Protein Array Technology Research Articles

Low Expression of the Novel Tumor Suppressor Trim62 Is An Independent Adverse Prognostic Factor for Survival In Acute Myeloid

Abstract 563▪FN2▪This icon denotes a clinically relevant abstract Background:The role of TRIM (tripartite motif) proteins in human cancer is unknown except for TRIM19 (PML, promyelocytic leukemia gene), which acquires oncogenic activity when fused to RARα (retinoic acid receptor alpha) and causes acute promyelocytic leukemia. We have confirmed a putative role of TRIM62 as a tumor suppressor by genetically deleting the TRIM62 locus in a mouse model. TRIM62 maps to chromosome 1p35.1, a genomic region frequently associated with loss of heterozygosity in human cancer, including leukemia. Methodology:We assessed TRIM62 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Slides were probed with 195 antibodies against apoptosis, cell cycle, signaling, integrins, and phosphatases among other functional protein groups. Patients were segregated according to TRIM62 levels into TRIM62LOW (lower two tertiles) and TRIM62HIGH (highest tertile). Results:The level of TRIM62 protein in AML samples was significantly below (p=0.0048) that of CD34+ cells obtained from healthy volunteers., The level of expression was similar in the paired PB and BM derived AML samples (p=0.2433). Cases of AML-M4 and –M5 exhibited lower expression of TRIM62 than other FAB subtypes.,Stratification according to specific cytogenetic abnormalities (p=0.9286), standard cytogenetic risk criteria (favorable, intermediate, poor; p=0.6824), or mutational status (FLT3, NPM1, RAS, p53, or IDH1/2) failed to show statistically significant differences in TRIM62 expression.Overall survival (OS) was significantly different in TRIM62LOW and TRIM62HIGH for all patients (40.4 vs. 75.6 weeks, p=0.00002), and this difference was greater among patients with cytogenetically normal AML (CN-AML; 47 vs. 125.3 weeks, p=0.00001; Figure 1‘A). TRIM62HIGH patients had significantly longer OS compared with TRIM62LOW patients, regardless of whether they had received standard cytarabine-based chemotherapy (p=0.00003) or epigenetic therapy (i.e. hypomethylating agents with or without histone deacetylase inhibitors; p=0.02). TRIM62LOW was associated with a trend toward shorter remission duration compared with TRIM62HIGH for all cases (38 vs. 63 weeks, p=0.06) and in CN-AML patients (47.7 vs. 58.1 weeks, p=0.09). By multivariate analysis, TRIM62HIGH was identified as an independent favorable risk factor for survival both in the whole cohort (p=0.0085) and in patients with CN-AML (p=0.0014). Notably, TRIM62LOW discriminated a subset of patients with a distinct poor prognosis among those with FLT3MUT (36 vs. 128.5 weeks, p=0.006), FLT3WT (66 vs. 129 weeks, p=0.001), NPM1MUT (34.6 vs. 253.1 weeks, p=0.0026), NPM1WT (51.7 vs. 102.6 weeks, p=0.017), and RASWT (46.3 vs. 110, p=0.0002). Among patients with the most favorable prognosis (FLT3WTNPM1MUT CN-AML), TRIM62HIGH identified a subset of patients for whom stem cell transplant is not recommended: OS for FLT3WTNPM1MUTTRIM62HIGH vs. FLT3WTNPM1MUTTRIM62LOW patients was 100% vs. 35%, after 6 years of follow-up (Figure 1B). When patients with FLT3WTNPM1mutant were excluded from the analysis, TRIM62 still stratified two distinct groups of patients with AML regarding OS, both for the whole cohort (p=0.03) and for those with CN-AML (p=0.0006). By assessing the level of 195 other proteins on the same RPPA chip, we showed that TRIM62 levels correlated significantly and positively (R>0.2) with proteins involved in leukemia stem cell pathways (β-catenin and Notch), migration/homing (fibronectin, integrin β3), hypoxia (VHL, HIF1α), and the p53 pathway (MDM2, p21). [Display omitted] Conclusions:TRIM62 loss, consistent with a role as a tumor suppressor, represents a powerful independent adverse prognostic factor in AML. TRIM62HIGH identifies a population of CN-AML patients with superior outcomes compared with TRIM62LOW amongst those with CN-AML even after stratification by NPM1 and FLT3 mutational status. Disclosures:No relevant conflicts of interest to declare.

