Cristina Fhied

Establishment of a multi-analyte serum biomarker panel to identify lymph node metastases in non-small cell lung cancer.

Introduction: In non-small cell lung cancer (NSCLC), the presence of locoregional lymph node metastases remains the most important prognostic factor and significantly guides treatment regimens. Unfortunately, currently-available noninvasive staging modalities have limited accuracy. The objective of this study was to create a multianalyte blood test capable of discriminating a patient’s true (pathologic) nodal status preoperatively. Methods: Pretreatment serum specimens collected from 107 NSCLC patients with localized disease were screened with 47 biomarkers implicated in disease presence or progression. Multivariate statistical algorithms were then used to identify the optimal combination of biomarkers for accurately discerning each patient’s nodal status. Results: We identified 15 candidate biomarkers that met our criteria for statistical relevance in discerning a patient’s preoperative nodal status. A ‘random forest’ classification algorithm was used with these parameters to define a 6-analyte panel, consisting of macrophage inflammatory protein-1α, carcinoembryonic antigen, stem cell factor, tumor necrosis factor-receptor I, interferon-γ, and tumor necrosis factor-α, that was the optimum combination of biomarkers for identifying a patient’s pathologic nodal status. A Classification and Regression Tree analysis was then created with this panel that was capable of correctly classifying 88% of the patients tested, relative to the pathologic assessments. This value is in contrast to our observed 85% classification rate using conventional clinical methods. Conclusions: This study establishes a serum biomarker panel with efficacy in discerning preoperative nodal status. With further validation, this blood test may be useful for assessing nodal status (including occult disease) in NSCLC patients facing tumor resection therapy.

Development and validation of a plasma biomarker panel for discerning clinical significance of indeterminate pulmonary nodules.

Introduction: The recent findings of the National Lung Screening Trial showed 24.2% of individuals at high risk for lung cancer having one or more indeterminate nodules detected by low-dose computed tomography–based screening, 96.4% of which were eventually confirmed as false positives. These positive scans necessitate additional diagnostic procedures to establish a definitive diagnosis that adds cost and risk to the paradigm. A plasma test able to assign benign versus malignant pathology in high-risk patients would be an invaluable tool to complement low-dose computed tomography–based screening and promote its rapid implementation. Methods: We evaluated 17 biomarkers, previously shown to have value in detecting lung cancer, against a discovery cohort, comprising benign (n = 67) cases and lung cancer (n = 69) cases. A Random Forest method based analysis was used to identify the optimal biomarker panel for assigning disease status, which was then validated against a cohort from the Mayo Clinic, comprising patients with benign (n = 61) or malignant (n = 20) indeterminate lung nodules. Results: Our discovery efforts produced a seven-analyte plasma biomarker panel consisting of interleukin 6 (IL-6), IL-10, IL-1ra, sIL-2R&agr;, stromal cell-derived factor-1&agr;+&bgr;, tumor necrosis factor &agr;, and macrophage inflammatory protein 1 &agr;. The sensitivity and specificity of our panel in our validation cohort is 95.0% and 23.3%, respectively. The validated negative predictive value of our panel was 93.8%. Conclusion: We developed a seven-analyte plasma biomarker panel able to identify benign nodules, otherwise deemed indeterminate, with a high degree of accuracy. This panel may have clinical utility in risk-stratifying screen-detected lung nodules, decrease unnecessary follow-up imaging or invasive procedures, and potentially avoid unnecessary morbidity, mortality, and health care costs.

Biomarkers of the insulin-like growth factor pathway predict progression and outcome in lung cancer.

