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1.  Erlotinib in African Americans with Advanced Non-Small Cell Lung Cancer: A Prospective Randomized Study with Genetic and Pharmacokinetic Analysis 
Prospective studies focusing on EGFR inhibitors in African Americans with NSCLC have not been previously performed. In this phase II randomized study, 55 African Americans with NSCLC received erlotinib 150mg/day or a body weight adjusted dose with subsequent escalations to the maximum allowable, 200mg/day, to achieve rash. Erlotinib and OSI-420 exposures were lower compared to previous reports, consistent with CYP3A pharmacogenetics implying higher metabolic activity. Tumor genetics revealed only two EGFR mutations, EGFR amplification in 17/47 samples, 8 KRAS mutations and 5 EML4-ALK translocations. Although absence of rash was associated with shorter time to progression (TTP), disease control rate, TTP, and 1-year survival were not different between the two dose groups, indicating the dose-to-rash strategy failed to increase clinical benefit. Observed low incidence of toxicity and low erlotinib exposure suggest standardized and maximum allowable dosing may be suboptimal in African Americans.
PMCID: PMC4180036  PMID: 24781527
EGFR; Erlotinib; African American; Pharmacokinetics; Pharmacogenetics
2.  Overexpression of Rad51C splice variants in colorectal tumors 
Oncotarget  2014;6(11):8777-8787.
Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3).
Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT.
Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.
PMCID: PMC4496183  PMID: 25669972
Rad51C isoform overexpression; Promoter methylation; Colorectal tumors
3.  A Randomized Phase II Study to Assess the Efficacy of Pemetrexed or Sunitinib or Pemetrexed Plus Sunitinib in the Second-Line Treatment of Advanced Non–Small-Cell Lung Cancer 
Second-line chemotherapy for advanced non-small cell lung cancer (NSCLC) improves survival modestly but new strategies are needed. This trial was designed to evaluate an antivascular endothelial growth factor strategy with or without standard chemotherapy in previously treated NSCLC.
Patients with stage IIIB/IV NSCLC with performance status 0 to 1 progressive after first-line chemotherapy were eligible for randomization to pemetrexed, sunitinib, or the combination. Patients were stratified by performance status, stage, and sex. Primary objective was 18-week progression-free survival (PFS) rate; secondary objectives included response, overall survival (OS), and toxicity. Target accrual was 225. The study was terminated early because of decreasing accrual rates.
Between April 2008 and September 2011, 130 patients were registered and randomized; of this, 125 patients were treated. Baseline characteristics in the three arms were well balanced. Toxicity was higher in the sunitinib-containing arms. The 18-week PFS rate in the pemetrexed, sunitinib, and combination arms was 54% (95% confidence interval [CI], 40–71), 37% (95% CI, 25–54), and 48% (95% CI, 35–66), respectively (p= 0.25). Median PFS in the pemetrexed, sunitinib, and combination arms in months was 4.9 (2.1–8.8), 3.3 (2.3–4.2), and 3.7 (2.5–5.8), respectively (p= 0.18). There was an overall statistically significant difference in OS between the three arms: median OS in months was 10.5 (8.3–20.2) for pemetrexed, 8.0 (6.8–13.5) for sunitinib, and 6.7 (4.1–10.1) for the combination (p= 0.03).
Pemetrexed had a superior toxicity profile to either sunitinib or the combination of pemetrexed and sunitinib. The 18-week PFS rate was not significantly different between the arms. OS was significantly better with pemetrexed alone compared with the two sunitinib-containing arms, with the doublet performing worst for OS.
PMCID: PMC4284815  PMID: 24419419
CALGB 30704; Lung cancer
4.  Strong Inverse Correlation Between MicroRNA-125b and Human Papillomavirus DNA in Productive Infection 
Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, — 99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.
PMCID: PMC4284817  PMID: 20736742
human papillomavirus; microRNA; in situ hybridization; L2
5.  Randomized Phase II Study of Palifermin for Reducing Dysphagia in Patients Receiving Concurrent Chemoradiotherapy for Locally Advanced Unresectable Non-small Cell Lung Cancer 
Dysphagia is a common, dose-limiting toxicity of combined chemoradiotherapy (CT/RT) in patients with locally advanced non-small cell lung cancer (NSCLC). This study assessed the efficacy and safety of palifermin in reducing dysphagia from CT/RT followed by consolidation chemotherapy (CT).