High P53 Protein Expression Level Independent of Mutational Status Is An Adverse Prognostic Factor for Survival in Acute Myeloid Leukemia

Abstract 1490 Background:The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML). In AML, p53 mutations have been associated with poor risk cytogenetics (i.e. complex karyotype, −5/−7). However, the function of p53 can also be compromised by protein stabilization and/or expression. The implications of p53 protein expression have not been studied in AML. Methodology:We assessed p53 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts (719 samples). Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1: 2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status was determined by Sanger sequencing of exons 5 through 9 of the p53 gene. Results:Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). Cases of AML-M3 and –M6 exhibited higher expression of p53 than other FAB subtypes. p53 expression directly correlated with age (p=0.01) and CD34 (p=0.001) and inversely correlated with WBC (p=0.007), BM (p=0.0001) and PB (p=0.0001) blasts, platelets (p=0.007), HLA-DR (p=0.01), CD19 (p=0.02), and survival (p=0.01). High p53 (p53high) expression level was more associated with unfavorable cytogenetics than with favorable or intermediate cytogenetics (p=0.00001). When all cytogenetic abnormalities were considered, pts with −5 had the highest levels of p53 (p=0.00001). Pts with RAS mutations, but not those with FLT3-ITD, NPM1, or IDH1/2, had lower levels of p53 protein. When pts were divided according to the level of p53 protein expression p53high was associated with lower complete remission (CR) rates (51% vs 56%; p=??) and higher relapsed rates (82% vs 62%; p=??). The median overall survival (OS) of pts with p53high and p53low were 29.8 vs. 51 wks (p=0.009). Most cases with p53high had unfavorable cytogenetics and the effect on OS was predominantly seen in that subpopulation with p53high and p53low pts living a medina of 23.4 vs. 36 wks (p=0.07), respectively. In order to determine whether the poor outcomes associated with p53high were due to the presence of a higher rate of p53 mutations among pts with p53high, we determined the p53 mutational status of 55 pts. p53high was highly correlated with the presence of p53 mutations as the latter were detected in 17/40 pts with p53high but in only 1/16 pts with p53low. Importantly, the presence of p53high, both in the presence (29 wks) or in the absence (24 wks) of p53 mutations, was associated with significantly worse overall survival compared with pts with p53low (56 wks; p=0.05, Figure 1). [Display omitted] Multivariate analysis indicated that p53 is a significant independent risk factor for survival in AML. The final model included: age (p=0.000001), favorable cytogenetics (0.01), unfavorable cytogenetics (p=0.00001), WBC (p=0.0005), albumin (p=0.0003), FLT3-ITD (P=0.04), and P53 (P=0.02).p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), c-MET (p=0.01), FoxO3a (p=0.004), KIT (p=0.001), p38p180p182 (p0.02), BAD (p=0.0001), cleaved PARP (p=0.002), cleaved PARP (p=0.01), TCF4 (p=0.02), fibronectin (p=0.02), and hsp70 (p=0.003), and negatively with AKTp473 (p=0.01), ERK (p=0.002), mTOR (p=0.005), PI3Kp85 (p=0.002), PKCδ (p=0.00002), GAB2 (p=0.00005), beclin (p=0.007), JMJD6 (p=0.001), Gata3 (p=0.02), p21 (p=0.01), and Mdm2 (p=0.001). Conclusions:Our results suggest that high levels of p53 protein constitute a powerful marker of short survival in AML. This effect is independent of p53 mutational status. The poor outcome of pts with high level of expression of p53 in the absence of p53 mutations suggests that the p53 pathway may be functionally perturbed in a much higher proportion of pts with AML than previously recognized. These data support the use of p53 protein expression levels in prognostication and in the development of targeted therapeutics. Disclosures:No relevant conflicts of interest to declare.

Abstract A185: Identification of drug-associated proteins in NSCLC xenograft models by reverse-phase-protein-arrays.

