Tumor-specific Proteins Research Articles

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy.

Immunotherapy holds tremendous promise for improving cancer treatment1. Administering radiotherapy with immunotherapy has been shown to improve immune responses and can elicit an “abscopal effect”2. Unfortunately, response rates for this strategy remain low3. Herein, we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NPs formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent upon NP surface properties. We showed that AC-NPs deliver tumor specific proteins to antigen-presenting cells and significantly improve the efficacy of αPD-1 treatment using the B16F10 melanoma model, generating up to 20% cure rate as compared to 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+/Treg and CD8+/Treg ratios. Our work presents a novel strategy for improving cancer immunotherapy with nanotechnology.

Quantitative Shotgun Proteomics Unveils Candidate Novel Esophageal Adenocarcinoma (EAC)-specific Proteins

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches.

Melanoma antigens and related immunological markers.

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.

Ultra-High Mass Resolution MALDI Imaging Mass Spectrometry of Proteins and Metabolites in a Mouse Model of Glioblastoma

MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

Size-Selective Harvesting of Extracellular Vesicles for Strategic Analyses Towards Tumor Diagnoses.

Extracellular vesicles (EV), typified by exosomes or microvesicles, are expected to be effective diagnostic markers for cancers. The sizes of the vesicles range from 20 to 1000nm, but the size-dependent variations of the contents of EVs are still poorly understood. We succeeded in the size-selective harvesting of the vesicles by utilizing the molecular weight-dependent characteristics of a variety of polyethylene glycols (PEG) as precipitating reagents and analyzed the antigens displayed on the surfaces of the vesicles and the miRNAs included in the vesicles from each size group. As a result, the relatively larger (<100nm) particles precipitated by PEG5k clearly exhibited the greatest amount of epithelial cell adhesion molecule (EpCAM), from both breast cancer (MCF-7) and colon cancer (HCT116) cells, and a larger quantity of microRNA (miRNA) specific to breast cancer cells (miRNA155 for MCF-7) seemed to be contained in the PEG-precipitated particles. The results demonstrated that the quantities of both the tumor-specific miRNA and protein were similarly distributed among the several classes of the size-sorted EVs and that the size-selective harvesting of EVs may be informative for strategic analyses towards the diagnoses of cancers.

The Targetable Epigenetic Tumor Protein EZH2 is Enriched in Intraocular Medulloepithelioma.

PurposeIntraocular medulloepithelioma (IM), the second most common primary neuroepithelial tumor of the eye, can lead to blindness in the affected eye and in rare cases, is deadly. Intraocular medulloepithelioma lacks targetable biomarkers for potential pharmacologic therapy. The purpose of this study was to identify actionable, tumor-specific proteins for potential diagnostic or therapeutic strategies. We hypothesize that the tumor-specific epigenetic enzyme EZH2 is selectively expressed in IM.MethodsWe conducted a retrospective case series study of five IM from five eyes of four children and one adult. Hematoxylin and eosin (H&E) stains of sections from formalin-fixed, paraffin-embedded blocks of IM tumors were used to localize IM tumor cells in each case. Using an EZH2-specific antibody for immunohistochemistry, we semiquantitatively calculated the proportion of IM tumor cells positive for EZH2, and also assayed for EZH2 staining intensity.ResultsWe found that EZH2 was expressed in all IM cases but this protein was absent in nontumor ciliary body or retinal tissues. However, not all IM tumor cells expressed EZH2. Similar to retinoblastoma, moderately to poorly differentiated (primitive appearing) IM tumor cells strongly expressed EZH2; expression was weaker or absent in areas of well-formed neuroepithelial units.ConclusionsTo our knowledge, this is the first study to identify an actionable tumor-specific maker, EZH2, in IM. Our findings point to the possibility of exploring the potential of EZH2 inhibitors, already in clinical trials for other cancers, for IM.

New approach of delivering cytotoxic drugs towards CAIX expressing cells: A concept of dual-target drugs

Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies.

The Sulfamate Small Molecule CAIX Inhibitor S4 Modulates Doxorubicin Efficacy.

Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 –CAIX high and HT29 –CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 µM, p = 0.0003). Similar results were observed for HT29—CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 µM, p<0.0001) and in the HT29 –CAIX low cells (IC50 is 0.09 µM, p<0.0001). In vivo doxorubicin treatment was only effective in the MDA-MB-231 xenografts, but the efficacy of doxorubicin was decreased when combined with S4. In conclusion, the efficacy of doxorubicin treatment can be increased when combined with the selective sulfamate CAIX inhibitor S4 in vitro in certain cell lines. Nevertheless, in xenografts S4 did not enhance doxorubicin efficacy in the FaDu and HT29 tumor models and decreased doxorubicin efficacy in the MDA-MB-231 tumor model. These results stress the importance of better understanding the role of CAIX inhibitors in intratumoral pH regulation before combining them with standard treatment modalities, such as doxorubicin.

