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1.  Assessment of FANCD2 nuclear foci formation in paraffin embedded tumors; a potential patient enrichment strategy for treatment with DNA interstrand crosslinking agents 
A major mechanism of DNA repair related to homologous recombination is the Fanconi Anemia pathway (FA). FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin following DNA damage, or during S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients’ tumor tissue, which could also be practically incorporated to large scale screening. In order to develop this method, we first used Western immunoblot to detect FANCD2 protein mono-ubiquitination in fresh tumor specimens of ovarian cancer patients undergoing surgery, and stained formalin fixed paraffin embedded (FFPE) tumor tissue simultaneously with DAPI, FANCD2 and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 FFPE tumor samples using the FA triple staining immunofluorescence (FATSI) method. The ratios of FANCD2 foci negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin embedded tissues renders the reported method suitable for large scale screening to select patients for treatment with DNA interstrand crosslinking agents, PARP inhibitors or their combination.
doi:10.1016/j.trsl.2012.09.003
PMCID: PMC3755957  PMID: 23063585
patient selection; DNA repair foci
2.  Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma  
The Journal of Clinical Investigation  2014;124(4):1582-1586.
Targeted cancer therapies often induce “outlier” responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.
doi:10.1172/JCI72763
PMCID: PMC3973082  PMID: 24569458
3.  Phase II Trials of Single Agent Anti-VEGF Therapy for Patients with Chronic Lymphocytic Leukemia (CLL) 
Leukemia & lymphoma  2010;51(12):2222-2229.
Between 2005 and 2008, we conducted separate phase II clinical testing of 3 distinct anti-VEGF therapies for patients with relapsed/refractory CLL. Collectively, 46 patients were accrued to trials of single-agent anti-VEGF antibody (bevacizumab, n=13) or 1 of 2 receptor tyrosine kinase inhibitors (AZD2171, n=15; sunitinib malate, n=18). All patients have completed treatment. Patients received a median of 2 cycles of bevacizumab, AZD2171, or sunitinib malate. All 3 trials were closed early due to lack of efficacy. No complete or partial remissions were observed. Individually and collectively, these studies indicate single-agent anti-VEGF therapy has minimal clinical activity for patients with relapsed/refractory CLL.
doi:10.3109/10428194.2010.524327
PMCID: PMC3928074  PMID: 21054149
chronic lymphocytic leukemia; angiogenesis; vascular endothelial growth factor (VEGF); therapy; bevacizumab; receptor tyrosine kinase inhibitor
4.  Inhibition of Ras-Effector Interaction by Cyclic Peptides 
MedChemComm  2012;4(2):378-382.
A combinatorial library of 6 × 106 cyclic peptides was synthesized in the one bead-two compound format, with each bead displaying a unique cyclic peptide on its surface and a linear peptide encoding tag in its interior. Screening of the library against K-Ras identified compounds that bound K-Ras with submicromolar affinity and disrupted its interaction with effector proteins.
doi:10.1039/C2MD20329D
PMCID: PMC3621770  PMID: 23585920
5.  A Phase II Study of Modulated-Capecitabine and Docetaxel in Chemonaive Patients with Advanced Non-Small Cell Lung Cancer (NSCLC) 
Introduction
This phase II single-arm trial of docetaxel and capecitabine in previously untreated non-small cell lung cancer (NSCLC) patients was designed to evaluate response rate of this regimen based on promising efficacy data from phase II testing in pre-treated NSCLC patients. The trial also evaluated the correlation between peripheral blood dihydropyrimidine dehydrogenase (DPD) expression and efficacy/toxicity.
Methods
Patients with advanced NSCLC (metastatic, including malignant pleural effusion) without prior chemotherapy were enrolled. Baseline DPD screening was performed; patients with baseline DPD level < 0.07 nmol/min/mg protein were considered ineligible for the study. Treatment included a 28-day cycle of docetaxel 36 mg/m2 days 1, 8, 15 and capecitabine 1250 mg/m2/day in divided doses on days 5–18. Overall response rate (RR) was the primary endpoint with a target RR of 50%. Correlative studies included evaluation of DPD activity levels in peripheral blood and correlation with clinical responses.