This randomized, double-blind, phase II trial enrolled adults with unresectable stage III NSCLC. Subjects received weekly paclitaxel (50 mg/m2) and carboplatin (AUC 2.0) with concurrent daily radiation (RT) of 6000 to 6600 cGy, followed by consolidation CT. Palifermin (n = 49) or placebo (n = 46) was administered before starting concurrent CT/RT and once weekly for 6 weeks. The primary end points were the incidence of grade ≥2 dysphagia and safety.
The incidence of grade ≥2 and ≥3 dysphagia was numerically lower in palifermin subjects versus placebo subjects (61% versus 70%; p = 0.36; 22% versus 28%, p = 0.50, respectively). Mean duration of dysphagia (grade ≥2) was 25 days for palifermin subjects and 32 days for placebo subjects (p = 0.32). The incidence of adverse events was similar in the two treatment groups, and median overall survival and progression-free survival were not adversely affected by palifermin treatment (overall survival: 513 versus 319 days; progression-free survival: 262 versus 235 days for palifermin versus placebo arms, respectively). The palifermin arm received more doses of CT per study design and significantly more patients received RT doses ≥6000 cGy (84% versus 61%, p = 0.01).
The results of this exploratory trial suggest that additional larger studies may be warranted to further evaluate the effect of palifermin on dysphagia, exposure to CT/RT, and long-term survival.
PMCID: PMC4284819  PMID: 22011667
Non-small cell; Lung; Dysphagia
7.  Y-chromosome status identification suggests a recipient origin of posttransplant non–small cell lung carcinomas: chromogenic in situ hybridization analysis☆,☆☆ 
Human pathology  2014;45(5):1065-1070.
Owing to the need of lifelong immunosuppression, solid-organ transplant recipients are known to have an increased risk of posttransplant malignancies including lung cancer. Posttransplant neoplastic transformation of donor-derived cells giving rise to hematopoietic malignancies, Kaposi sarcoma, and basal cell carcinoma in nongraft tissues has been reported. The goal of this study was to assess the cell origin (donor versus recipient derived) of posttransplant non–small cell lung carcinomas (NSCLCs) in kidney and heart transplant recipients. An institutional database search identified 2557 kidney and heart transplant recipients in 8 consecutive years. Among this cohort, 20 (0.8%) renal and 18 (0.7%) heart transplant recipients developed NSCLC. The study cohort comprised 6 of 38 NSCLCs arising in donor-recipient sex-mismatched transplant patients. The tumor cell origin was evaluated by chromogenic in situ hybridization with Y-chromosome probe on formalin-fixed, paraffin-embedded tissues. Y-chromosome was identified in 97% ± 1% (range from 92% to 99%) of all types of nucleated cells in male control tissues. In all 5 NSCLCs from male recipients of female donor organ, Y-chromosome was identified in 97% ± 2% (range from 92% to 100%) of tumor cells, statistically equivalent to normal control (P < .001). No Y-chromosome was identified in NSCLC cells from a female recipient of male kidney. These findings suggest a recipient derivation of NSCLC arising in kidney and heart transplant recipients. A combination of histologic evaluation and chromogenic in situ hybridization with Y-chromosome analysis allows reliable determination of tissue origin in sex-mismatched solid-organ transplant recipients and may aid in management of posttransplant malignancy in such cases.