Abstract At present, most non-small cell lung cancer (NSCLC) treatments are not adapted to the individual response of a patient. The stratification of patients for the most efficient response to conventional chemotherapeutics and targeted therapies will improve established therapy schemes and patient's perspectives. Our project aims at unraveling the influence of specific signaling molecules on the response to common NSCLC drugs. We searched for predictive markers for the response of NSCLC tumors to certain drugs and novel combinations of treatments, which may be useful for therapy decisions towards a more efficient personalized treatment. Expression of 77 proteins described to be involved in EGFR signaling (e.g. MAPK, JAK/STAT, PI3K/AKT pathways) and NSCLC disease in general were quantified in 53 patient derived NSCLC xenograft models using the reverse-phase protein array technology (RPPA). The tumor models are characterized by different response rates upon treatment with different chemotherapeutics and EGFR-targeted therapies. The response rates were obtained directly in the xenograft models. After RPPA analysis we associated the protein expression with the response to established chemotherapeutics (e.g. Vinorelbine, Paclitaxel, Carboplatin) and EGFR-targeted therapies (Cetuximab, Erlotinib). Another goal is to analyze the protein expression changes after treatment in dependence on the response rate. Therefore, protein profiles were raised in NSCLC xenograft models before and after treatment with Cetuximab, a chimeric (mouse/human) monoclonal antibody that inhibits EGFR. The major goal in this experimental subset is to find links between the activities of specific proteins and the response to Cetuximab treatment. A similar experiment with Erlotinib (an EGFR inhibitor) treated samples is ongoing. Statistical analysis indicated significant associations between the expression of distinct proteins and the response rate to certain drugs. Proteins of the ErbB signaling pathway were differentially expressed in Carboplatin responders and non-responders. MEK1 upregulation was observed upon Cetuximab treatment in responders. We revealed an association between higher P-SRC expression and increased Gemcitabine response rates. This result fits to observations that describe coherence between SRC inhibition and reduced suppression of cell growth and survival in the presence of Gemcitabine in cancer cell lines. We are now strengthening our results in various validation experiments. First, we selected seven target proteins based on RPPA analysis. Predictive targets for drug sensitivity are being analyzed to investigate the dependency between target protein activity, downstream signaling and response to distinct drugs in tumor cell lines in a pharmacogenomic approach. Therefore, we knocked down target genes in several NSCLC cell lines and unraveled changes in the drug sensitivity. Microarray and protein analyzes were carried out to study the mechanisms of target-drug association by investigating the downstream signaling upon target knockdown. In summary, our study suggests predictive markers for the response of NSCLC tumors to certain drugs, which may be useful for therapy decisions towards a more efficient personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A185.

Expression profiling of apoptotic proteins in human breast cancer cells treated with AT-101 by protein array technology

Expression profiling of apoptotic proteins in human breast cancer cells treated with AT-101 by protein array technology

Development of a Novel Proteomic Approach for Mitochondrial Proteomics from Cardiac Tissue from Patients with Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting approximately 2.2 million Americans. Because several studies have suggested that changes in mitochondrial function and morphology may contribute to AF, we developed a novel proteomic workflow focused on the identification of differentially expressed mitochondrial proteins in AF patients. Right human atrial tissue was collected from 20 patients, 10 with and 10 without AF, and the tissue was subjected to hydrostatic pressure cycling-based lysis followed by label-free mass spectrometric (MS) analysis of mitochondrial enriched isolates. Approximately 5% of the 700 proteins identified by MS analysis were differentially expressed between the AF and non-AF samples. We chose four differentially abundant proteins for further verification using reverse phase protein microarray analysis based on their known importance in energy production and regulatory association with atrial ion channels: four and a half LIM, destrin, heat shock protein 2, and chaperonin-containing TCP1. These initial study results provide evidence that a workflow to identify AF-related proteins that combines a powerful upfront tissue cell lysis with high resolution MS for discovery and protein array technology for verification may be an effective strategy for discovering candidate markers in highly fibrous tissue samples.

Nuclear Chk1 prevents premature mitotic entry

Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Transplantomics and Biomarkers in Organ Transplantation: A Report From the First International Conference

Transplantomics and Biomarkers in Organ Transplantation: A Report From the First International Conference

Low-Level Laser Irradiation Alters Cardiac Cytokine Expression Following Acute Myocardial Infarction: A Potential Mechanism for Laser Therapy

Low-level laser irradiation (LLLI) has the potential of exerting cardioprotective effect following myocardial infarction (MI). The authors hypothesized that LLLI could influence the expression of cardiac cytokines and contribute to the reversal of ventricular remodeling. LLLI regulates the expression of cytokines after tissue damage. However, little is known concerning the alteration of the cardiac cytokine expression profile after LLLI. MI was created by coronary ligation. The surviving rats were divided randomly into laser and control groups. 33 rats were exposed to a diode laser (635 nm, 5 mW, CW, laser, beam spot size 0.8 cm(2), 6 mW/cm(2), 150 sec, 0.8 J, 1J/cm(2)) as laser group. Another 33 rats received only coronary ligation and served as control group. 28 rats received a thoracotomy without coronary ligation (sham group). One day after laser irradiation, 5 rats from each group were sacrificed and the heart tissues were analyzed by cytokine antibody arrays. Enzyme-linked immunosorbent assay (ELISA) was performed to confirm its reliability. Two weeks after MI, cardiac function and structure were evaluated by echocardiography and histological study. Cytokine antibody array indicated 4 cytokines were significantly changed after laser therapy. ELISA confirmed that granulocyte-macrophage colony stimulating factor and fractalkine were the cytokines involved in the response to therapeutic laser irradiation. However, there was no difference in cytokine release between various groups at 2 weeks after MI. Although LLLI did not improve the damaged heart function, it did reduce the infarct area expansion. The antibody-based protein array technology was applied for screening the cytokine expression profile following MI, with or without laser irradiation. The expression of multiple cytokines was regulated in the acute phase after LLLI. Our results revealed a potential novel mechanism for applying laser therapy to the treatment of heart disease.