Results
Twenty-eight patients received 86 cycles of treatment (median 3 cycles) and were evaluable for response. The RR was 18% (5 patients); RR did not meet the pre-specified efficacy endpoint and the trial was stopped. 14 patients had stable disease (SD - 50%) and 4 pts had SD > 12 weeks. Median time to progression was 3.3 months (95% CI 1.5 – 4.6 months). Median overall survival was 10.5 months (95% CI: 3.2 – 15 months). Main toxicities included fatigue, stomatitis and leukopenia. DPD levels ranged from 0.06 to 0.26 nmol/min/mg. The majority of responders (4/5) had DPD levels ≤ 0.1 nmol/min/mg. Most of the responders (4/5) experienced grade 3 toxicities including leukopenia, dehydration, fatigue, and diarrhea. None of the patients (0/4) with higher DPD levels (>0.2 nmol/min/mg) had a response.
Conclusion
The response rate for the regimen did not demonstrate sufficient activity and further study of this regimen in this setting is not indicated. Interestingly, the results suggest that low DPD expression may be associated with response to capecitabine but also with increased toxicity.
doi:10.1016/j.lungcan.2012.09.013
PMCID: PMC3549571  PMID: 23079156
non-small cell lung cancer; dihydropyrimidine deficiency; capecitabine
6.  Phase I dose escalation study of KOS-1584, a novel epothilone, in patients with advanced solid tumors 
Cancer chemotherapy and pharmacology  2011;69(2):10.1007/s00280-011-1724-7.
Purpose
First-in-man study of KOS-1584, a second generation epothilone.
Methods
Patients with advanced solid malignancies received KOS-1584 every 3 weeks until disease progression. Using a modified Fibonacci dose escalation scheme, one patient was enrolled at each dose level until the first instance of grade 2 toxicity. Thereafter, a standard 3 + 3 design was utilized.
Results
Sixty-six patients in 14 cohorts were dosed from 0.8 to 48 mg/m2. Diarrhea, arthralgias, and encephalopathy were dose-limiting toxicities (DLTs) at doses ≥36 mg/m2. At the recommended phase II dose (RP2D), the most common adverse effects were peripheral neuropathy (low grade), fatigue, arthralgias/myalgias, and diarrhea (31, 6%). The incidence of neutropenia was low. The overall clearance, volume of distribution, and half-life of KOS-1584 were 11 ± 6.17 L/h/m2, 327 ± 161 L/m2, and 21.9 ± 8.75 h, respectively. The half-life for the seco-metabolite (KOS-1891) was 29.6 ± 13.8 h. KOS-1584 exhibited linear pharmacokinetics. A dose-dependent increase in microtubulin bundle formation was observed at doses ≥27 mg/m2. Two patients achieved partial responses and 24 patients had stable disease (SD).
Conclusions
The RP2D of KOS-1584 is 36 mg/m2. The lack of severe neurologic toxicity, diarrhea, neutropenia, or hypersensitivity reactions; favorable pharmacokinetic profile; and early evidence of activity support further evaluation.
doi:10.1007/s00280-011-1724-7
PMCID: PMC3865603  PMID: 21874318
Epothilone; KOS-1584; Phase I; Solid tumors
7.  Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors 
Diagnostic Pathology  2013;8:201.
Background
Protein arginine methyltransferase-5 (PRMT5) is a chromatin-modifying enzyme capable of methylating histone and non-histone proteins, and is involved in a wide range of cellular processes that range from transcriptional regulation to organelle biosynthesis. As such, its overexpression has been linked to tumor suppressor gene silencing, enhanced tumor cell growth and survival.
Material and methods
Quantitative real-time polymerase chain reaction, Western immunoblot and immunohistochemistry were used to characterize PRMT5 expression in lung cancer cell lines and human tumors. Clinicopathological findings of tissue microarray based samples from 229 patients with non-small cell lung carcinomas (NSCLC) and 133 cases with pulmonary neuroendocrine tumors (NET) were analyzed with regard to nuclear and cytoplasmic PRMT5 expression.
Results
There was statistically significant difference in PRMT5 messenger RNA expression between tumors and nonneoplastic lung tissues. Immunoblot experiments showed abundant expression of PRMT5 and its symmetric methylation mark H4R3 in lung carcinoma but not in non-neoplastic human pulmonary alveolar and bronchial epithelial cell lines. More than two thirds of lung tumors expressed PRMT5. High levels of cytoplasmic PRMT5 were detected in 20.5% of NSCLC and in 16.5% of NET; high levels of nuclear PRMT5 were detected in 38.0% of NSCLC and 24.0% of NET. Cytoplasmic PRMT5 was associated with high grade in both NSCLC and pulmonary NET while nuclear PRMT5 was more frequent in carcinoid tumors (p < 0.05).
