Andrea L. Russo

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: A preliminary report

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short‐term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post‐mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post‐mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol–chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein‐1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty‐three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = −5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post‐mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi‐quantitative RT‐PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

Differential DNA Hypermethylation of Critical Genes Mediates the Stage-Specific Tobacco Smoke-Induced Neoplastic Progression of Lung Cancer

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non–small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fishers exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fishers exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer–associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

In vitro and In vivo Radiosensitization of Glioblastoma Cells by the Poly (ADP-Ribose) Polymerase Inhibitor E7016

Purpose: Poly (ADP-ribose) polymerase (PARP) inhibitors are undergoing clinical evaluation for cancer therapy. Because PARP inhibition has been shown to enhance tumor cell sensitivity to radiation, we investigated the in vitro and in vivo effects of the novel PARP inhibitor E7016. Experimental Design: The effect of E7016 on the in vitro radiosensitivity of tumor cell lines was evaluated using clonogenic survival. DNA damage and repair were measured using γH2AX foci and neutral comet assay. Mitotic catastrophe was determined by immunostaining. Tumor growth delay was evaluated in mice for the effect of E7016 on in vivo (U251) tumor radiosensitivity. Results: Cell lines exposed to E7016 preirradiation yielded an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 from 1.4 to 1.7. To assess DNA double-strand breaks repair, γH2AX measured at 24 hours postirradiation had significantly more foci per cell in the E7016/irradiation group versus irradiation alone. Neutral comet assay further suggested unrepaired double-strand breaks with significantly greater DNA damage at 6 hours postirradiation in the combination group versus irradiation alone. Mitotic catastrophe staining revealed a significantly greater number of cells staining positive at 24 hours postirradiation in the combination group. In vivo, mice treated with E7016/irradiation/temozolomide had an additional growth delay of six days compared with the combination of temozolomide and irradiation. Conclusions: These results indicate that E7016 can enhance tumor cell radiosensitivity in vitro and in vivo through the inhibition of DNA repair. Moreover, enhanced growth delay with the addition of E7016 to temozolomide and radiotherapy in a glioma mouse model suggests a potential role for this drug in the treatment of glioblastoma multiforme.

Vorinostat enhances the radiosensitivity of a breast cancer brain metastatic cell line grown in vitro and as intracranial xenografts

Vorinostat (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor, is currently undergoing clinical evaluation as therapy for cancer. We investigated the effects of vorinostat on tumor cell radiosensitivity in a breast cancer brain metastasis model using MDA-MB-231-BR cells. In vitro radiosensitivity was evaluated using clonogenic assay. Cell cycle distribution and apoptosis was measured using flow cytometry. DNA damage and repair was evaluated using γH2AX. Mitotic catastrophe was measured by immunostaining. Growth delay and intracranial xenograft models were used to evaluate the in vivo tumor radiosensitivity. Cells exposed to vorinostat for 16 hours before and maintained in the medium after irradiation had an increase in radiosensitivity with a dose enhancement factor of 1.57. γH2AX, as an indicator of double-strand breaks, had significantly more foci per cell in the vorinostat plus irradiation group. Mitotic catastrophe, measured at 72 hours, was significantly increased in cells receiving vorinostat plus irradiation. Irradiation of s.c. MDA-MB-231-BR tumors in mice treated with vorinostat resulted in an increase in radiation-induced tumor growth delay. Most importantly, animals with intracranial tumor implants lived the longest after combination treatment. These results indicate that vorinostat enhances tumor cell radiosensitivity in vitro and in vivo. There was a greater than additive improvement in survival in our intracranial model. Combining vorinostat with radiation may be a potential treatment option for patients with breast cancer who develop brain metastases. [Mol Cancer Ther 2009;8(6):1589–95]

Loss of Heterozygosity Patterns Provide Fingerprints for Genetic Heterogeneity in Multistep Cancer Progression of Tobacco Smoke–Induced Non–Small Cell Lung Cancer

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non-small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P >0.05, Fishers exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P <0.05, Fishers exact test) occur at the later stages of non-small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P <0.05, Fishers exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.

Mutational analysis and clinical correlation of metastatic colorectal cancer

Early identification of mutations may guide patients with metastatic colorectal cancer toward targeted therapies that may be life prolonging. The authors assessed tumor genotype correlations with clinical characteristics to determine whether mutational profiling can account for clinical similarities, differences, and outcomes.

