David P Carbone

Publications of David P Carbone

• Identifying Barriers Associated With Enrollment of Patients With Lung Cancer into Clinical Trials.

  Authors: Leora Horn, Vicki L Keedy, Norma Campbell, Gabe Garcia, Amy Hayes, Brady Spencer, David P Carbone, Alan Sandler, David H Johnson

  Clinical lung cancer. 05/2012;

  BACKGROUND: Enrollment of patients with lung cancer into clinical trials is required to accelerate the pace of new therapy development and contribute to a better understanding of the biologicalBACKGROUND: Enrollment of patients with lung cancer into clinical trials is required to accelerate the pace of new therapy development and contribute to a better understanding of the biological characteristics of cancer. METHODS: We conducted a retrospective chart review of all patients seen by the thoracic medical oncology team at the Vanderbilt Ingram Cancer Center (VICC) from November 2005 to November 2008 to determine the barriers associated with patient enrollment in to clinical trials. RESULTS: One thousand forty-three patient charts were audited: 32% of patients were eligible for enrollment, and 14% enrolled in a study. There were no significant differences in protocol availability or eligibility by sex, smoking status, or age. Patients living further from the cancer center were significantly less likely to have a study protocol available (P = .009), but if a protocol was available they were more likely to be eligible for enrollment (P < .001). Significantly more protocols were available for patients with non-small-cell lung cancer (NSCLC) compared with those who had small-cell lung cancer (SCLC) (63% vs. 48%; P < .001). Patients with advanced disease were more likely to have a protocol available (P < .001) and enter a study (P = .031). The most common reasons for patients not being eligible for enrollment were poor performance status (32%) and presence of comorbid disease (27%). The most common reasons for potentially eligible patients not enrolling in a study included preference for treatment closer to home (49%) and patient refusal (43%). CONCLUSION: Additional strategies are required to increase accrual of patients into lung cancer trials, including development of protocols for early-stage disease and modifying eligibility and performance status criteria for this unique patient population.

• Smoking Attenuates Transforming Growth Factor-β-Mediated Tumor Suppression Function through Downregulation of Smad3 in Lung Cancer.

  Authors: Debangshu Samanta, Adriana L Gonzalez, Nagaraj Nagathihalli, Fei Ye, David P Carbone, Pran K Datta

  Cancer prevention research (Philadelphia, Pa.). 03/2012; 5(3):453-63.

  Epidemiologic studies have shown that most cases of lung cancers (85%-90%) are directly attributable to cigarette smoking. Although much information has been gained about the effects of cigaretteEpidemiologic studies have shown that most cases of lung cancers (85%-90%) are directly attributable to cigarette smoking. Although much information has been gained about the effects of cigarette smoking on various signaling pathways causing lung cancer, nothing is known about the effect of cigarette smoking on the TGF-β-induced tumor suppressor function in lung cancer. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated with cigarette smoke condensate (CSC) and dimethyl sulfoxide (as a control) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in a decrease in Smad3 and Smad4 complex formation and TGF-β-mediated transcription due to reduced expression of Smad3. Long-term CSC treatment reduced apoptosis, increased cell viability, decreased TGF-β-mediated growth inhibition, and enhanced tumorigenicity. The decrease in apoptosis is due to the upregulation of Bcl-2, which is a downstream target of Smad3. Re-expression of Smad3 in the CSC-treated cells restored TGF-β signaling, increased apoptosis, and decreased cell viability and tumorigenicity. Withdrawal of CSC treatment resulted in the restoration of Smad3 expression, reduction in cell viability, and increased TGF-β-mediated growth inhibition. Expression of Smad3 is lower in lung tumors of current smokers than that observed in never-smokers. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity partly by abrogating TGF-β-mediated growth inhibition and apoptosis by reducing expression of Smad3. Cancer Prev Res; 5(3); 453-63. ©2012 AACR.

• Added Value of a Serum Proteomic Signature in the Diagnostic Evaluation of Lung Nodules.