PMCID: PMC4271837  PMID: 24746212
Post–solid-organ transplantation lung cancer; Chromogenic in situ hybridization for Y-chromosome
8.  A pilot test of a combined tobacco dependence treatment and lung cancer screening program 
Lung cancer screening with computed tomography has demonstrated a significant reduction in mortality. While these findings are important for the lung cancer research field, the most important risk factor for lung cancer, i.e. smoking, should not be ignored. We performed a pilot study to examine the feasibility of delivering a program that included both tobacco dependence treatment and lung cancer screening. The objectives of this study were to: (1) estimate the proportion of smokers who complied with a 12-week treatment protocol that included both tobacco dependence treatment and lung cancer screening, (2) obtain preliminary estimates of abstinence and quit attempts at 4 and 6 months, and (3) obtain preliminary estimates of the cognitive social health information processing (C-SHIP) constructs and how they change following the intervention. In this randomized pilot study, 18 volunteers completed a 12-week protocol: half received the tobacco dependence treatment program before a CT scan (BCT) and the other received the CT scan first, followed by the treatment program (ACT). The treatment protocol included both nurse-delivered telephone counseling and either nicotine replacement therapy or varenicline. Only one person did not complete all follow-up evaluations. At 4 months post enrollment, the carbon monoxide confirmed quit rates were 33.3% in the BCT arm and 22.2% in the ACT arm (27.8% overall), and all but one had made a 24-h attempt to quit. At 6 months the confirmed abstinence decreased to 22.1% in the BCT arm and 11.1% in the ACT arm (16.7% overall), and 72.2% of participants had made a 24-h quit attempt. These preliminary results suggest that it might be better to deliver treatment before the screening test. Future randomized trials with a larger sample size are needed to confirm these findings.
PMCID: PMC4272196  PMID: 22088938
Lung cancer screening; Smoking cessation; Nicotine replacement therapy; Varenicline; Epidemiology; Public health
9.  Phase I/II Study of Inhaled Doxorubicin Combined with Platinum-Based Therapy for Advanced Non–Small Cell Lung Cancer 
We have shown the feasibility of administering inhaled doxorubicin to patients with cancer. This study evaluated inhaled doxorubicin combined with cisplatin and docetaxel in patients with non–small cell lung cancer. The principal objective was to determine safety and, secondarily, efficacy.
Experimental Design
Patients who had chemo-naïve advanced non–small cell lung cancer were enrolled in the study. Adequate organ and pulmonary function was required: diffusing capacity for carbon monoxide/forced expiratory volume in 1 second/forced vital capacity ≥50%, resting/exercise O2 saturation ≥90%/85%. In phase I, doxorubicin was escalated: dose level 1 (6 mg/m2) and level 2 (7.5 mg/m2). Escalation was permitted if ≤2 of 6 patients experienced pulmonary dose-limiting toxicity (grade 2 Radiation Therapy Oncology Group lung morbidity; resting O2 saturation of <85%; decrease in diffusing capacity for carbon monoxide, forced vital capacity, or forced expiratory volume in 1 second of ≥20% from baseline or ≤30% of predicted; or grade 3 Common Terminology Criteria for Adverse Events version 3.0 pulmonary toxicity). Doses of cisplatin and docetaxel were 75 mg/m2. Treatments and pulmonary function tests were repeated every 21 days, with up to eight cycles for responding patients.
Twenty-eight patients were treated at level 1 and eight patients at level 2. Doxorubicin was escalated to 7.5 mg/m2, however, after two patients developed pulmonary dose-limiting toxicity; the remainder were treated at 6.0 mg/m2. Twenty-four evaluable patients received at least two courses or had progressive disease following the first course at the phase II dose. Toxicity was associated with i.v. chemotherapy although one patient had delayed pulmonary toxicity responding to corticosteroids and oxygen. Seven (29%) evaluable patients responded (six partial responses and one complete response) and 13 (54%) patients had stable disease for up to eight cycles.
Although this combination was safe, the primary objective was not met and will not be pursued further.
PMCID: PMC4262532  PMID: 20371682
10.  Detection of extracellular RNAs in cancer and viral infection via tethered cationic lipoplex nanoparticles containing molecular beacons 
Analytical chemistry  2013;85(23):11265-11274.
Non-invasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses, and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick™ isolation kit were first used to isolate exosomes from the cell culture medium and human serum respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g. qRT-PCR. Here we demonstrate this concept using lentivirus and serum from lung cancer patients.