Urine Analysis and Protein Networking Identify Met as a Marker of Metastatic Prostate Cancer

Purpose: Metastatic prostate cancer is a major cause of death of men in the United States. Expression of met, a receptor tyrosine kinase, has been associated with progression of prostate cancer. Experimental Design: To investigate met as a biomarker of disease progression, urinary met was evaluated via ELISA in men with localized (n = 75) and metastatic (n = 81) prostate cancer. Boxplot analysis was used to compare the distribution of met values between each group. We estimated a receiver operating characteristic curve and the associated area under the curve to summarize the diagnostic accuracy of met for distinguishing between localized and metastatic disease. Protein-protein interaction networking via yeast two-hybrid technology supplemented by Ingenuity Pathway Analysis and Human Interactome was used to elucidate proteins and pathways related to met that may contribute to progression of disease. Results: Met distribution was significantly different between the metastatic group and the group with localized prostate cancer and people with no evidence of cancer (P < 0.0001). The area under the curve for localized and metastatic disease was 0.90, with a 95% confidence interval of 0.84 to 0.95. Yeast two-hybrid technology, Ingenuity Pathway Analysis, and Human Interactome identified 89 proteins that interact with met, of which 40 have previously been associated with metastatic prostate cancer. Conclusion: Urinary met may provide a noninvasive biomarker indicative of metastatic prostate cancer and may be a central regulator of multiple pathways involved in prostate cancer progression.

Clinical implementation of integrated whole-genome copy number and mutation profiling for glioblastoma

BACKGROUND Multidimensional genotyping of formalin-fixed paraffin-embedded (FFPE) samples has the potential to improve diagnostics and clinical trials for brain tumors, but prospective use in the clinical setting is not yet routine. We report our experience with implementing a multiplexed copy number and mutation-testing program in a diagnostic laboratory certified by the Clinical Laboratory Improvement Amendments. METHODS We collected and analyzed clinical testing results from whole-genome array comparative genomic hybridization (OncoCopy) of 420 brain tumors, including 148 glioblastomas. Mass spectrometry-based mutation genotyping (OncoMap, 471 mutations) was performed on 86 glioblastomas. RESULTS OncoCopy was successful in 99% of samples for which sufficient DNA was obtained (n = 415). All clinically relevant loci for glioblastomas were detected, including amplifications (EGFR, PDGFRA, MET) and deletions (EGFRvIII, PTEN, 1p/19q). Glioblastoma patients ≤40 years old had distinct profiles compared with patients >40 years. OncoMap testing reliably identified mutations in IDH1, TP53, and PTEN. Seventy-seven glioblastoma patients enrolled on trials, of whom 51% participated in targeted therapeutic trials where multiplex data informed eligibility or outcomes. Data integration identified patients with complete tumor suppressor inactivation, albeit rarely (5% of patients) due to lack of whole-gene coverage in OncoMap. CONCLUSIONS Combined use of multiplexed copy number and mutation detection from FFPE samples in the clinical setting can efficiently replace singleton tests for clinical diagnosis and prognosis in most settings. Our results support incorporation of these assays into clinical trials as integral biomarkers and their potential to impact interpretation of results. Limited tumor suppressor variant capture by targeted genotyping highlights the need for whole-gene sequencing in glioblastoma.

Low-Dose Involved-Field Radiation in the Treatment of Non-Hodgkin Lymphoma: Predictors of Response and Treatment Failure

PURPOSE To investigate clinical and pathologic factors significant in predicting local response and time to further treatment after low-dose involved-field radiation therapy (LD-IFRT) for non-Hodgkin lymphoma (NHL). METHODS AND MATERIALS Records of NHL patients treated at a single institution between April 2004 and September 2011 were retrospectively reviewed. Low-dose involved-field radiation therapy was given as 4 Gy in 2 fractions over 2 consecutive days. Treatment response and disease control were determined by radiographic studies and/or physical examination. A generalized estimating equation model was used to assess the effect of tumor and patient characteristics on disease response. A Cox proportional hazards regression model was used to assess time to further treatment. RESULTS We treated a total of 187 sites in 127 patients with LD-IFRT. Histologies included 66% follicular, 9% chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma, 10% marginal zone, 6% mantle cell lymphoma (MCL), and 8% other. Median follow-up time was 23.4 months (range, 0.03-92.2 months). The complete response, partial response, and overall response rates were 57%, 25%, and 82%, respectively. A CLL histology was associated with a lower response rate (odds ratio 0.2, 95% confidence interval 0.1-0.5, P=.02). Tumor size, site, age at diagnosis, and prior systemic therapy were not associated with response. The median time to first recurrence was 13.6 months. Those with CLL and age ≤ 50 years at diagnosis had a shorter time to further treatment for local failures (hazard ratio [HR] 3.63, P=.01 and HR 5.50, P=.02, respectively). Those with CLL and MCL had a shorter time to further treatment for distant failures (HR 11.1 and 16.3, respectively, P<.0001). CONCLUSIONS High local response rates were achieved with LD-IFRT across most histologies. Chronic lymphocytic leukemia and MCL histologies and age ≤ 50 years at diagnosis had a shorter time to further treatment after LD-IFRT.

Advancing age within established Gleason score categories and the risk of prostate cancer‐specific mortality (PCSM)

Study Type – Prognosis (case series)