  Authors: Chad V Pecot, Ming Li, Xueqiong J Zhang, Rama Rajanbabu, Ciara Calitri, Aaron Bungum, James R Jett, Joe B Putnam, Carol Callaway-Lane, Steve Deppen, Eric L Grogan, David P Carbone, John A Worrell, Karel G M Moons, Yu Shyr, Pierre P Massion

  Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 02/2012;

  BACKGROUND: Current management of lung nodules is complicated by nontherapeutic resections and missed chances for cure. We hypothesized that a serum proteomic signature may add diagnostic informationBACKGROUND: Current management of lung nodules is complicated by nontherapeutic resections and missed chances for cure. We hypothesized that a serum proteomic signature may add diagnostic information beyond that provided by combined clinical and radiographic data.METHODS: Cohort A included 265 and cohort B 114 patients. Using multivariable logistic regression analysis we calculated the area under the receiver operating characteristic curve (AUC) and quantified the added value of a previously described serum proteomic signature beyond clinical and radiographic risk factors for predicting lung cancer using the integration discrimination improvement (IDI) index.RESULTS: The average computed tomography (CT) measured nodule size in cohorts A and B was 37.83 versus 23.15 mm among patients with lung cancer and 15.82 versus 17.18 mm among those without, respectively. In cohort A, the AUC increased from 0.68 to 0.86 after adding chest CT imaging variables to the clinical results, but the proteomic signature did not provide meaningful added value. In contrast, in cohort B, the AUC improved from 0.46 with clinical data alone to 0.61 when combined with chest CT imaging data and to 0.69 after adding the proteomic signature (IDI of 20% P = 0.0003). In addition, in a subgroup of 100 nodules between 5 and 20 mm in diameter, the proteomic signature added value with an IDI of 15% (P ≤ 0.0001).CONCLUSIONS: The results show that this serum proteomic biomarker signature may add value to the clinical and chest CT evaluation of indeterminate lung nodules.Impact: This study suggests a possible role of a blood biomarker in the evaluation of indeterminate lung nodules. Cancer Epidemiol Biomarkers Prev; 1-7. ©2012 AACR.

• ROS1 Rearrangements Define a Unique Molecular Class of Lung Cancers.

  Authors: Kristin Bergethon, Alice T Shaw, Sai-Hong Ignatius Ou, Ryohei Katayama, Christine M Lovly, Nerina T McDonald, Pierre P Massion, Christina Siwak-Tapp, Adriana Gonzalez, Rong Fang [......] Haiquan Chen, Keith D Wilner, Eunice L Kwak, Jeffrey W Clark, David P Carbone, Hongbin Ji, Jeffrey A Engelman, Mari Mino-Kenudson, William Pao, A John Iafrate

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 01/2012;

  PURPOSEChromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known aboutPURPOSEChromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement. PATIENTS AND METHODSUsing a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.ResultsOf 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response. CONCLUSIONROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.

• Role of insulin-like growth factor-1 signaling pathway in cisplatin-resistant lung cancer cells.

  Authors: Yunguang Sun, Siyuan Zheng, Artour Torossian, Christina K Speirs, Stephen Schleicher, Nicholas J Giacalone, David P Carbone, Zhongming Zhao, Bo Lu

  International journal of radiation oncology, biology, physics. 12/2011; 82(3):e563-72.

  The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatinThe development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. Cisplatin-R cells illustrated greater expression of the markers CD133 and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.

• Insulin-like growth factor receptor expression is associated with aggressive phenotypes and has therapeutic activity in biliary tract cancers.

  Authors: Hirokazu Ohashi, Yasushi Adachi, Hiroyuki Yamamoto, Hiroaki Taniguchi, Katsuhiko Nosho, Hiromu Suzuki, Yoshiaki Arimura, Kohzoh Imai, David P Carbone, Yasuhisa Shinomura

  Cancer science. 11/2011; 103(2):252-61.

  Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeuticInsulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeutic target have not been studied comprehensively in biliary tract carcinomas (BTC). We investigated the immunohistochemical expression of elements of the IGF axis, matrilysin, overexpression of p53 and the methylation status of the IGFBP-3 promoter in 80 surgically resected BTC. We also assessed the effect of IGF-IR blockade on signal transduction, proliferation and survival in three BTC cell lines using a new tyrosine kinase inhibitor, BMS-536924, and dominant negative IGF-IR (IGF-IR/dn). The effects of IGF-IR blockade was also studied in nude mouse xenograft models. IGF-I was expressed in 60% and IGF-II in 50% of tumors. High expression was associated with tumor size. IGF-IR was expressed in 69% of the cases and was associated with advanced stage and matrilysin expression. Hypermethylation of the IGFBP-3 promoter was detected in 41% of BTC and was inversely correlated with p53 expression. BMS-536924 blocked autophosphorylation of IGF-IR and both Akt and ERK activation by both IGF-I and insulin. BMS-536924 suppressed proliferation and tumorigenicity in vitro in a dose-dependent fashion. This inhibitor upregulated chemotherapy-induced apoptosis in a dose-dependent fashion. Moreover, IGF-IR blockade was effective against tumors in mice. IGF-IR might identify a subset of BTC with a particularly aggressive phenotype and is a candidate therapeutic target in this disease. BMS-536924 might have significant therapeutic utility.

• Elucidating the mechanism of regulation of transforming growth factor β Type II receptor expression in human lung cancer cell lines.

  Authors: Sunil K Halder, Yong-Jig Cho, Arunima Datta, Govindaraj Anumanthan, Amy-Joan L Ham, David P Carbone, Pran K Datta

  Neoplasia (New York, N.Y.). 10/2011; 13(10):912-22.

  Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive andLung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII). However, little is known about the mechanism of down-regulation of TβRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-β-induced tumor suppressor function. Here we show that HDIs restore TβRII expression and that DNA hypermethylation has no effect on TβRII promoter activity in lung cancer cell lines. TGF-β-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TβRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TβRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TβRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TβRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TβRII in lung cancer and that TGF-β tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TβRII expression.

• Adjuvant therapy in non-small cell lung cancer: future treatment prospects and paradigms.

  Authors: David P Carbone, Enriqueta Felip

  Clinical lung cancer. 08/2011; 12(5):261-71.

  There is a high risk of relapse after curative-intent resection for even early-stage non-small cell lung cancer (NSCLC), and thus adjuvant chemotherapy has been explored with the goal of eliminatingThere is a high risk of relapse after curative-intent resection for even early-stage non-small cell lung cancer (NSCLC), and thus adjuvant chemotherapy has been explored with the goal of eliminating occult metastases and consequently reducing the risk of recurrence. Although adjuvant chemotherapy confers a survival advantage of approximately 5% at 5 years and is now generally accepted for patients with stage II-IIIA disease, adjuvant therapy for patients with stage I disease is more controversial. In this review we describe approaches to improve treatment outcomes and ongoing research into new therapies in the adjuvant setting. In the future it is likely that patient selection on the basis of gene signatures and biomarkers will be of increasing importance in determining optimal treatment for individual patients. New targeted therapies such as epidermal growth factor receptor (EGFR) inhibitors, angiogenesis inhibitors, and anticancer immunotherapies are showing activity in the advanced disease setting and are being studied for incorporation into multimodal adjuvant treatment approaches. It is hoped that such advances and a changing treatment paradigm will ultimately result in greater survival for patients with early NSCLC.

• Resuscitating cancer immunosurveillance: selective stimulation of DLL1-Notch signaling in T cells rescues T-cell function and inhibits tumor growth.

  Authors: Yuhui Huang, Luping Lin, Anil Shanker, Anshu Malhotra, Li Yang, Mikhail M Dikov, David P Carbone

  Cancer research. 08/2011; 71(19):6122-31.

  Deficiencies in immune function that accumulate during cancer immunoediting lead to a progressive escape from host immunosurveillance. Therapies that correct or overcome these defects could have aDeficiencies in immune function that accumulate during cancer immunoediting lead to a progressive escape from host immunosurveillance. Therapies that correct or overcome these defects could have a powerful impact on cancer management, but current knowledge of the types and mechanisms of immune escape is still incomplete. Here, we report a novel mechanism of escape from T-cell immunity that is caused by reduction in levels of the Delta family Notch ligands DLL1 and DLL4 in hematopoietic microenvironments. An important mediator of this effect was an elevation in the levels of circulating VEGF. Selective activation of the DLL1-Notch signaling pathway in bone marrow precursors enhanced T-cell activation and inhibited tumor growth. Conversely, tumor growth led to inhibition of Delta family ligand signaling through Notch in the hematopoietic environment, resulting in suppressed T-cell function. Overall, our findings uncover a novel mechanism of tumoral immune escape and suggest that a soluble multivalent form of DLL1 may offer a generalized therapeutic intervention to stimulate T-cell immunity and suppress tumor growth.