Abstract 3307: Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients

Abstract Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis. Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques. Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples. Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics. Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3307.

Abstract 848: Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer

Abstract Introduction Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test detecting blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection. Materials and methods RNA and proteins were isolated from CRC cell line SW480 before and after siRNA-mediated down-modulation of the splicing machinery: SF3B1, U2AF1, and SRSF1. To identify splice variants, mRNA was sequenced (Illumina HiSeq) and analyzed. RNA-seq analysis included quality checks (FASTQ, RSeQC), reads mapping (STAR), differential gene expression (DESeq2) and differential expression of splicing isoforms between conditions (MATS). Results from the in silico analysis were validated by qRT-PCR. Proteins were analyzed by in-depth tandem mass spectrometry (QExactive). A proteogenomic data analysis pipeline was established to enrich the sequence database, against which the mass spectra are searched, with the predicted protein splice variants and identify protein isoforms. To further extend the splice-variant database, PacBio sequencing of full-length transcripts is being performed for the SW480 siSF3B1- and control-samples. Results Differential expression analysis on RNA and protein level proved that the knock-down experiments were performed successfully. The RNA-seq analysis revealed hundreds of mRNA splice variants, including positive controls described in literature. For example, down-modulation of SF3B1 resulted in quantitative mRNA changes of spliced isoforms of ADD3 both in RNA-seq and RT-PCR data. The proteomics experiment yielded over 6000 proteins per sample, among which a number of protein isoforms resulting from alternative splicing. Conclusions and discussion We established a proteogenomic pipeline for the analysis of alternative splicing and provided experimental proof of concept. We expect, however, that the true complexity of RNA variant information remains highly underestimated. We therefore are performing PacBio long-read RNA-seq to validate our approach and to identify additional (novel) splicing events. In future studies we will apply the mRNA-seq based proteogenomic pipeline for detection of protein alterations to a series of adenomas at low- and high-risk of progression, and CRCs to validate the isoforms in a clinically relevant setting. Novel findings will be evaluated for their performance as screening markers for CRC. This work was financially supported by KWF project nr: VU 2013-6025. Citation Format: Malgorzata A. Komor, Annemieke C. Hiemstra, Thang V. Pham, Sander R. Piersma, Robert P. Sebra, Bo W. Han, Meredith Ashby, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond JA Fijneman. Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 848.

Network Modeling Identifies Patient-specific Pathways in Glioblastoma.

Glioblastoma is the most aggressive type of malignant human brain tumor. Molecular profiling experiments have revealed that these tumors are extremely heterogeneous. This heterogeneity is one of the principal challenges for developing targeted therapies. We hypothesize that despite the diverse molecular profiles, it might still be possible to identify common signaling changes that could be targeted in some or all tumors. Using a network modeling approach, we reconstruct the altered signaling pathways from tumor-specific phosphoproteomic data and known protein-protein interactions. We then develop a network-based strategy for identifying tumor specific proteins and pathways that were predicted by the models but not directly observed in the experiments. Among these hidden targets, we show that the ERK activator kinase1 (MEK1) displays increased phosphorylation in all tumors. By contrast, protein numb homolog (NUMB) is present only in the subset of the tumors that are the most invasive. Additionally, increased S100A4 is associated with only one of the tumors. Overall, our results demonstrate that despite the heterogeneity of the proteomic data, network models can identify common or tumor specific pathway-level changes. These results represent an important proof of principle that can improve the target selection process for tumor specific treatments.

THBS1 is induced by TGFB1 in the cancer stroma and promotes invasion of oral squamous cell carcinoma.