Conclusion
The observed findings support the role of PRMT5 in lung tumorigenesis and reflect its functional dichotomy in cellular compartments.
Virtual slide
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1611895162102528
doi:10.1186/1746-1596-8-201
PMCID: PMC3933389  PMID: 24326178
Protein arginine methyltransferase-5; Lung carcinoma; Neuroendocrine tumors
8.  In Vivo Quantification of Active Decitabine-Triphosphate Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia 
The AAPS Journal  2012;15(1):242-249.
Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.
doi:10.1208/s12248-012-9427-5
PMCID: PMC3535094  PMID: 23180159
acute myeloid leukemia; decitabine; metabolite; quantification method; triphosphate
9.  Randomized Phase II Trial of Erlotinib Alone or With Carboplatin and Paclitaxel in Patients Who Were Never or Light Former Smokers With Advanced Lung Adenocarcinoma: CALGB 30406 Trial 
Journal of Clinical Oncology  2012;30(17):2063-2069.
Purpose
Erlotinib is clinically effective in patients with non–small-cell lung cancer (NSCLC) who have adenocarcinoma, are never or limited former smokers, or have EGFR mutant tumors. We investigated the efficacy of erlotinib alone or in combination with chemotherapy in patients with these characteristics.
Patients and Methods
Patients with advanced NSCLC (adenocarcinoma) who were epidermal growth factor receptor tyrosine kinase inhibitor and chemotherapy naive never or light former smokers (smokers of > 100 cigarettes and ≤ 10 pack years and quit ≥ 1 year ago) were randomly assigned to continuous erlotinib or in combination with carboplatin and paclitaxel (ECP) for six cycles followed by erlotinib alone. The primary end point was progression-free survival (PFS). Tissue collection was mandatory.
Results
PFS was similar (5.0 v 6.6 months; P = .1988) in patients randomly assigned to erlotinib alone (arm A; n = 81) or to ECP (arm B; n = 100). EGFR mutation analysis was possible in 91% (164 of 181) of patients, and EGFR mutations were detected in 40% (51 of 128) of never smokers and in 42% (15 of 36) of light former smokers. In arm A, response rate (70% v 9%), PFS (14.1 v 2.6 months), and overall survival (OS; 31.3 v 18.1 month) favored EGFR-mutant patients. In arm B, response rate (73% v 30%), PFS (17.2 v 4.8 months), and OS (38.1 v 14.4 months) favored EGFR-mutant patients. Incidence of grades 3 to 4 hematologic (2% v 49%; P < .001) and nonhematologic (24% v 52%; P < .001) toxicity was greater in patients treated with ECP.
Conclusion
Erlotinib and erlotinib plus chemotherapy have similar efficacy in clinically selected populations of patients with advanced NSCLC. EGFR mutations identify patients most likely to benefit.
doi:10.1200/JCO.2011.40.1315
PMCID: PMC3397694  PMID: 22547605
10.  Bortezomib for Patients with Advanced Stage Bronchioloalveolar Carcinoma (BAC): A California Cancer Consortium Phase II Study (NCI # 7003) 
Background
Bronchioloalveolar carcinoma (BAC), a subtype of non-small cell lung cancer (NSCLC), is a difficult disease to treat with low response rates with cytotoxic chemotherapy. Bortezomib, a proteasome inhibitor, has demonstrated objective responses in BAC patients in early phase clinical trials. We conducted a phase II study of bortezomib inpatients with advanced stage BAC.
Methods
Patients with advanced BAC, adenocarcinoma with BAC features or BAC with adenocarcinoma features and less than two prior regimens were eligible. Prior epidermal growth factor receptor (EGFR) inhibitor therapy was allowed. Bortezomib was administered intravenously at 1.6 mg/m2 on days 1 and 8 of every 21 days cycle until disease progression or unacceptable toxicity. The primary endpoint was response rate. The Simon two-stage design was utilized.
Results
Forty-two patients were enrolled and the study was halted early for slow accrual. Patient characteristics were: female 55%, median age 68 years, and ECOG performance status of 0 and 1 in 31 and 11 patients respectively. Twenty-six(62%)patients had received prior therapy with an EGFR inhibitor. A median of 4 cycles of therapy were administered. Objective responses were noted in 5% while 57% had disease stabilization. The median progression-free survival and overall survival were 5.5 months and 13.6 months respectively. Grade 3 diarrhea and fatigue were noted in 3 and 5 patients respectively.