BACKGROUND Insulin-like growth factor 1 (IGF-I), IGF binding proteins (IGFBP) 1 to 7, and C-peptide have been postulated to predict survival in non-small cell lung cancer (NSCLC). Studying serum levels in NSCLC patients treated with surgical resection may provide information on the aggressiveness of tumors and be predictive of disease recurrence. METHODS Immunobead assays were used to measure pretreatment serum levels of IGF-I, IGFBP1 to IGFBP7, and C-peptide in 100 NSCLC patients. Of these, 59 had no metastatic progression (T1 to T4 N0 M0), whereas 41 had positive lymph nodes (T1 to T4 N1 to N3 M0). Data were analyzed using the Mann-Whitney two-sided rank sum test or Kaplan-Meier curves. RESULTS Low serum IGFBP5 levels correlated strongly with a positive nodal status (p < 0.001) and any incidence of disease recurrence (p = 0.003). Low serum levels of IGFBP5 also predicted poor recurrence-free survivals in the overall cohort (p ≤ 0.001) and in patients with no nodal metastases (p = 0.027). Conversely, a high serum level of IGFBP7 correlated with positive nodal status (p = 0.008), but was not prognostic for recurrence-free survival. No significant correlations were found for IGFBP5 or IGFBP7 for sex, age, race, smoking history, tumor histology, or fasting state. CONCLUSIONS IGFBP5 and IGFBP7 had value as biomarkers for identifying NSCLC progression and patient outcome.

Enhancement of a multianalyte serum biomarker panel to identify lymph node metastases in non-small cell lung cancer with circulating autoantibody biomarkers

We recently reported the development of a multianalyte serum algorithm to identify nodal status in non‐small cell lung cancer (NSCLC) patients facing an anatomic resection with curative intent. This study aims to enhance the overall performance characteristics of this test by adding autoantibody biomarkers identified through immunoproteomic discovery. More specifically, we used sera from 20 NSCLC patients to probe 2‐D immunoblots of HCC827 lysates for tumor‐associated autoantigens. Relevant differences in immunoreactivity associated with pathological nodal status were then identified via tandem mass spectrometry. Identified autoantigens were then developed into Luminex immunobead assays alongside a series of autoantigen targets relevant to early‐disease detection. These assays were then used to measure circulating autoantibody levels in the identical cohort of NSCLC patients used in our original study. This strategy identified 11 autoantigens found primarily in patients with disease progression to the locoregional lymph nodes. Custom Luminex‐based “direct‐capture” assays (25 total; including autoantibody targets relevant to early‐disease detection) were assembled to measure autoantibody levels in sera from 107 NSCLC patients. Multivariate classification algorithms were then used to identify the optimal combination of biomarkers when considered collectively with our original 6‐analyte serum panel. The new algorithm resulting from this analysis consists of TNF‐α, TNF‐RI, MIP‐1α and autoantibodies against Ubiquilin‐1, hydroxysteroid‐(17‐β)‐dehydrogenase, and triosephosphate isomerase. The inclusion of autoantibody biomarkers provided a dramatic improvement in the overall test performance characteristics, relative to the original test panel, including an 11% improvement in the classification efficiency.

Value of circulating insulin-like growth factor-associated proteins for the detection of stage I non-small cell lung cancer.

OBJECTIVE Circulating biomarkers related to insulin-like growth factor (IGF) signaling are associated with disease progression in multiple carcinomas, but their potential diagnostic value for lung cancer screening has been inadequately examined. We evaluated 9 circulating IGF-related factors for their ability to assign clinical significance to indeterminate pulmonary nodules identified via computed tomography-based radiologic studies. METHODS Patients (n = 224 stage I non-small cell lung cancer; n = 123 benign) were enrolled by Rush University and the Mayo Clinic and had pretreatment serum evaluated for levels of IGF-1, IGF-2, and insulin-like growth factor binding proteins (IGFBPs) 1-7. The Mann-Whitney rank-sum test and receiver-operator characteristics curves were used to assess differences in biomarker concentrations relevant to malignant versus benign pathology. These targets were used to help refine our companion blood test for assigning clinical significance to computed tomography-detected solitary nodules (discovery cohort, n = 94) and were validated against an independent cohort from the Mayo Clinic (n = 81). RESULTS Patients with benign pulmonary nodules were found to have serum concentrations of IGFBP-3, IGFBP-5, IGF-1, and IGF-2 that were higher (P = .001, P < .001, P = .002, and P = .011, respectively) than those with non-small cell lung cancer, with distinct associations with histologic subtypes observed. Refinement of our multianalyte classification algorithm using IGF-related factors provided a new panel consisting of interleukin-6, interleukin-1 receptor antagonist, interleukin-10, stromal cell-derived factor-1(α + β), IGFBP-4, IGFBP-5, and IGF-2 with improved assay performance-achieving a (validated) negative predictive value of 100%. CONCLUSIONS Our findings suggest a divergent role for IGF signaling in the biology of benign and malignant pulmonary nodules. Upon further validation, these observations may help identify cases of false positives resulting from computed tomography-based screening studies.