New targets of pemphigus vulgaris antibodies identified by protein array technology

We performed partial evaluation of pemphigus vulgaris (PV) autoantibody profile using the protein array technology. The sera from seven patients with acute PV and five healthy donors were probed for the presence of autoantibodies characteristic of the organ-non-specific autoimmune disorders rheumatoid arthritis, lupus erythematosus, scleroderma, diabetes and some other autoimmune disorders, but not to desmosomal proteins. The array targeted 785 human genes amplified using Mammalian Gene Clone Collection with gene-specific primers containing 20-bp nucleotide extension complementary to ends of linear pXT7 vector. The array identified PV antibodies significantly (P<0.05) differentially reactive with 16 antigens, most of which were cell-surface proteins, such as CD2, CD31, CD33, CD36, CD37, CD40, CD54, CD66c and CD84 molecules, nicotinamide/nicotinic acid mononucleotide adenylyltransferase, immunoglobulin heavy chain constant region gamma 2 and others. Reactivity with Fc-IgG helps explain an ability of the chimeric desmoglein constructs to absorb out all disease-causing PV antibodies. Anti-M(1) muscarinic receptor antibody was also identified, consistent with the facts that while blockade of this receptor causes keratinocyte detachment, its activation is therapeutic in PV. Further proteomics analysis of PV antibodies should help elucidate the immunopathogenic mechanisms underlying keratinocyte detachment and blistering.

Use of protein array technology to investigate receptor tyrosine kinases activated in hepatocellular carcinoma

Receptor tyrosine kinases (RTKs) play a role in various processes, including cell growth, differentiation, apoptosis and carcinogenesis. RTKs are activated in various types of cancers, including breast, stomach, colon, pancreas and liver cancer and hepatocellular carcinoma (HCC). In the present study, protein array technology was used to analyze the expression status of various RTKs activated in HCC. The expression of activated RTKs was examined in the HCC cell lines, Alex, HuH7, Li-7, Hep3B, HLE and HLF; in the human normal hepatocyte cell line, hNHeps; and in human HCC and adjacent non-cancerous tissues. Of the 42 different phospho-RTKs, 15 (ErbB2, ErbB3, ErbB4, FGFR2α, FGFR3, insulin R, Mer, PDGFRβ, c-Ret, ROR2, Tie, TrkA, VEGFR3, EphA1 and EphA4) were activated in some of the cancer cell lines studied. Among these, only ErbB2 was activated in all the HCC cell lines examined. Also, in vitro experiments were performed in subcutaneous HCC-bearing athymic nude mice to determine the therapeutic effects of inhibiting ErbB2 activation using the ErbB2-targeting drug trastuzumab. The results revealed that trastuzumab markedly suppressed the growth of HCC. These data suggest that ErbB2 is activated in HCC and that trastuzumab may play a role in the treatment of this disease. In addition, the use of protein array technology is proposed as a tool for detecting the expression of activated RTKs and identifying an effective RTK-based therapy.

Abnormal Expression of Friend Leukemia Virus Integration 1 (Fli1) Protein Is An Adverse Prognostic Factor In AML.