PMCID: PMC4121114  PMID: 24102152
lipoplex nanoparticles; exosome; extracellular RNA; cancer detection; viral infection
11.  MicroRNA-34a is an important component of PRIMA-1-induced apoptotic network in human lung cancer cells 
Restoration of p53 function in tumor cells would be an attractive strategy for lung cancer therapy because p53 mutations are found in more than 50% of lung cancers. The small molecule PRIMA-1 has been shown to restore the tumor suppression function of p53 and to induce apoptosis in human tumor cells. The mechanism of apoptosis induced by PRIMA-1 remains unclear. We investigated the effects of PRIMA-1 in apoptosis with Western immunoblot analysis, TaqMan microRNA real-time PCR, cell viability analysis and flow cytometry using human lung cancer cell lines containing mutant (H211 and H1155), wild-type (A549) or null (H1299) p53. PRIMA-1 induced massive apoptosis in the H211 and H1155 cells, but was less toxic to the A549 and H1299 cells. Western immunoblot analysis showed cleavage of PARP in H211 and H1155 cells but not in A549 and H1299 cells following treatment with PRIMA-1. In addition, p53 protein was also phosphorylated in H211 and H1155 cells. TaqMan microRNA assay showed that the expression of microRNA-34a was increased in the H211 and H1155 cells posttreatment. Knockdown microRNA-34a decreased the rate of apoptosis caused by PRIMA-1. The above results suggest that microRNA-34a is one of the important components of PRIMA-1-induced apoptotic network in the cancer cells harboring mutant p53.
PMCID: PMC4153870  PMID: 19921694
microRNA-34; PRIMA-1; apoptosis; lung cancer
12.  Clinical and Metabolic Parameters in Non-Small Cell Lung Carcinoma and Colorectal Cancer Patients with and without KRAS Mutations 
Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those carrying transversions, suggesting that age may determine the type of KRAS mutation in CRC patients.
PMCID: PMC4198984  PMID: 25158139
KRAS; non-small cell lung carcinoma; colorectal cancer; transition; transversion
13.  Increased NQO1 but Not c-MET and Survivin Expression in Non-Small Cell Lung Carcinoma with KRAS Mutations 
Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor)-targeted therapy has been used in the treatment of LC (lung cancer), mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog) driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(P)H:quinone oxidoreductase), also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin) in 108 patients with non-small cell lung carcinoma (NSCLC). NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1) oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2) selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3) since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.
PMCID: PMC4199031  PMID: 25222473
lung cancer; non-small cell lung carcinoma; oxidative stress; KRAS; mutation; NQO1; DNA methyl transferase; ERK1/2; c-MET; survivin
14.  Efficacy of bevacizumab plus erlotinib versus erlotinib alone in advanced non-small-cell lung cancer after failure of standard first-line chemotherapy (BeTa): a double-blind, placebo-controlled, phase 3 trial 
Lancet  2011;377(9780):1846-1854.
Bevacizumab and erlotinib target different tumour growth pathways with little overlap in their toxic-effect profiles. On the basis of promising results from a phase 1/2 trial assessing safety and activity of erlotinib plus bevacizumab for recurrent or refractory non-small-cell lung cancer (NSCLC), we aimed to assess efficacy and safety of this combination in a phase 3 trial.
In our double-blind, placebo-controlled, randomised phase 3 trial (BeTa), we enrolled patients with recurrent or refractory NSCLC who presented to 177 study sites in 12 countries after failure of first-line treatment. Patients were randomly allocated in a one-to-one ratio to receive erlotinib plus bevacizumab (bevacizumab group) or erlotinib plus placebo (control group) according to a computer-generated randomisation sequence by use of an interactive voice response system. The primary endpoint was overall survival in all enrolled patients. Patients, study staff, and investigators were masked to treatment assignment. We assessed safety by calculation of incidence of adverse events and tissue was collected for biomarker analyses. This trial is registered with, number NCT00130728.
Overall survival did not differ between 317 controls and 319 patients in the bevacizumab group (hazard ratio [HR] 0·97, 95% CI 0·80–1·18, p=0·7583). Median overall survival was 9·3 months (IQR 4·1–21·6) for patients in the bevacizumab group compared with 9·2 months (3·8–20·2) for controls. Progression-free survival seemed to be longer in the bevacizumab group (3·4 months [1·4–8·4]) than in the control group (1·7 months [1·3–4·1]; HR 0·62, 95% CI 0·52–0·75) and objective response rate suggested some clinical activity of bevacizumab and erlotinib. However, these secondary endpoint differences could not be defined as significant because the study prespecified that the primary endpoint had to be significant before testing of secondary endpoints could be done, to control type I error rate. In the bevacizumab group, 130 (42%) of 313 patients with safety data had a serious adverse event, compared with 114 (36%) controls. There were 20 (6%) grade 5 adverse events, including two arterial thromboembolic events, in the bevacizumab group, and 14 (4%) in the control group.