Conclusions
Bortezomib is tolerated well and is associated with modest anti-cancer activity in advanced BAC, including inpatients that progressed on EGFR inhibitor therapy.
doi:10.1097/JTO.0b013e318225924c
PMCID: PMC3220078  PMID: 21716143
bortezomib; proteasome inhibition; BAC; bronchioloalveolar carcinoma; NSCLC
11.  The Search for an Optimal DNA, RNA, and Protein Detection by in situ Hybridization, Immunohistochemistry, and Solution-Based Methods 
Methods (San Diego, Calif.)  2010;52(4):281-286.
Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and Bovine Papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was “gentle” fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as “gentle” cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or -if one does not have unfixed tissues - solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.
doi:10.1016/j.ymeth.2010.09.005
PMCID: PMC3375896  PMID: 20888418
in situ hybridization; immunohistochemistry; fixation; microRNA; real-time PCR; EBV; Papillomavirus
12.  Multi-Institutional Phase II Study of Selumetinib in Patients With Metastatic Biliary Cancers 
Journal of Clinical Oncology  2011;29(17):2357-2363.
Purpose
Biliary cancers (BCs) carry a poor prognosis, but targeting the RAS/RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is of significance. Selumetinib is an inhibitor of MEK1/2, so this trial was designed to determine the safety and efficacy of selumetinib in BC.
Patients and Methods
This was a multi-institutional phase II study of selumetinib at 100 mg given orally twice per day to patients with advanced BC. The primary end point was response rate. All patients were required to provide tissue before enrolling. The levels of phosphorylated ERK (pERK) and AKT (pAKT) were assessed by immunohistochemistry. Tumors were genotyped for the presence of BRAF- and/or RAS-activating mutations.
Results
Twenty-eight eligible patients with a median age of 55.6 years were enrolled. Thirty-nine percent of patients had received one prior systemic therapy. Three patients (12%) had a confirmed objective response. Another 17 patients (68%) experienced stable disease (SD), 14 of whom (56%) experienced prolonged SD (> 16 weeks). Patients gained an average nonfluid weight of 8.6 pounds. Median progression-free survival was 3.7 months (95% CI, 3.5 to 4.9) and median overall survival was 9.8 months (95% CI, 5.97 to not available). Toxicities were mild, with rash (90%) and xerostomia (54%) being most frequent. Only one patient experienced grade 4 toxicity (fatigue). All patients had tissue available for analysis. No BRAF V600E mutations were found. Two patients with short-lived SD had KRAS mutations. Absence of pERK staining was associated with lack of response.
Conclusion
Selumetinib displays interesting activity and acceptable tolerability in patients with metastatic BC. Our results warrant further evaluation of selumetinib in patients with metastatic BC.
doi:10.1200/JCO.2010.33.9473
PMCID: PMC3107751  PMID: 21519026
13.  Risk Factors for Tumor Lysis Syndrome in patients with Chronic Lymphocytic Leukemia Treated with the Cyclin Dependent Kinase Inhibitor, Flavopiridol 
Tumor lysis syndrome (TLS) has been described in over 40% of patients with chronic lymphocytic leukemia (CLL) treated with the cyclin dependent kinase inhibitor, flavopiridol. We conducted a retrospective analysis to determine predictive factors for TLS. In 116 patients, the incidence of TLS was 46% (95% CI: 36%-55%). In univariable analysis, female gender, greater number of prior therapies, Rai stages III-IV, adenopathy ≥ 10 cm, splenomegaly, del(11q), decreased albumin, and increased absolute lymphocyte count, white blood cell count (WBC), β2-microglobulin, and lactate dehydrogenase (LDH) were associated (p<0.05) with TLS. In multivariable analysis, female gender, adenopathy ≥ 10 cm, elevated WBC, increased β2-microglobulin, and decreased albumin were associated with TLS (p<0.05). With respect to patient outcomes, 49% and 44% of patients with and without TLS, respectively, responded to flavopiridol (p=0.71). In a multivariable analysis controlling for number of prior therapies, cytogenetics, Rai stage, age, and gender, progression-free survival (PFS) was inferior in patients with TLS (p=0.01). Female patients and patients with elevated β2-microglobulin, increased WBC, adenopathy ≥ 10 cm, and decreased albumin were at highest risk and should be monitored for TLS with flavopiridol. TLS does not appear to be predictive of response or improved PFS in patients receiving flavopiridol.
doi:10.1038/leu.2011.109
PMCID: PMC3162125  PMID: 21606960
chronic lymphocytic leukemia; flavopiridol; tumor lysis syndrome
14.  Phase I trial of non-cytotoxic suramin as a modulator of docetaxel and gemcitabine therapy in previously treated patients with non-small cell lung cancer 
Cancer chemotherapy and pharmacology  2010;66(6):1019-1029.