AbstractAbstract 1674 Background.Friend leukemia virus integration 1 (Fli1), a member of the Ets transcription factor family, is expressed in hematopoietic cells and tissues and aberrantly expressed in cancer. Specifically, it is aberrantly-expressed in retrovirus-induced hematological tumors in mice, and is found to be rearranged in human Ewing's sarcoma and related primitive neuroectodermal tumors characterized by a t(11;22)(q24;q12) translocation. Our ongoing studies have shown that Fli1 is essential for embryonic development: Loss of Fli1 results in embryonic lethality due in part to absence of megakaryocytes and aberrant vasculogenesis. Loss of Fli1 also negatively impacts HSC differentiation, consistent with its role in the regulation of important stem cell regulatory genes. To date, limited attention has been directed towards elucidating the potential role of Fli1 in human hematological tumors cancers, including AML. Methodology.We therefore evaluated the level of Fli1 expression in AML using Reverse Phase Protein Array (RPPA) technology to measure Fli1 expression (along with 195 other antibodies) in marrow and blood samples from 511 newly diagnosed AML patients and 21 APL patients. Results.Compared to Fli1 protein levels in normal bone marrow CD34+ cells (n=11), expression was above normal in 32% of patients and below normal in 5%. Elevated Fli1 was not associated with any WHO classification, but was more common in FAB M1. Fli1 levels did not correlate with age, gender, performance status, or any single cytogenetic abnormality, but was negatively correlated with an antecedent hematologic disorder (p=0.002) and positively correlated with infection at the time of diagnosis (p=0.008). Levels were higher in patients with a NPM1 mutation (p=0.0005) or a FLT3-ITD abnormality (p< 0.00001). Expression was significantly lower at relapse compared to diagnosis in 49 paired samples (p= .02). Expression levels were similar in blood and marrow in 140 paired specimens (p=.65). Fli1levels were negatively correlated with 10 proteins (R >0.3) (integrinβ3, SRC, SRCp416, SRCp527, FAK, BCLXL AKTp308, FOXO3A, IGFBP2 and Fibronectin) and positively correlated (R >0.3) with 19 proteins (SMAD4, CREB, CREBp133, MTOR, ELKp383, TSC2, SSPB2, GAB2, PTEN and PTENp, HSP90, AKT, NPM1, NURR77, PRKR, TNK1, TAZp, BAX and DLX1). Fli1levels were not associated with remission attainment (p=.44), but remission duration (p=0.02) and overall survival (p=0.03) were significantly shorter for those with low (RemDur 22.6 and OS 45.2, weeks) or high Fli1(RemDur 40.3 and OS 35.4 weeks) compared to patients with normal levels of Fli1(RemDur 51.1 and OS 59.4 weeks). High Fli1 levels were prognostic in univariate analysis and was an independent prognostic factor in multivariate analysis along with age, unfavorable cytogenetics, favorable cytogenetics, gender, serum creatinine and FLT3mutation. Conclusions.Both low and high expression of Fli1 protein are associated with poor outcome. While low levels of Fli1 may fail to properly regulate Fli1 target genes, elevated levels of Fli1 may not only affect expression of Fli1 target genes, but also aberrantly affect the expression of additional genes not normally responsive to Fli1. The correlation between high levels of FLi1 and high levels of AKT, mTOR, TSC2 and inactivated pPTEN suggest that targeting this signaling pathway might interfere with Fli1 expression. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Differential Immunogenicity and Clinical Relevance of Kidney Compartment Specific Antigens after Renal Transplantation

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n = 1,785) and decline in graft function over the subsequent year (n = 105). There was an enrichment of immunogenic antigens in the renal cortex (p = 0.01) with post-transplant time, and for glomerular specific targets (p = 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design.

Immunoproteomics Profiling of Blood Stage Plasmodium vivax Infection by High-Throughput Screening Assays

Completed genome sequences and stage-specific transcriptomes of the intraerythrocytic developmental cycle of Plasmodium vivax offers the opportunity to profile immune responses against P. vivax infection using innovative screening approaches. To detect the immune responses to blood stage-specific proteins, we applied a protein array technology to screen the sera of vivax malaria patients. Herein, a set of genes from the P. vivax blood stage was cloned using the In-Fusion cloning method and expressed by a wheat germ cell-free system. A total of 94 open reading frames (ORFs) were cloned and 89 (95%, 89/94) proteins were expressed, which were screened with sera from P. vivax-infected patients and healthy individuals using protein arrays. A total of 18 (19.1%, 18/94) highly immunoreactive proteins were identified, including 7 well-characterized vivax vaccine candidates. The remaining 11 ORFs have not been previously described as immunologically reactive. These novel immunoproteomes of the vivax malaria blood stage will be further studied as potential vaccine candidates. In this first report, high-throughput screening assays have been applied to investigate blood stage-specific immunoproteomes from vivax malaria. These methods may be used to determine immunodominant candidate antigens from the P. vivax genome.

Genomic and proteomic approaches identify Rab25 as a potential prognostic marker for luminal B breast tumors