Addition of bevacizumab to erlotinib does not improve survival in patients with recurrent or refractory NSCLC.
PMCID: PMC4134127  PMID: 21621716
15.  A phase I study of prolonged infusion of triapine in combination with fixed dose rate gemcitabine in patients with advanced solid tumors 
Investigational new drugs  2012;31(3):685-695.
Prolonged exposure of cancer cells to triapine, an inhibitor of ribonucleotide reductase, followed by gemcitabine enhances gemcitabine activity in vitro. Fixed-dose-rate gemcitabine (FDR-G) has improved efficacy compared to standard-dose. We conducted a phase I trial to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of prolonged triapine infusion followed by FDR-G.
Experimental Design
Triapine was given as a 24-hour infusion, immediately followed by FDR-G (1000 mg/m2 over 100-minute). Initially, this combination was administered days 1 and 8 of a 21-day cycle (Arm A, triapine starting dose 120 mg); but because of myelosuppression, it was changed to days 1 and 15 of a 28-day cycle (Arm B, starting dose of triapine 75 mg). Triapine steady-state concentrations (Css) and circulating ribonucleotide reductase M2-subunit (RRM2) were measured.
Thirty-six patients were enrolled. The MTD was determined to be triapine 90 mg (24-hour infusion) immediately followed by gemcitabine 1000 mg/m2 (100-minute infusion), every 2 weeks of a 4-week cycle. DLTs included grade 4 thrombocytopenia, leukopenia and neutropenia. The treatment was well tolerated with fatigue, nausea/vomiting, fever, transaminitis, and cytopenias being the most common toxicities. Among 30 evaluable patients, 1 had a partial response and 15 had stable disease. Triapine PK was similar, although more variable, compared to previous studies using doses normalized to body-surface-area. Steady decline in circulating levels of RRM2 may correlate with outcome.
This combination was well tolerated and showed evidence of preliminary activity in this heavily pretreated patient population, including prior gemcitabine failure.
PMCID: PMC3646991  PMID: 22847785
Triapine; Gemcitabine; Phase I; Clinical Trial
16.  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.
PMCID: PMC3755957  PMID: 23063585
patient selection; DNA repair foci
17.  Molecular pathways and therapeutic targets in lung cancer 
Oncotarget  2014;5(6):1392-1433.
Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review.
PMCID: PMC4039220  PMID: 24722523
lung cancer; targeted therapy; immune therapy
18.  A Phase II Study of Modulated-Capecitabine and Docetaxel in Chemonaive Patients with Advanced Non-Small Cell Lung Cancer (NSCLC) 
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.
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.
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.
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.
PMCID: PMC3549571  PMID: 23079156
non-small cell lung cancer; dihydropyrimidine deficiency; capecitabine
19.  Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer 
Frontiers in Oncology  2014;4:368.
The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.
PMCID: PMC4271581  PMID: 25566506
lung cancer; Fanconi anemia; pathway dysfunction; therapeutic target; FATSI
20.  Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors 
Diagnostic Pathology  2013;8:201.
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.
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).
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:
PMCID: PMC3933389  PMID: 24326178
Protein arginine methyltransferase-5; Lung carcinoma; Neuroendocrine tumors
21.  A dose escalation and pharmacodynamic study of triapine and radiation in patients with locally advanced pancreas cancer 
Triapine (Vion Pharmaceuticals), a novel inhibitor of the M2 subunit of ribonucleotide reductase (RR), is a potent radiosensitizer. This NCI/CTEP-sponsored phase I study assessed the safety/tolerability of triapine in combination with radiation (RT) in patients with locally advanced pancreas cancer (LAPCA).
We evaluated 3 dose levels of triapine (24 mg/m2, 48 mg/m2, 72 mg/m2) administered with 50.4 Gy of RT in 28 fractions. Patients with LAPCA received triapine thrice weekly, every other week during the course of RT. Dose-limiting toxicity (DLT) was assessed during and for 4 weeks following completion of RT. Dynamic contrast enhanced (DCE)-MRI and serum RR levels were evaluated as potential predictors for early response.
