HG) and is associated with a better prognosis compared with wild-type (WT) in glioma patients. The majority of lower-grade gliomas express IDH1(R132), whereas this mutation is rare in grade IV gliomas. The aim of this study was to noninvasively investigate metabolic and physiologic changes associated with the IDH1 mutation in a mouse glioma model. Using a 7T magnet, we compared MRI and proton magnetic resonance spectroscopy (MRS) in U87 glioma cells overexpressing either the mutated IDH1(R132) or IDH1 wild-type (IDH1(WT)) gene in a mouse flank xenograft model. Flank tumors overexpressing IDH1(R132) showed a resonance at 2.25 ppm corresponding to the 2-HG peak described for human IDH1(R132) gliomas. WT tumors lacked this peak in all cases. IDH1 mutant tumors demonstrated significantly reduced glutamate by in vivo MRS. There were no significant differences in T(2), apparent diffusion coefficient (ADC), or perfusion values between the mutant and IDH1(WT) tumors. The IDH1(R132) mutation results in 2-HG resonance at 2.25 ppm and a reduction of glutamate levels as determined by MRS. Our results establish a model system where 2-HG can be monitored noninvasively, which should be helpful in validating 2-HG levels as a prognostic and/or predictive biomarker in glioma.

Fibroblast growth factor family aberrations in cancers: clinical and molecular characteristics

Fibroblast growth factor ligands and receptors (FGF and FGFR) play critical roles in tumorigenesis, and several drugs have been developed to target them. We report the biologic correlates of FGF/FGFR abnormalities in diverse malignancies. The medical records of patients with cancers that underwent targeted next generation sequencing (182 or 236 cancer-related genes) were reviewed. The following FGF/FGFR genes were tested: FGF3, 4, 6, 7, 10, 12, 14, 19, 23 and FGFR1, 2, 3, and 4. Of 391 patients, 56 (14.3%) had aberrant FGF (N = 38, all amplifications) and/or FGFR (N = 22 including 5 mutations and one FGFR3-TACC3 fusion). FGF/FGFR aberrations were most frequent in breast cancers (26/81, 32.1%, p = 0.0003). In multivariate analysis, FGF/FGFR abnormalities were independently associated with CCND1/2, RICTOR, ZNF703, RPTOR, AKT2, and CDK8 alterations (all P < 0.02), as well as with an increased median number of alterations (P < 0.0001). FGF3, FGF4, FGF19 and CCND1 were co-amplified in 22 of 391 patients (5.6%, P < 0.0001), most likely because they co-localize on the same chromosomal region (11q13). There was no significant difference in time to metastasis or overall survival when comparing patients harboring FGF/FGFR alterations versus those not. Overall, FGF/FGFR was one of the most frequently aberrant pathways in our population comprising patients with diverse malignancies. These aberrations frequently co-exist with anomalies in a variety of other genes, suggesting that tailored combination therapy may be necessary in these patients.

Cyclin-dependent kinase pathway aberrations in diverse malignancies: clinical and molecular characteristics

Aberrations in the cyclin-dependent kinase (CDK) pathways that regulate the cell cycle restriction point contribute to genomic instability and tumor proliferation, and can be targeted by recently developed CDK inhibitors. We therefore investigated the clinical correlates of CDK4/6 and CDKN2A/B abnormalities in diverse malignancies. Patients with various cancers who underwent molecular profiling by targeted next generation sequencing (Foundation Medicine; 182 or 236 cancer-related genes) were reviewed. Of 347 patients analyzed, 79 (22.8%) had aberrant CDK 4/6 or CDKN2A/B. Only TP53 mutations occurred more frequently than those in CDK elements. Aberrations were most frequent in glioblastomas (21/26 patients; 81%) and least frequent in colorectal cancers (0/26 patients). Aberrant CDK elements were independently associated with EGFR and ARID1A gene abnormalities (P < 0.0001 and p = 0.01; multivariate analysis). CDK aberrations were associated with poor overall survival (univariate analysis; HR[95% CI] = 2.09 [1.35–4.70]; p = 0.004). In multivariate analysis, PTEN and TP53 aberrations were independently associated with poorer survival (HR = 4.83 and 1.92; P < 0.0001 and p = 0.01); CDK aberrations showed a trend toward worse survival (HR = 1.67; p = 0.09). There was also a trend toward worse progression-free survival (PFS) with platinum-containing regimens in patients with abnormal CDK elements (3.5 versus 5.0 months, p = 0.13). In conclusion, aberrations in the CDK pathway were some of the most common in cancer and independently associated with EGFR and ARID1A alterations. Patients with abnormal CDK pathway genes showed a trend toward poorer survival, as well as worse PFS on platinum-containing regimens. Further investigation of the prognostic and predictive impact of CDK alterations across cancers is warranted.