Abstract Estrogen receptor (ER) +ve luminal B subtype of breast tumors initially respond well to tamoxifen treatment. However, over longer periods of time, their frequent recurrence contributes to the overall high mortality rate of hormone receptor +ve breast cancer patients. Recently, with the development of PARP inhibitors, there is a potential line of treatment for the triple negative subtype. But a huge dearth exists in therapeutically targeting luminal B tumors since these tumors are also insensitive to chemotherapy. Our current research addresses this urgent need by utilizing a systems biology approach to identify potential therapeutic targets and diagnostic markers for luminal B subtype of breast cancer. The endocytic pathway, as demonstrated by our laboratory is a frequent target of genomic aberrations in cancer, and plays a critical role in maintenance of cellular polarity, stem cell function, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. Derailed endocytosis is therefore a hallmark of cancer. The Rab GTPases along with their effectors are critical regulators of this endocytic machinery and can have a huge impact on the cellular itinerary of growth factor as well as integrin receptor signaling. A broad spectrum analysis of distribution of all the Rab family members across 52 breast cancer cell lines reflected a dichotomous distribution of Rab25, with higher mRNA and protein expression in ER+ cell lines. Rab 25 is of immense interest to us because of its unique substitution of leucine for glutamine at position 71 which typically decreases the intrinsic GTPase activity, resulting in a dominant, constitutively active protein, akin to a transforming RAS mutation. Here we evaluate the role of Rab25 as a valuable predictive and prognostic marker for ER+ breast cancers, with critical implication in disease management and predicting response to therapy. Using high throughput data analysis from publicly available mRNA expression datasets, we report that Rab25 and its effector Rab coupling protein (RCP) are frequently coamplified and coordinately elevated in ER +ve breast cancers. Specifically in the luminal B subtypes, they are significantly associated with a markedly worsened outcome. Compatible with Rab25 and RCP being drivers of the amplicons, their respective mRNA levels are highly correlated across multiple tumor sets, most prominently in luminal B tumors (Soritiou p&amp;lt;0.0015; Pawitan p&amp;lt;0.01). Patients with low RNA levels of both Rab25 and RCP have a very good outcome, those with high level of either Rab25 or RCP have an intermediate outcome and patients with coordinate elevation of Rab25 and RCP mRNA have a bad outcome. Additionally, using our Reverse Phase Protein Array (RPPA) technology, we found that at protein level Rab25 and RCP correlate with poorer outcome in luminal B tumors. In vitro, using MCF7 cell line, which is a prototype of luminal B subtype, we observed an altered EGFR stability and signaling in Rab25 overexpressing stable clones which could be rescued with shRNA knock-down of Rab25, confirming specificity of Rab25 action. In MCF7 cells overexpressing Rab25, we found higher proliferation index as well as increased cell migration and invasion potentials. When injected as xenografts in mice, Rab25 overexpressing cells formed larger tumors than their wildtype controls, strongly suggesting a role for Rab25 in promoting tumor progression in luminal B tumors. The proposed studies will have a marked impact on our understanding of the mechanisms by which Rab25 and RCP contribute to breast cancer aggressiveness, and also of the therapeutic and clinical potential of Rab25 and the Rab25: RCP complex as a target and marker of the behavior of breast tumors.

The use of protein array to identify targetable receptor tyrosine kinases for treatment of human colon cancer

Several studies have reported that activated receptor tyrosine kinases (RTKs) are highly expressed in colon cancer and may promote tumor growth and survival. However, there is little information available as to the function and signaling of RTKs in colon cancers. In the present study, we performed protein array technology to determine the expression status of various RTKs that are activated in colon cancer compared to normal colonic cells and tissues. Of the 42 different phospho-RTKs, 5 (ErbB2, FGFR1, FGFR2a, FGFR3 and MSPR) were activated in Caco-2, SW480, WiDr, Lovo colon cancer cell lines and cancerous tissues. In order to determine the effect of inhibition of RTKs, especially ErbB2, athymic nude mice bearing xenograft tumors were treated with the ErbB2-targeting drug trastuzumab alone, or in combination with 5-Fluorouracil (5-FU). Similar to the treatment of 5-FU alone, trastuzumab suppressed the growth of colon cancer. Combination therapy of trastuzumab and 5-FU inhibited tumor growth significantly compared to the treatment of 5-FU alone or trastuzumab alone. In addition, xenograft tumors were also analyzed by phospho-MAPK protein array. The activity of Akt3/PKBgamma was inhibited with 5-FU alone and trastuzumab, indicating that trastuzumab may inhibit colon cancer growth through ErbB2-Akt3/PKBgamma signaling. These data demonstrate that ErbB2 could be an important candidate for colon cancer therapy and the addition of trastuzumab to 5-FU therapy might augment the clinical response in colon cancer patients. Therefore, the analysis of phospho-RTK expression by protein array as a useful tool might identify novel therapies for individual patients with colon cancer.

Progression of lupus-like disease drives the appearance of complement-activating IgG antibodies in MRL/lpr mice

Nucleic acids are known to induce complement activation, which results in the masking and removal of apoptotic cells exposing nuclear components. Dysregulation of these events is characteristic of SLE, a systemic autoimmune disease characterized by the appearance of ANAs. In this study, we aimed to investigate the relationship between development of ANAs and their effect on complement activation by nucleic acids. We used protein array technology to characterize complement activation by murine mAbs and polyclonal antibodies against various forms of nucleic acid. Serum samples from MRL/lpr mice were collected, starting before the onset of the disease till 6 months of age. Binding of IgG and its subclasses to dsDNA, ssDNA, RNA, plasmid DNA and nucleosome complexes was determined, along with C3 fixation. We show that complement C3 binding to various forms of nucleic acid that serve as targets in lupus is absent in normal serum. The addition of dsDNA-specific mAbs to normal serum results in the deposition of complement C3 to nucleic acids. In MRL/lpr mice, IgG antibodies against various nuclear antigens appear with ageing and disease progression. C3 binding to the antigens is somewhat delayed and suggests that accumulation or maturation of pathogenic antibodies is required for inducing C3 binding to ICs containing nucleic acids. C3 deposition on nuclear antigens, therefore, reflects the state of disease progression in this murine model of SLE.