Twelve patients were treated. Four patients (1 non-evaluable [NE]) were enrolled at dose level 1 (DL1), three patients at DL2, and five patients (2NE) at DL3. No DLTs were observed and the MTD was not reached. Two patients (17%) achieved PR and 6 patients (50%) had SD. One patient underwent R0 resection following therapy. 92% of patients (100% on DL3) experienced freedom from local tumor progression. 75% of patients who eventually progressed developed metastases without local progression. RR levels did not appear to predict outcome. In 4 patients with available data, DCE-MRI may predict early response or resistance to therapy.
The combination of triapine at 72 mg/m2 three times weekly every other week and standard RT is tolerable with interesting activity in patients with LAPCA.
PMCID: PMC3477302  PMID: 22818416
Triapine; radiation; pancreas cancer
22.  Phase II study of dasatinib in patients with previously treated malignant mesothelioma (Cancer and Leukemia Group B 30601): a brief report 
Journal of Thoracic Oncology  2012;7(4):755-759.
We conducted a phase II trial of dasatinib in malignant mesothelioma (MM) patients to evaluate its toxicity and efficacy as a second-line treatment.
Material and Methods
Patients with unresectable MM and no symptomatic effusions were given dasatinib 70 mg twice daily as part of a 28-day cycle. We also measured plasma VEGF and PDGFβ and serum CSF-1 and mesothelin-related protein at baseline and during therapy.
Forty-six patients were enrolled in this study. Fifty percent of the first 12 patients enrolled experienced ≥ grade 3 treatment-related adverse events, and therefore, the starting dose was reduced to 50 mg twice daily. Grade 3 and 4 toxicities included fatigue (11%) and pleural effusion (9%). The overall disease control rate was 32.6%, and PFS at 24 weeks was 23% (95% CI: 13.5%, 40.0%). Survival was markedly longer in patients with lower pre-treatment CSF-1 levels and in patients whose CSF-1 levels decreased from baseline during therapy.
Single-agent dasatinib has no activity in MM and is associated with pulmonary toxicities that prohibit its use in an unselected MM population.
PMCID: PMC3308128  PMID: 22425926
dasatinib; mesothelioma; SRC kinase
23.  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.
PMCID: PMC3375896  PMID: 20888418
in situ hybridization; immunohistochemistry; fixation; microRNA; real-time PCR; EBV; Papillomavirus
24.  Altered p16INK4 and RB1 Expressions Are Associated with Poor Prognosis in Patients with Nonsmall Cell Lung Cancer 
Journal of Oncology  2012;2012:957437.
p16INK4 and RB1 are two potent cell cycle regulators to control the G1/S transition by interacting with CDK4/6, E2F, and D-type cyclins, respectively. Depending on the tumour type, genetic alterations resulting in the functional inactivation have frequently been reported in both genes. By contrast, much less is known regarding the overexpression of these proteins in the tumor cells. In this study, expressions of p16INK4 RB1, and CDKN2A copy number variances (CNV) in the tumor cells were assessed by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively, in 73 nonsmall cell lung cancer (NSCLC) with known 5-year survivals. The histologic type (P = 0.01), p16INK4 (P = 0.004), and RB1 (P < 0.001) were predictive of survivals. The CDKN2A CNV (P < 0.05) was also significant when compared to those cases without CNV. Therefore, among the molecular genetic prognostic factors, expressions of RB1 and p16INK4 in the tumor cells were the most strongly predictive of adverse outcomes in stage I and II nonsquamous NSCLC.
PMCID: PMC3350860  PMID: 22619677
25.  A Novel Classification of Lung Cancer into Molecular Subtypes 
PLoS ONE  2012;7(2):e31906.
The remarkably heterogeneous nature of lung cancer has become more apparent over the last decade. In general, advanced lung cancer is an aggressive malignancy with a poor prognosis. The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer, however, has created new opportunities for targeted therapy and improved outcome. In this paper, we define “molecular subtypes” of lung cancer based on specific actionable genetic aberrations. Each subtype is associated with molecular tests that define the subtype and drugs that may potentially treat it. We hope this paper will be a useful guide to clinicians and researchers alike by assisting in therapy decision making and acting as a platform for further study. In this new era of cancer treatment, the ‘one-size-fits-all’ paradigm is being forcibly pushed aside—allowing for more effective, personalized oncologic care to emerge.
PMCID: PMC3283716  PMID: 22363766

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