Development of a bead-based immunoassay to routinely measure vimentin autoantibodies in the clinical setting.

INTRODUCTION Vimentin is an intermediate filament protein generally expressed in the cytosol of many adult cell types, including leukocytes, fibroblasts and endothelial cells. Several tissue and/or injury-specific isoforms of vimentin are known to exist that may trigger autoimmune responses due to aberrant structural or conformational variations. Such scenarios include allograft rejection and certain autoimmune diseases, such as rheumatoid arthritis. The primary objective for this study was to develop a Luminex immunobead assay to quantitate circulating levels of vimentin antibodies and, secondarily, to appraise the feasibility of these autoantibodies as a biomarker for clinical diagnosis. METHODS Recombinant human vimentin was conjugated to MagPlex® beads using standard carbodiimide/NHS chemistry and coupling efficiency tested and assay parameters determined using a commercial anti-vimentin polyclonal antibody. A limited number of serum samples (n=71) were then tested to evaluate the diagnostic value for future biomarker development efforts. RESULTS Findings from repeated testing of three distinct batches of assays provide assay range parameters of 0.18-15μg/mL, median inter-assay recovery parameter within 1% of completion, and inter-assay variation (%CV) at 7%. The assay was found to be stable at several conditions with less than 5% loss in a month. Preliminary evaluation of the assay demonstrates significantly (p=0.022) higher circulating levels of anti-vimentin antibodies in 51 cases of renal allograft rejection relative to 20 cases of age-matched controls. CONCLUSION A direct capture assay for vimentin autoantibodies was developed and analytically validated. Preliminary evaluation of this assay against patient materials was promising and justifies additional testing with larger cohorts in future studies.

Development of bead based multiplexed immunoassay for evaluation of midkine, syndecan-1, and ANGPTL4 in patient serum

ABSTRACT Background: Angiogenesis is associated with tumor progression in a range of malignancies. Herein, we develop custom immunobead assays for several mechanistically important targets and evaluated these against sera from cohorts of non-small cell lung cancer (NSCLC) patients. Methods: Antigen “capture” antibodies for midkine, syndecan-1, and ANGPTL4 were independently conjugated to MagPlex® Microspheres using standard carbodiimide/NHS-based chemistry. These reagents served as the basis for quantitative sandwich assay assembly using biotinylated detection antibodies and R-phycoerythrin-conjugated streptavidin reporter system. Standard curves were created using dilution series of recombinant target proteins with assay performance characteristics calculated, accordingly. Finally, we evaluated a range of serum samples from NSCLC patients (n = 32) to verify assay performance. Results: Multiplexed assays for midkine, syndecan-1, and ANGPTL4 were developed with three orders of magnitude in dynamic range, excellent intra- and inter-assay precision, and accuracy parameters (<10%, and <15% variability, respectively). Detection and quantifications limits were suitable for the three assays to efficiently evaluate sera across a range of disease stages with a four-fold dilution factor. Conclusion: We successfully developed and analytically validated a 3-plex immunobead assay for quantifying midkine, syndecan-1, and ANGPTL4 in patient sera. This multiplexed assay will provide an important tool for future studies delineating the role of angiogenesis in lung cancer progression.