Abstract 818: Correlation of mRNA and protein expression of CA125 and HE4 in ovarian cancer tissues using real-time RT-PCR and reverse phase protein array technologies

Abstract BACKGROUND: Epithelial ovarian cancer is the fifth most frequent cause of cancer deaths among women in the United States. Prognosis is improved when it is detected in early stages but poor as the disease progresses. Monitoring for ovarian cancer currently uses the serum marker CA125, although it is not very effective in detecting early stage ovarian cancers. Recently, the biomarker HE4 has shown clinical utility in the detection of ovarian cancer. However, it is likely that additional markers are needed to detect early stage ovarian cancer. OBJECTIVE: This study investigated whether reverse phase protein array (RPPA) technology would complement real time RT-PCR and serve as an additional tool to screen for biomarkers. RPPA is a sensitive, high throughput, quantitative assay that measures small amounts of proteins simultaneously on a single slide. METHODS: The mRNA expression levels of CA125 and HE4 were measured using real-time quantitative RT-PCR from 27 normal ovary tissue specimens and 54 epithelial ovarian tumors. The protein levels for these biomarkers were assessed in tissue lysates from the same normal ovary and ovarian tumor specimens by immunoblotting and reverse phase protein arrays. RESULTS: CA125 mRNA was elevated in 76% (41/54) of the tumors at levels greater than 10 fold relative to the average expression in normal ovary tissue. Fifty-nine percent (32/54) of epithelial tumors expressed CA125 mRNA at greater than 100 fold compared to normal ovarian tissue. HE4 mRNA was over expressed in 84% (42/50) of the tumors at levels 10 fold greater and in 68% of the tumors at 100 fold greater than the average normal ovary tissue. Protein expression levels of the CA125 and HE4 biomarkers were measured by immunostaining tissue lysates on RPPAs. As observed for CA125 and HE4 mRNAs, there were statistically significant differences in the protein levels detected between normal ovary and ovarian tumor for both biomarkers. CA125 protein was detected in 81% of tumors at 2.5 fold greater than, and in 75% of the tumors at 5 fold greater than, the average of the normal ovary tissue. Seventy-seven percent of tumors had HE4 protein detected at levels greater than 2.5 fold and 30% greater than 5 fold compared to normal ovary tissue. Immunoblotting validated the RPPA results for HE4 protein expression in a subset of the tissue lysates. Importantly, a significant correspondence between the mRNA and protein levels of each biomarker was demonstrated with correlation coefficients of 0.88 and 0.78 for CA125 and HE4, respectively. CONCLUSION: In this dataset, there is a robust correlation between mRNA and protein levels for HE4 and CA125. As RPPA yielded similar results compared to immunoblotting, is quantitative, and has higher throughput, RPPA is a valuable method for high throughput analysis of protein expression in tissue lysates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 818.

Abstract 1981: MYC downregulation and chemoresistance in non-small cell lung cancer (NSCLC): Evidence from the Biomarker-Based Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) program

Abstract Background: Primary or acquired resistance to platinum-based therapy remains an important issue in the treatment of unresectable non-small cell lung cancer (NSCLC). Our objective was to identify factors of resistance to platinum-based therapy. Experimental procedures: Gene expression profiling from the BATTLE program (pretreated resistant tumors stage III/IV, N=32) and a control group from 2 independent publicly available datasets (never treated tumors stage III/IV, N=45) were compared. Gene expression profiling was generated using the same platform (U133 Plus 2.0 Array). Pathway and gene set analyses were used to define networks and pathways associated with resistance. For validation, we used: i-an independent set of 38 never treated NSCLC stage III/IV, ii-a set of 53 NSCLC cell lines tested for cisplatin sensitivity with proteomic profiling generated by Reverse Phase Protein Array (RPPA) technology using 177 well-characterized antibodies, iii-the comparison of two pairs of NSCLC cell lines (H1437, H460) that were made resistant by iterative exposure to cisplatin, and iv-transfection experiments. Results: A total of 3,963 probesets were found to be differentially expressed between BATTLE samples and the control group with a p-value &amp;lt; 0.001 (two-tailed t-test). DNA repair gene sets were upregulated in BATTLE samples compared to never treated tumors. Network analysis found that MYC as well as many of its downstream regulated genes were significantly downregulated in BATTLE samples. In particular, a high proportion of MYC target genes associated with apoptosis were downregulated. Using real time PCR, MYC gene expression was significantly lower in 15 BATTLE samples compared with an independent set of 38 never treated stage III/IV NSCLC (p-value=0.01). The proteomic profiling of 53 NSCLC cell lines showed that MYC expression was one of the top proteins associated with sensitivity to cisplatin. An inverse correlation was observed between sensitivity to cisplatine (IC50) and MYC protein expression evaluated by RPPA (total MYC: r=-0.41, p-value=0.003; phosphorylated MYC: r=-0.30, p-value=0.03). MYC gene expression by real time PCR was lower in resistant H1437 and H460 cell lines compared to parental cell lines. Finally, transfection of H226 cell lines with a vector expressing MYC improved sensitivity to cisplatin compared to the control. Conclusion: This is the first gene expression profiling analysis of NSCLC samples pretreated and resistant to chemotherapy included in a prospective clinical trial. MYC down regulation may play an important role in NSCLC resistance to platinum-based chemotherapy. Mechanisms of MYC down regulation should be further explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1981.