• The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status.

  Authors: Masanori Ii, Hua Li, Yasushi Adachi, Hiroyuki Yamamoto, Hirokazu Ohashi, Hiroaki Taniguchi, Yoshiaki Arimura, David P Carbone, Kohzoh Imai, Yasuhisa Shinomura

  Clinical cancer research : an official journal of the American Association for Cancer Research. 06/2011; 17(15):5048-59.

  Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown successful targeting therapy forInsulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown successful targeting therapy for colorectal, pancreatic, gastric, and esophageal carcinomas using recombinant adenoviruses expressing dominant negative IGF-IR. Mutation in k-ras is one of key factors in gastrointestinal cancers. In this study, we sought to evaluate the effect of a new monoclonal antibody for IGF-IR, figitumumab (CP-751,871), on the progression of human gastrointestinal carcinomas with/without k-ras mutation. We assessed the effect of figitumumab on signal transduction, proliferation, and survival in six gastrointestinal cancer cell lines with/without k-ras mutation, including colorectal and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. Combination effects of figitumumab and chemotherapy were also studied. Then figitumumab was evaluated in the treatment of xenografts in nude mice. Figitumumab blocked autophosphorylation of IGF-IR and its downstream signals. The antibody suppressed proliferation and tumorigenicity in all cell lines. Figitumumab inhibited survival by itself and up-regulated chemotherapy (5-FU and gemcitabine) induced apoptosis. Moreover, the combination of this agent and chemotherapy was effective against tumors in mice. The effect of figitumumab was not influenced by the mutation status of k-ras. Figitumumab reduced expression of IGF-IR but not insulin receptor in these xenografted tumors. The drug did not affect murine body weight or blood concentrations of glucose, insulin, IGF binding protein 3, and growth hormone. IGF-IR might be a good molecular therapeutic target and figitumumab may thus have therapeutic value in human gastrointestinal malignancies even in the presence of k-ras mutations.

• Lung cancer diagnosis from proteomic analysis of preinvasive lesions.

  Authors: S M Jamshedur Rahman, Adriana L Gonzalez, Ming Li, Erin H Seeley, Lisa J Zimmerman, Xueqiong J Zhang, M Lisa Manier, Sandra J Olson, Ronak N Shah, Alison N Miller, Joe B Putnam, York E Miller, Wilbur A Franklin, William J Blot, David P Carbone, Yu Shyr, Richard M Caprioli, Pierre P Massion

  Cancer research. 04/2011; 71(8):3009-17.

  Early detection may help improve survival from lung cancer. In this study, our goal was to derive and validate a signature from the proteomic analysis of bronchial lesions that could predict theEarly detection may help improve survival from lung cancer. In this study, our goal was to derive and validate a signature from the proteomic analysis of bronchial lesions that could predict the diagnosis of lung cancer. Using previously published studies of bronchial tissues, we selected a signature of nine matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) mass-to-charge ratio features to build a prediction model diagnostic of lung cancer. The model was based on MALDI MS signal intensity (MALDI score) from bronchial tissue specimens from our 2005 published cohort of 51 patients. The performance of the prediction model in identifying lung cancer was tested in an independent cohort of bronchial specimens from 60 patients. The probability of having lung cancer based on the proteomic analysis of the bronchial specimens was characterized by an area under the receiver operating characteristic curve of 0.77 (95% CI 0.66-0.88) in this validation cohort. Eight of the nine features were identified and validated by Western blotting and immunohistochemistry. These results show that proteomic analysis of endobronchial lesions may facilitate the diagnosis of lung cancer and the monitoring of high-risk individuals for lung cancer in surveillance and chemoprevention trials.

• Lung cancer in 2010: One size does not fit all.

  Authors: Christine M Lovly, David P Carbone

  Nature reviews. Clinical oncology. 02/2011; 8(2):68-70.