Differential expression of circulating biomarkers of tumor phenotype and outcomes in previously treated non-small cell lung cancer patients receiving erlotinib vs. cytotoxic chemotherapy

Background The objective of this study was to identify serum biomarkers capable of predicting clinical outcomes in previously-treated NSCLC patients with wild-type for EGFR activating mutations or insufficient tissue for mutation status determination. Methods Sixty-six Luminex immunoassays representative of biological themes that emerged from a re-analysis of transcriptome data from the Cancer Genome Atlas (TCGA) were evaluate against pretreatment serum specimens from previously-treated advanced NSCLC patients received either cytotoxic chemotherapy (n=32) or erlotinib (n=79). Known EGFR mutation positive cases were excluded from analysis. Associations of biomarkers with outcome parameters and their differential interaction with treatment for survival outcomes were assessed using multivariate Cox PH analyses. Results Our EMT-based transcriptomic analysis revealed a range of biological processes associated with angiogenesis, apoptosis, cachexia, inflammation, and metabolism emerging as those most highly associated with patient outcome. These processes were evaluated via surrogate serum biomarkers. A treatment-biomarker interaction analysis revealed that higher pretreatment levels of c-Met signaling biomarkers (i.e. HGF levels), pro-inflammatory/ pro-cachexia (e.g. IL-8, sIL-2Rα, FGF-2) processes and a pro-angiogenic (e.g. TGF-α, IL-8, VEGF) milieu were associated with inferior survival (HR=0.35, 0.29, 0.58, 0.50, 0.61, 0.45, respectively; all p<0.05) for patients receiving chemotherapy, relative to erlotinib. In contrast, high levels of decoy receptor for IL-1, sIL-1RII, and a high tissue vimentin/E-cadherin ratio were associated with a poor OS (HR=3.78; p=0.00055) in the erlotinib cohort. Conclusions Contemporary precision medicine initiatives that pair patient tumor characteristics with the optimal therapy type may maximize the use of agents targeting EGFR in the treatment of NSCLC.BACKGROUND The objective of this study was to identify serum biomarkers capable of predicting clinical outcomes in previously-treated NSCLC patients with wild-type for EGFR activating mutations or insufficient tissue for mutation status determination. METHODS Sixty-six Luminex immunoassays representative of biological themes that emerged from a re-analysis of transcriptome data from the Cancer Genome Atlas (TCGA) were evaluate against pretreatment serum specimens from previously-treated advanced NSCLC patients received either cytotoxic chemotherapy (n=32) or erlotinib (n=79). Known EGFR mutation positive cases were excluded from analysis. Associations of biomarkers with outcome parameters and their differential interaction with treatment for survival outcomes were assessed using multivariate Cox PH analyses. RESULTS Our EMT-based transcriptomic analysis revealed a range of biological processes associated with angiogenesis, apoptosis, cachexia, inflammation, and metabolism emerging as those most highly associated with patient outcome. These processes were evaluated via surrogate serum biomarkers. A treatment-biomarker interaction analysis revealed that higher pretreatment levels of c-Met signaling biomarkers (i.e. HGF levels), pro-inflammatory/ pro-cachexia (e.g. IL-8, sIL-2Rα, FGF-2) processes and a pro-angiogenic (e.g. TGF-α, IL-8, VEGF) milieu were associated with inferior survival (HR=0.35, 0.29, 0.58, 0.50, 0.61, 0.45, respectively; all p<0.05) for patients receiving chemotherapy, relative to erlotinib. In contrast, high levels of decoy receptor for IL-1, sIL-1RII, and a high tissue vimentin/E-cadherin ratio were associated with a poor OS (HR=3.78; p=0.00055) in the erlotinib cohort. CONCLUSIONS Contemporary precision medicine initiatives that pair patient tumor characteristics with the optimal therapy type may maximize the use of agents targeting EGFR in the treatment of NSCLC.