Purpose
In preclinical models, non-cytotoxic suramin (concentrations <50 μM) potentiates the activity of multiple chemotherapeutic agents. The present study evaluated the safety and tolerability of suramin in combination with docetaxel or gemcitabine in previously chemotherapy-treated patients with advanced non-small cell lung cancer.
Methods
Patients received suramin intravenously in combination with either docetaxel on day 1 or gemcitabine on days 1 and 8, of each 21-day treatment cycle. After 3 cycles, patients with partial response (PR) or better continued on the same combination, whereas patients with stable disease (SD) or worse crossed-over to the other combination. Pharmacokinetic analyses were performed before and after each treatment.
Results
Eighteen patients received a total of 79 courses (37 suramin plus docetaxel, 42 suramin plus gemcitabine). The dose-limiting toxicity (DLT) was febrile neutropenia, observed in three of six patients treated with suramin and docetaxel 75 mg/m2. No DLTs were observed with suramin plus docetaxel 56 mg/m2 or suramin plus gemcitabine 1,250 mg/m2. Common adverse events included neutropenia, thrombocytopenia, anemia, fatigue, nausea, vomiting, skin rash, hyperglycemia, and electrolyte abnormalities. The target plasma suramin concentration range of 10–50 μM was achieved in 90% of treatments. Discernable antitumor activity was noted in 11 patients (2 PR, 9 SD).
Conclusions
Non-cytotoxic suramin, in combination with docetaxel 56 mg/m2 or gemcitabine 1,250 mg/m2, was reasonably well-tolerated with a manageable toxicity profile. Target plasma concentrations were correctly predicted by our previously described dosing nomogram. The observed preliminary evidence of antitumor activity encourages evaluation of this strategy in efficacy trials.
doi:10.1007/s00280-010-1252-x
PMCID: PMC2919610  PMID: 20107799
Suramin; Docetaxel; Gemcitabine; Chemosensitizer; Modulator; Non-small cell lung cancer
15.  Phase II Clinical Trial of Sorafenib in Metastatic Medullary Thyroid Cancer 
Journal of Clinical Oncology  2010;28(14):2323-2330.
Purpose
Mutations in the RET proto-oncogene and vascular endothelial growth factor receptor (VEGFR) activity are critical in the pathogenesis of medullary thyroid cancer (MTC). Sorafenib, a multikinase inhibitor targeting Ret and VEGFR, showed antitumor activity in preclinical studies of MTC.
Patients and Methods
In this phase II trial of sorafenib in patients with advanced MTC, the primary end point was objective response. Secondary end points included toxicity assessment and response correlation with tumor markers, functional imaging, and RET mutations. Using a two-stage design, 16 or 25 patients were to be enrolled onto arms A (hereditary) and B (sporadic). Patients received sorafenib 400 mg orally twice daily.
Results
Of 16 patients treated in arm B, one achieved partial response (PR; 6.3%; 95% CI, 0.2% to 30.2%), 14 had stable disease (SD; 87.5%; 95% CI, 61.7% to 99.5%), and one was nonevaluable. In a post hoc analysis of 10 arm B patients with progressive disease (PD) before study, one patient had PR of 21+ months, four patients had SD ≥ 15 months, four patients had SD ≤ 6 months, and one patient had clinical PD. Median progression-free survival was 17.9 months. Arm A was prematurely terminated because of slow accrual. Common adverse events (AEs) included diarrhea, hand-foot-skin reaction, rash, and hypertension. Although serious AEs were rare, one death was seen. Tumor markers decreased in the majority of patients, and RET mutations were detected in 10 of 12 sporadic MTCs analyzed.
Conclusion
Sorafenib is reasonably well tolerated, with suggestion of clinical benefit for patients with sporadic MTC. Caution should be taken because of the rare but fatal toxicity potentially associated with sorafenib.
doi:10.1200/JCO.2009.25.0068
PMCID: PMC2881718  PMID: 20368568
16.  Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease 
Journal of Clinical Oncology  2009;27(35):6012-6018.
Purpose
Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated.