  Advances in non-small-cell lung cancer over the past decade have resulted in new treatments with minimal toxic effects and dramatic clinical benefits. 2010 saw continued advancement in ourAdvances in non-small-cell lung cancer over the past decade have resulted in new treatments with minimal toxic effects and dramatic clinical benefits. 2010 saw continued advancement in our understanding of the molecular genetics of lung cancer and of specific targeted inhibitors with remarkable clinical benefit in selected populations.

• Inhibition of JAK2 signaling by TG101209 enhances radiotherapy in lung cancer models.

  Authors: Yunguang Sun, Luigi Moretti, Nicholas J Giacalone, Stephen Schleicher, Christina K Speirs, David P Carbone, Bo Lu

  Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 02/2011; 6(4):699-706.

  Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. We tested whether TG101209, aPersistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. TG101209 inhibited STAT3 activation and survivin expression and sensitized HCC2429 (dose enhancement ratio = 1.34, p = 0.002) and H460 (dose enhancement ratio = 1.09, p = 0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with KRAS-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared with radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to KRAS mutation status for future clinical trials testing combination of TG101209 and radiotherapy.

• Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry.

  Authors: Haixia Zhang, Qinfeng Liu, Lisa J Zimmerman, Amy-Joan L Ham, Robbert J C Slebos, Jamshedur Rahman, Takefume Kikuchi, Pierre P Massion, David P Carbone, Dean Billheimer, Daniel C Liebler

  Molecular & cellular proteomics : MCP. 02/2011; 10(6):M110.006593.

  Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high costLiquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

• Insulin-like growth factor-I receptor blockade reduces tumor angiogenesis and enhances the effects of bevacizumab for a human gastric cancer cell line, MKN45.

  Authors: Hua Li, Yasushi Adachi, Hiroyuki Yamamoto, Yongfen Min, Hirokazu Ohashi, Masanori Ii, Yoshiaki Arimura, Takao Endo, Choon-Taek Lee, David P Carbone, Kohzoh Imai, Yasuhisa Shinomura

  Cancer. 01/2011; 117(14):3135-47.

  Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for tumorigenicity and tumor progression of gastrointestinal cancers. The authors previously reported the success of therapyInsulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for tumorigenicity and tumor progression of gastrointestinal cancers. The authors previously reported the success of therapy for gastrointestinal cancers using adenoviruses that expressed dominant-negative IGF-IR (IGF-IR/dn). In addition, it has been demonstrated that IGF-IR signaling affects vascular endothelial growth factor (VEGF) expression in some other types of tumors. The objective of the current study was to evaluate this interaction by studying the roles of IGF-IR in tumor angiogenesis and lymphangiogenesis and their implications for targeted therapy in gastric cancer. The impact of IGF signals on the expression of VEGF-A and VEGF-C in a human gastric cancer cell, MKN45, and vascular formation were assessed. The effects of IGF-IR/dn with or without bevacizumab on angiogenesis, lymphangiogenesis, and tumor suppression in mouse xenografts were assessed. IGFs induced the expression of VEGF ligands and up-regulated in vitro vascular vessel formation. IGF-IR/dn reduced VEGF expression, reduced the activation of both protein kinase B (Akt) and mitogen-activated protein kinase (MAPK), and reduced vascular formation, indicating that IGF-IR/dn inhibited tumor growth in mice by inhibiting both angiogenesis and lymphangiogenesis. However, IGF-IR/dn did not affect either blood sugar or body weight in these mice. The combination of IGF-IR/dn and bevacizumab was highly effective against these xenograft tumors, and only this combination resulted in the complete regression of 43% of tumors, reduced the expression of VEGF, and induced apoptosis. The current results indicated that IGF-IR is involved in angiogenesis and lymphangiogenesis through the modulation of VEGF ligand expression in the gastric cancer cell line MKN45. Targeting IGF-IR in combination with agents that block the VEGF pathway may have therapeutic utility for gastric cancer therapy.

• A candidate targeting molecule of insulin-like growth factor-I receptor for gastrointestinal cancers.

  Authors: Yasushi Adachi, Hiroyuki Yamamoto, Hirokazu Ohashi, Takao Endo, David-P Carbone, Kohzoh Imai, Yasuhisa Shinomura

  World journal of gastroenterology : WJG. 12/2010; 16(46):5779-89.

  Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage. One new group of targets is tyrosine kinase receptors, which can be treated by severalAdvances in molecular research in cancer have brought new therapeutic strategies into clinical usage. One new group of targets is tyrosine kinase receptors, which can be treated by several strategies, including small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs). Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal (GI) carcinomas. The concept of targeting specific carcinogenic receptors has been validated by successful clinical application of many new drugs. Type I insulin-like growth factor (IGF) receptor (IGF-IR) signaling potently stimulates tumor progression and cellular differentiation, and is a promising new molecular target in human malignancies. In this review, we focus on this promising therapeutic target, IGF-IR. The IGF/IGF-IR axis is an important modifier of tumor cell proliferation, survival, growth, and treatment sensitivity in many malignant diseases, including human GI cancers. Preclinical studies demonstrated that downregulation of IGF-IR signals reversed the neoplastic phenotype and sensitized cells to anticancer treatments. These results were mainly obtained through our strategy of adenoviruses expressing dominant negative IGF-IR (IGF-IR/dn) against gastrointestinal cancers, including esophagus, stomach, colon, and pancreas. We also summarize a variety of strategies to interrupt the IGFs/IGF-IR axis and their preclinical experiences. Several mAbs and TKIs targeting IGF-IR have entered clinical trials, and early results have suggested that these agents have generally acceptable safety profiles as single agents. We summarize the advantages and disadvantages of each strategy and discuss the merits/demerits of dual targeting of IGF-IR and other growth factor receptors, including Her2 and the insulin receptor, as well as other alternatives and possible drug combinations. Thus, IGF-IR might be a candidate for a molecular therapeutic target in human GI carcinomas.

• Anti-vascular endothelial growth factor treatment in combination with chemotherapy delays hematopoietic recovery due to decreased proliferation of bone marrow hematopoietic progenitor cells.

  Authors: Sergey V Novitskiy, Ildiko Csiki, Yuhui Huang, David H Johnson, Eva M Harth, David P Carbone, Mikhail M Dikov

  Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 09/2010; 5(9):1410-5.

  Targeting of cancer by chemotherapy in combination with anti-vascular endothelial growth factor (VEGF) therapy has demonstrated not only the clinical efficacy but also a higher risk of seriousTargeting of cancer by chemotherapy in combination with anti-vascular endothelial growth factor (VEGF) therapy has demonstrated not only the clinical efficacy but also a higher risk of serious hematologic complications including neutropenia. The purpose of the study was to elucidate the molecular mechanisms responsible for the development of neutropenia during the combination treatment. Mouse model and in vitro studies were undertaken to determine the effect of interference with VEGF signaling by VEGF-specific agents or a multitargeted VEGF receptor (VEGFR) tyrosine kinase inhibitor on proliferation of hematopoietic progenitor cell (HPC) and repopulation of the hematopoietic compartment after myeloablation. The studies demonstrated that blockage of VEGFR1 or VEGFR2 signaling decreased HPC proliferation and impaired repopulation of the hematopoietic compartment after myelosuppression by slowing the progression of HPC through the cell cycle. The combination of cytotoxic drugs and VEGFR tyrosine kinase inhibitor had an additive inhibitory effect and decreased proliferation of HPC significantly stronger than either agent alone. Signaling through both VEGFR1 and VEGFR2 is required for normal reconstitution of the hematopoietic compartment after cytotoxic chemotherapy.

• VeriStrat classifier for survival and time to progression in non-small cell lung cancer (NSCLC) patients treated with erlotinib and bevacizumab.

  Authors: David P Carbone, J Stuart Salmon, Dean Billheimer, Heidi Chen, Alan Sandler, Heinrich Roder, Joanna Roder, Maxim Tsypin, Roy S Herbst, Anne S Tsao, Hai T Tran, Thao P Dang

  Lung cancer (Amsterdam, Netherlands). 09/2010; 69(3):337-40.

