Hypermethylation of regulatory sequences at the locus of the a%-el glutathione S-trnsferase gene GSTPI was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTPI expression was found to accompany human prostatic MATERIALS AND METHODS Immunohistochemical Staiing for GSTP1. Formalin-fixed paraffin-embedded prostatic tissue sections were stained with anti-GSTP1 antiserum (1:100 dilution; Oncor) using an immunoperoxidase method (Vectastain ABC Kit; Vector Laboratories), with either 3,3'-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as the peroxidase substrate. As a control for the specificity of staining, tissue sections prepared from normal human liver and normal human kidney were also stained with the anti-GSTP1 antiserum. Each ofthe tissues exhibited the characteristic staining pattern previously described (14).Immunoblot Analysis for GSTP1 Polypeptides. Salinewashed cultured prostatic carcinoma cells (15-19) were collected by centrifugation at 14,000 x g for 10 min at room temperature and then lysed in a protein extraction buffer [2% SDS/101% (vol/vol) glycerol/10 mM dithiothreitol in 62 mM Tris'HCl, pH 7.8] by heating to 950C for 10 min. The DNA content of each cell extract was estimated by using a diphenylamine assay (20). Equivalent extracts from each of the cultured cell lines were subjected to immunoblot analysis for GSTP1 and DNA topoisomerase I polypeptides, using specific antisera (rabbit anti-GSTP1 from Oncor; human antitopoisomerase I provided by W. C. Earnshaw, Johns Hopkins University) as previously described (21).Northern Blot Analyss for GSTPI mRNA. Total RNAs were extracted from cultured cells by the method of Chomczynski and Sacchi (22) and then quantitated by using an orcinol assay (23). Purified RNAs (20 ug) were electrophoresed on 1.5% agarose gels in the presence of2.2 M formaldehyde, transferred to Zeta-Probe (Bio-Rad) filters, and then assessed for GSTPI and TOP] mRNA levels by hybridization with specific 32P-labeled cDNA probes (prepared using the Random Primers DNA Labeling System, GIBCO/BRL). Following hybridization at 50CC for 16 hr (in 50%o formamide/7% SDS/0.5% nonfat dry milk/heat-denatured salmon sperm DNA at 200 pg/ml/300 mM NaCl/2 mM EDTA/20 mM sodium phosphate, pH 7.4), blots were washed with 0.3x SSC (lx SSC is 150mM NaCl/15 mM sodium citrate, pH 7.0) and 0.3% SDS for 60 mi at 650C before being exposed to X-Omat AR (Kodak) film at -700C. Plasmid pGSTii1 containing the entire GSTPI coding sequence (24) was obtained from the American Type Culture Collection; plasmid phTOPl-D1 containing 704 bp of human TOP) cDNA (25) was provided by

RTK and BRAF fusions are rare but potentially druggable resistance mechanisms to EGFR TKIs. Detection of RTK and BRAF fusions should be part of comprehensive profiling panels to determine resistance to EGFR TKIs and direct appropriate combination therapeutic strategies.

Purpose: There is an unmet need for treatment options in hepatocellular carcinoma (HCC). Sorafenib is currently the only approved systemic treatment for HCC. Refametinib, an oral, allosteric MEK inhibitor, has demonstrated antitumor activity in combination with sorafenib in vitro and in vivo. A phase II study evaluated efficacy and safety of refametinib plus sorafenib in Asian patients with HCC (NCT01204177).Experimental Design: Eligible patients received twice-daily refametinib 50 mg plus twice-daily sorafenib 200 mg (morning)/400 mg (evening), with dose escalation to sorafenib 400 mg twice daily from cycle 2 if no grade !2 hand-foot skin reaction, fatigue, or gastrointestinal toxicity occurred. Primary efficacy endpoint: disease control rate. Secondary endpoints: time to progression, overall survival, pharmacokinetic assessment, biomarker analysis, safety, and tolerability.Results: Of 95 enrolled patients, 70 received study treatment. Most patients had liver cirrhosis (82.9%) and hepatitis B viral infection (75.7%). Disease control rate was 44.8% (primary efficacy analysis; n ¼ 58). Median time to progression was 122 days, median overall survival was 290 days (n ¼ 70). Best clinical responders had RAS mutations; majority of poor responders had wild-type RAS. Most frequent drug-related adverse events were diarrhea, rash, aspartate aminotransferase elevation, vomiting, and nausea. Dose modifications due to adverse events were necessary in almost all patients.Conclusions: Refametinib plus sorafenib showed antitumor activity in patients with HCC and was tolerated at reduced doses by most patients. Frequent dose modifications due to grade 3 adverse events may have contributed to limited treatment effect. Patients with RAS mutations appear to benefit from refametinib/sorafenib combination. Clin Cancer Res; 20(23); 5976-85. Ó2014 AACR.

Purpose: Cell cycle dysregulation resulting in expression of antiapoptotic genes and uncontrolled proliferation is a feature of undifferentiated nasopharyngeal carcinoma. The pharmacodynamic effects of seliciclib, a cyclin-dependent kinase (CDK) inhibitor, were studied in patients with nasopharyngeal carcinoma. Experimental Design: Patients with treatment-naI« ve locally advanced nasopharyngeal carcinoma received seliciclib at 800 mg or 400 mg twice daily on days 1 to 3 and 8 to 12. Paired tumor samples obtained at baseline and on day 13 were assessed by light microscopy, immunohistochemistry, and transcriptional profiling using real-time PCR low-density array consisting of a panel of 380 genes related to cell cycle inhibition, apoptosis, signal transduction, and cell proliferation. Results: At 800 mg bd, one patient experienced grade 3 liver toxicity and another had grade 2 vomiting; no significant toxicities were experienced in 13 patients treated at 400 mg bd. Seven of fourteen evaluable patients had clinical evidence of tumor reduction. Some of these responses were associated with increased tumor apoptosis, necrosis, and decreases in plasma EBV DNA posttreatment. Reduced protein expression of Mcl-1, cyclin D1, phosphorylated retinoblastoma protein pRB (T821), and significant transcriptional down-regulation of genes related to cellular proliferation and survival were shown in some patients posttreatment, indicative of cell cycle modulation by seliciclib, more specifically inhibition of cdk2/cyclin E, cdk7/cyclin H, and cdk9/cyclinT. Conclusions: Brief treatment with this regimen of seliciclib in patients with nasopharyngeal carcinoma is tolerable at 400 mg bd and associated with tumor pharmacodynamic changes consistent with cdk inhibition, and warrants further efficacy studies in this tumor.

Post-transplant lymphoproliferative disease (PTLD) is a B cell proliferative disorder that is associated with Epstein-Barr virus (EBV), an ubiquitous herpesvirus. EBV-seronegative organ transplant recipients are at highest risk. EBV infection in PTLD lesions exists in a latent rather than lytic state, making tumor regression in response to antiviral agents unlikely. Viral latency proteins drive proliferation of T cells but also allow T cells to target PTLD lesions for destruction. Augmentation of the cellular immune response via the infusion of EBV-specific cytotoxic T cells has yielded promising results in the prevention and treatment of PTLD in bone marrow transplant recipients. Efforts to extend this strategy to solid organ transplant recipients are ongoing.