Patients and Methods
Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy.
Results
Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P ≤ .01), resulted in improved tolerability and treatment delivery.
Conclusion
Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.
doi:10.1200/JCO.2009.22.6944
PMCID: PMC2793044  PMID: 19826119
17.  A Multi-institutional Phase II study of the efficacy and tolerability of Lapatinib in patients with advanced hepatocellular carcinomas 
Background
Hepatocellular carcinoma (HCC) is on the rise worldwide. HCC responds poorly to chemotherapy. Lapatinib is an inhibitor of EGFR and HER2/NEU both implicated in hepatocarcinogenesis. This trial was designed to determine the safety and efficacy of lapatinib in HCC.
Methods
A Fleming phase II design with a single stage of 25 patients with a 90% power to exclude a true response rate of < 10% and detect a true response rate of ≥30% was utilized. The dose of lapatinib was 1,500 mg/d administered orally in 28-day cycles. Tumor and blood specimens were analyzed for expression of HER2/NEU/CEP17 and status of downstream signal pathway proteins.
Results
Twenty-six patients with HCC enrolled on this study. 19% had one prior therapy. Most common toxicities were diarrhea (73%), nausea (54%) and rash (42%). No objective responses were observed. Ten (40%) patients had stable disease (SD) as their best response including 6 (23%) with SD lasting > 120 days. Median progression-free-survival was 1.9 months and median overall survival 12.6 months. Patients who developed a rash had a borderline statistically significant longer survival. Tissue and blood specimens were available on >90% of patients. No somatic mutations in EGFR (exons 18–21) were found. In contrast to our previous findings, we did not find evidence of HER2/NEU somatic mutations. PTEN, P-AKT and P70S6K expression did not correlate with survival.
Conclusions
Lapatinib is well-tolerated but appears to benefit only a subgroup of patients for whom predictive molecular or clinical characteristics are not yet fully defined.
doi:10.1158/1078-0432.CCR-09-0465
PMCID: PMC2774354  PMID: 19737952
18.  Expression of a Mutant p53 Results in an Age-Related Demographic Shift in Spontaneous Lung Tumor Formation in Transgenic Mice 
PLoS ONE  2009;4(5):e5563.
Background
Mutations in the P53 gene are among the most common genetic abnormalities in human lung cancer. Codon 273 in the sequence-specific DNA binding domain is one of the most frequently mutated sites.
Methodology
To investigate the role of mutant p53 in lung tumorigenesis, a lung specific p53(273H) transgenic mouse model was developed. Rates of lung cancer formation in the transgenic animals and their littermates were evaluated by necropsy studies performed in progressive age cohorts ranging from 4 to 24 months. In order to establish the influence of other common genetic abnormalities in lung tumor formation in the animals, K-Ras gene mutation and p16INK4a (p16) promoter methylation were evaluated in a total of 281 transgenic mice and 189 non-transgenic littermates.
Principal Findings
At the age extremes of 4–12 and 22–24 months no differences were observed, with very low prevalence of tumors in animals younger than 12 months, and a relatively high prevalence at age 22 months or older. However, the transgenic mice had a significant higher lung tumor rate than their non-transgenic counterparts during the age of 13–21 months, suggesting an age-related shift in lung tumor formation induced by the lung-specific expression of the human mutant p53. Histopathology suggested a more aggressive nature for the transgenic tumors. Older mice (>13 months) had a significantly higher rate of p16 promoter methylation (17% v 82%). In addition, an age related effect was observed for K-Ras codons 12 or 13 mutations, but not for codon 61 mutations.
Conclusions/Significance
These results would suggest that the mutant p53(273H) contributes to an acceleration in the development of spontaneous lung tumors in these mice. Combination with other genetic and epigenetic alterations occurring after the age of 13 months is intimately linked to its oncogenic potential.
doi:10.1371/journal.pone.0005563
PMCID: PMC2680060  PMID: 19440353
19.  Methylation of Adjacent CpG Sites Affects Sp1/Sp3 Binding and Activity in the p21Cip1 Promoter 
Molecular and Cellular Biology  2003;23(12):4056-4065.
DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C→W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. By analyzing luciferase activity of transfected p21Cip1 promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21Cip1 expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21Cip1 expression was reconstituted when cells were pretreated with 5-aza-2′-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21Cip1 expression in response to depsipeptide treatment.
doi:10.1128/MCB.23.12.4056-4065.2003
PMCID: PMC156121  PMID: 12773551

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