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1.  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 
Background
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.1097/JTO.0000000000000071
PMCID: PMC4284815  PMID: 24419419
CALGB 30704; Lung cancer
3.  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.
Summary
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.
doi:10.1016/j.humpath.2014.01.003
PMCID: PMC4271837  PMID: 24746212
Post–solid-organ transplantation lung cancer; Chromogenic in situ hybridization for Y-chromosome
4.  A phase I dose escalation study of a pharmacobiologically based scheduling of capecitabine and mitomycin C in patients with gastrointestinal malignancies 
Background
Mitomycin C (MMC) produces significant upregulation of thymidine phosphorylase, a principal determinant of the therapeutic index of capecitabine-based treatment, starting 4–6 days after treatment. On the basis of the time-dependency of this upregulation, we performed a phase I dose escalation study of capecitabine and MMC in patients with gastrointestinal malignancies.
Methods
A total of 29 patients with advanced gastrointestinal malignancies received MMC at 6 mg/m2 on day 1 and capecitabine escalated in four successive patient cohorts of doses 500–1,000 mg/m2/day twice daily on days 8–21, every 28 days. MMC was capped at 36 mg/m2.
Results
A total of 29 patients were enrolled and 90% had at least one prior treatment in the metastatic setting. There was one DLT, grade 3 hand and foot syndrome, at dose level four. The most common toxicity was fatigue (61%). No patients experienced grade 4 toxicities. Nine patients experienced prolonged stability of disease.
Conclusion
Capecitabine in combination with MMC in the proposed schedule is well-tolerated with evidence of preliminary activity. The recommended dose for phase II studies are MMC at 6 mg/m2 on day 1 of a 28-day cycle with the dose capped at 36 mg/m2, in combination with capecitabine at 1,000 mg/m2 twice daily on days 8–21.
doi:10.1007/s00280-009-1091-9
PMCID: PMC4261191  PMID: 19657639
Capecitabine; Mitomycin C; Gastrointestinal malignancies
5.  Phase I/II Study of Inhaled Doxorubicin Combined with Platinum-Based Therapy for Advanced Non–Small Cell Lung Cancer 
Purpose
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.
Results
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.
Conclusion
Although this combination was safe, the primary objective was not met and will not be pursued further.
doi:10.1158/1078-0432.CCR-09-3015
PMCID: PMC4262532  PMID: 20371682
6.  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.
doi:10.1002/ijc.25049
PMCID: PMC4153870  PMID: 19921694
microRNA-34; PRIMA-1; apoptosis; lung cancer
7.  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.
doi:10.3390/ijerph110908645
PMCID: PMC4198984  PMID: 25158139
KRAS; non-small cell lung carcinoma; colorectal cancer; transition; transversion
8.  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.
doi:10.3390/ijerph110909491
PMCID: PMC4199031  PMID: 25222473
lung cancer; non-small cell lung carcinoma; oxidative stress; KRAS; mutation; NQO1; DNA methyl transferase; ERK1/2; c-MET; survivin
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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.
doi:10.3389/fonc.2014.00368
PMCID: PMC4271581  PMID: 25566506
lung cancer; Fanconi anemia; pathway dysfunction; therapeutic target; FATSI
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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