  We applied an established and commercially available serum proteomic classifier for survival after treatment with erlotinib (VeriStrat) in a blinded manner to pretreatment sera obtained fromWe applied an established and commercially available serum proteomic classifier for survival after treatment with erlotinib (VeriStrat) in a blinded manner to pretreatment sera obtained from recurrent advanced NSCLC patients before treatment with the combination of erlotinib plus bevacizumab. We found that VeriStrat could classify these patients into two groups with significantly better or worse outcomes and may enable rational selection of patients more likely to benefit from this costly and potentially toxic regimen.

• Detection of tumor epidermal growth factor receptor pathway dependence by serum mass spectrometry in cancer patients.

  Authors: Christine H Chung, Erin H Seeley, Heinrich Roder, Julia Grigorieva, Maxim Tsypin, Joanna Roder, Barbara A Burtness, Athanassios Argiris, Arlene A Forastiere, Jill Gilbert, Barbara Murphy, Richard M Caprioli, David P Carbone, Ezra E W Cohen

  Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 02/2010; 19(2):358-65.

  We hypothesized that a serum proteomic profile predictive of survival benefit in non-small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitorsWe hypothesized that a serum proteomic profile predictive of survival benefit in non-small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) reflects tumor EGFR dependency regardless of site of origin or class of therapeutic agent. Pretreatment serum or plasma from 230 patients treated with cetuximab, EGFR-TKIs, or chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC) were analyzed by mass spectrometry. Each sample was classified into "good" or "poor" groups using VeriStrat, and survival analyses of each cohort were done based on this classification. For the CRC cohort, this classification was correlated with the tumor EGFR ligand levels and KRAS mutation status. In the EGFR inhibitor-treated cohorts, the classification predicted survival (HNSCC: gefitinib, P = 0.007 and erlotinib/bevacizumab, P = 0.02; CRC: cetuximab, P = 0.0065) whereas the chemotherapy cohort showed no survival difference. For CRC patients, tumor EGFR ligand RNA levels were significantly associated with the proteomic classification, and combined KRAS and proteomic classification provided improved survival classification. Serum proteomic profiling can detect clinically significant tumor dependence on the EGFR pathway in non-small cell lung cancer, HNSCC, and CRC patients treated with either EGFR-TKIs or cetuximab. This classification is correlated with tumor EGFR ligand levels and provides a clinically practical way to identify patients with diverse cancer types most likely to benefit from EGFR inhibitors. Prospective studies are necessary to confirm these findings.

• Genetic and proteomic features associated with survival after treatment with erlotinib in first-line therapy of non-small cell lung cancer in Eastern Cooperative Oncology Group 3503.

  Authors: Joseph M Amann, Ju-Whei Lee, Heinrich Roder, Julie Brahmer, Adriana Gonzalez, Joan H Schiller, David P Carbone

  Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 02/2010; 5(2):169-78.

  Serum proteomics and mutations in the epidermal growth factor receptor (EGFR) and KRAS have been associated with benefit after therapy with EGFR-targeted therapies in non-small cell lung cancer, butSerum proteomics and mutations in the epidermal growth factor receptor (EGFR) and KRAS have been associated with benefit after therapy with EGFR-targeted therapies in non-small cell lung cancer, but all three have not been evaluated in any one study. Pretreatment serum proteomics predicts survival in Western advanced non-small cell lung cancer patients with wild-type EGFR and independent of KRAS mutation status. We analyzed available biospecimens from Eastern Cooperative Oncology Group 3503, a single-arm phase II study of erlotinib in first-line advanced lung cancer, for proteomics signatures in the previously described serum matrix-assisted laser desorption ionization proteomic classifier (VeriStrat) as well as for KRAS and EGFR mutations. Out of 137 enrolled patients, analyzable biologic samples were available on 102. Nine of 41 (22%) demonstrated KRAS mutations and 3 of 41 (7%) harbored EGFR mutations. VeriStrat classification identified 64 of 88 (73%) as predicted to have "good" and 24 of 88 (27%) predicted to have "poor" outcomes. A statistically significant correlation of VeriStrat status (p < 0.001) was found with survival. EGFR mutations, but not KRAS mutations, also correlated with survival. The previously defined matrix-assisted laser desorption ionization predictor remains a potent and highly clinically significant predictor of survival after first-line treatment with erlotinib in patients with wild-type EGFR and independent of mutations in KRAS.