Abstract 2683: Monoclonal antibodies and assays for colorectal cancer monitoring in serum

Abstract Colorectal cancer (CRC) is the third most common cancer in both men and women. There were 148,810 new cases and 49,960 deaths anticipated in 2008. The 1-year and 5-year relative survival for CRC patients is 82% and 64% respectively. When CRC is detected early, at a localized stage, the 5-year survival is 90%. However only 39% of CRC are diagnosed at this stage, as only 44% of US adults over 50 undergo the recommended screening. Our aim is to develop rapid, easy-to-use and cost-effective serum-based immunoassays for CRC surveillance and recurrence monitoring. We have developed proprietary approaches to the discovery of biomarkers (Bm) using an in-house generated library of monoclonal antibodies (mAb), a large collection of clinical samples from patients with major cancers, and a proprietary multiplex screening platform, known as Matrix Protein Array Technology (MPAT). The MPAT enables us to screen a large number of clinical samples with a large number of antibodies. Briefly, a protein matrix comprising samples from cancer patients, benign and normal controls, is replica printed on the MPAT solid support. Each sample matrix is simultaneously interrogated with a different mAb. mAb-Bm reaction is detected using fluorescence-based Odyssey imaging system (Li-Cor), followed by spot quantification and statistical analysis to select mAb against Bm that are significantly and differentially expressed in cases versus controls. We have used this approach to identify CRC specific Bm and mAb. We further validated Bm expression by MPAT, using the mAb on tissue protein extracts derived from patients with CRC (173), benign and inflammatory conditions of the colon (23), and normal controls (240). The clinical sample set used also included breast, lung, and ovary cases and controls, amounting to a total of 1329 samples. Some mAb-based Bm showed excellent discriminatory power between cancer and control, with up to 90% sensitivity at 90% specificity, as determined by ROC curves using statistical package GB-STAT (Dynamic Microsystems). Then, immunohistochemistry on in-house and commercial tissue microarrays (75 cases) confirmed cancer specificity, and mAb specifically stained CRC tissues, with membrane, nuclear and cytoplasmic localization. Finally, some candidate Bm appeared to be secreted and overexpressed in CRC patient serum, when tested by Western blot. From the pool of serum-based Bm, we have developed a pair of capture-detection mAb in an ELISA sandwich assay, and compared performance to a carcinoembryonic antigen (CEA) standard assay. This serum-based immunodiagnostic assay would have tremendous applications in CRC monitoring, whether alone or in combination with CEA. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2683.

DNA-LINKED PROTEIN ARRAY FOR HIGH-THROUGHPUT PROTEOMICS: FROM SPATIALLY UNKNOWN DNA ARRAYS TO IDENTIFIABLE PROTEIN ARRAYS

Protein array technology has a tremendous potential in large-scale and high-throughput functional proteomics. The conventional protein array approach, which is based on one-to-one indexing between a known protein and an array position, is however, not applicable for decoding genetic information from unknown expressed protein arrays and thus less practicable for global proteome analysis. To address this issue, we have introduced a novel concept of DNA-linked protein array by adopting a strategy of one-to-one indexing between spatially unknown individual DNA arrays and their encoded protein array products. A set of test DNA arrays of DNA-binding (POU) domain protein, c-Myc-tagged POU domain protein, and green fluorescent protein were randomly patterned on a glass surface and subsequently converted into identifiable protein arrays by simultaneously producing and linking the encoded proteins to their encoding genes using a puromycin-assisted mRNA–protein fusion approach in situ. For concept validation, functional assay was performed by the screening of mRNA–protein fusion arrays against a mixture of anti-cMyc and anti-GFP antibodies. Moreover, this approach also provides an additional advantage to incorporate post-translational modifications into protein arrays. The concept developed here is expected to markedly enhance the application of protein microarrays to the study of functional proteomics.