MINI01.06: Circulating Angiogenesis Biomarkers are Predictive for Early Stage Non–Small Cell Lung Cancer Progression and Clinical Outcome: Topic: Pathology

progression of disease eventually occurs in due to acquired drug resistance due to incompletely understood mechanisms. EML4 and CLTCL are all involved in intracellular vesicle trafficking in conjunction with clathrindependent endocytosis (CDE), a process that activates signaling down-stream of EGF. We have previously shown that RALBP1, a xenobiotic-transporting ATPase is an anti-apoptotic protein that regulates the rate of CDE and mediates multidrug-resistance. RALBP1 knockout mice are deficient in CDE. RALBP1 is overexpressed in w10% of lung adenocarcinoma and is associated with poorer survival. We tested the hypothesis that RALBP1 over-expression mediates crizotinib resistance and targeting it could overcome CRZ resistance. Methods: RALBP1 expression in EML4-ALK translocations in H3122 (CRZ-sensitive) and H2228 (CRZresistant) cell lines were determined byWestern-blotting. MTT and colony forming assayswere used for cytotoxicity studies. Anti-RALBP1 antibodies that recognize a cell surface epitope of RALBP1 and inhibit its transport activity were used to inhibit RALBP1. Results: H3122 was relatively more sensitive to CRZ than H2228 (IC50 0.2 vs. 1.0 mM, respectively). The expression of RALBP1 protein was higher in the H2228 compared with H3122. Both cell lines were sensitive to anti-RALBP1 antibodies. Cell growth was inhibited by 50% and 85% respectively by the antibodies alone in H2228 and H3122 cell lines, respectively; CRZ had no additional effect.

Abstract 1730: Expression of serum biomarkers related to epithelial-to-mesenchymal transition in non-small cell lung cancer recurrence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Recurrent disease in stage I non-small cell lung cancer (NSCLC) is primarily attributed to metastatic dissemination at the time of surgery undetected by current staging practices. We hypothesized that metastatic progression is driven by epithelial-to-mesenchymal transition (EMT) resulting in differences in tumor-shed protein biomarkers. The objective of this study was to evaluate the difference in expression of these biomarkers in patients with recurrent stage 1 NSCLC. Methods: We used the Luminex immunobead platform, including the MILLIPLEX map human angiogenesis/growth factor magnetic bead panel, to evaluate 80 biomarkers related to EMT against a total of 75 patients who underwent a complete anatomic resection. Patients were divided into the following cohorts: a) stage I NSCLC without recurrence in 2 years (n=50), and b) stage I NSCLC with recurrence within 2 years of follow up (n=25). Peripheral blood was collected and processed using standard phlebotomy techniques. Specimens were obtained in compliance with institutional IRB standards and consent. The Mann-Whitney test and receiver operator characteristics (ROC) curves were used to assess differences in biomarker concentrations between cohorts. Results: Univariate analysis revealed 19 biomarkers with significant (ROC >0.6) differences in expression between the patient cohorts including: angiopoietin 2, MCP-1, MIP-IB, TNF-R1, IGFBP-5, VEGF-D, IGF-1, IGFBP-3, follistatin, sICAM-1, sE-SELECTIN, CYFRA 21.1, RANTES, IL-Ira, M-CSF, IGFBP-1, IGFBP-6, HB-EGF, and PGF. The Mann-Whitney test revealed five biomarkers highly significant (p<0.05) for recurrence in stage 1 NSCLC, including MCP-1, VEGF-D, follistatin, sICAM-1, and placental growth factor (PGF). Evaluation of these biomarkers with the Ingenuity Pathway Analysis (IPA) suite identified several highly significant (p<1x10−5) biological themes, including ten IPA-defined processes associated with development (e.g. embryonic development and cardiovascular system development), seven processes associated with pathological processes (e.g. cancer, cell death, and respiratory disease), and seven processes associated with metastasis (e.g. cellular movement, immune cell trafficking, and cell-to-cell signaling and interaction). Random Forest analysis generated a 6-analyte panel consisting of MCP-1, IP-10, sICAM-1, IGFBP2, RANTES, and IGFBP3 that provided 71.1% classification accuracy with 66.1% sensitivity and 73.3% specificity. Conclusions: Here we report observations concerning the expression of EMT pathway members that may provide key insights into the role of circulating biomarkers related to recurrence in stage 1 NSCLC. Upon further validation, these biomarkers may serve as convenient surrogates to help guide molecular diagnostics and treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1730. doi:1538-7445.AM2012-1730