THBS1 (thrombospondin-1) is the extracellular matrix (ECM) protein that affects diverse cellular activities. It constitutes the tumor stroma, but the role of THBS1 in oral squamous cell carcinoma (OSCC) development is unclear. The aim of this study was to clarify the relevance of THBS1 in the pathogenesis of OSCC. The expression of THBS1 was examined in 44 OSCC by immunohistochemical analysis and in 43 OSCC by cDNA microarray analysis. Cell culture experiments were conducted using human OSCC cell lines HSC3 and HO1N1 and mouse fibroblast ST2 cells to examine the effect of TGFB1 on THBS1 expression, and the effect of THBS1 on cellular behaviors. THBS1 was specifically induced in the tumor microenvironment of OSCC. THBS1 appeared to be produced mainly by the stromal cells, but also by OSCC cells. TGFB1 stimulated THBS1 expression in ST2, primary fibroblasts, and the OSCC cells. THBS1 promoted migration and invasion of HSC3 and HO1N1 in transwell migration assays. THBS1 stimulated the expression of MMP3 (matrix metalloprotease 3), MMP9, MMP11, and MMP13 in ST2 cells and MMP3, MMP11, and MMP13 in HO1N1 cells. The RGD peptide suppressed the THBS1-stimulated migration and upregulation of MMP11 and MMP13. THBS1 is a tumor-specific ECM protein that is induced by TGFB1 and promotes migration of cancer cells and stimulates the expression of MMPs partly through the integrin signaling, thereby favoring OSCC invasion.

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM)

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples.

Screening and identification of post-traumatic stress-related serum factors in children with Wilms' tumors.

Wilms' tumors are one of the most common malignant, solid intra-abdominal tumors observed in children. Although potential tumor markers have been found, inflammatory cytokines interfere with the process of specific protein identification. The present study was undertaken to identify post-traumatic stress-related factors of Wilms' tumors and to verify the accuracy of early-stage tumor-specific serum protein markers. Serum samples were screened for differentially-expressed proteins using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Potential markers were isolated and purified using solid-phase extraction (SPE) and SDS-PAGE. Following enzymatic digestion of the protein samples, the peptide fragments were detected with high performance liquid chromatography-mass spectrometry. The obtained peptide mass fingerprint was searched in the Swiss-Prot protein sequence database via the Mascot search engine. Differentially-expressed proteins were verified using western blot analysis. Differentially-expressed proteins with a mass/charge of 5,816 were screened out using SELDI-TOF-MS, and significant differences between the tumor and control groups, and the trauma and control groups were observed. Target proteins were isolated and purified using SPE and SDS-PAGE. Thioredoxin 1 (Trx1) was found to be differentially expressed. In the serum of children with Wilms' tumors, there was an increase in the level of the post-traumatic stress-related inflammatory factor, Trx1, as compared with the normal control group. Thus, the results of this study indicate that Trx1 presents a potential post-traumatic stress-related factor of Wilms' tumors.

Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

Role of the Immune Response in Disease Progression and Therapy in Multiple Myeloma.

Multiple myeloma (MM) is a hematologic cancer derived from malignant plasma cells within the bone marrow. Unlike most solid tumors, which originate from epithelial cells, the myeloma tumor is a plasma cell derived from the lymphoid cell lineage originating from a post-germinal B-cell. As such, the MM plasma cell represents an integral component of the immune system in terms of both antibody production and antigen presentation, albeit not efficiently. This fundamental difference has significant implications when one considers the implications of immunotherapy. In the case of lymphoid malignancies such as myeloma, immune-based strategies must take into consideration this important difference, potentially necessitating immunotherapy targeted toward MM to be altered from that targeted at solid tumors. Typically, the immune system "surveys" cells within our body and is able to recognize and attack cancerous cells that may arise. However, some cancer cells are able to evade immune surveillance and continue to flourish, causing disease. The major mechanism leading to an effective tumor-specific response is one that enables effective antigen processing and presentation with subsequent T-cell activation, expansion, and effective trafficking to the tumor site. Plasma cells employ several mechanisms to escape immune surveillance which include altered interactions with T-cells, DCs, bone marrow stromal cells (BMSC's), and natural killer cells (NK Cells) that can be mediated by immunosuppressive cells such as and myeloid-derived suppressor cells (MDSC's) and cytokines such as IL-10, TGFβ, and IL-6 as well as down-regulation of the antigen processing machinery. Many therapies have been developed to reestablish a functional immune system in MM patients. These include adoptive T-cell therapies to deliver more tumor-specific T-cells, vaccines to increase the tumor-specific precursor frequency of the endogenous T-cell population, immunomodulatory agents (IMiDs) such as thalidomide and lenalidomide to enhance global endogenous immunity, immunostimulatory cytokines, and antibodies to specifically target tumor-specific cell-surface proteins or cytokines. This review will dissect these various approaches currently being explored in MM as well as highlight some future directions for myeloma-specific immune-based strategies.

Abstract A33: Utility of multiplexed biomarker analysis in non-small-cell lung cancer patients with discrepancies between FISH and immunohistochemical results of ALK testing

Abstract Background: The clinical implications in Non-small cell lung cancer (NSCLC) patients with discrepancy between fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) assessing ALK status are unclear. Materials and Methods: From 1131 archived tissue samples, we identified 41 NSCLC patients with discrepant results showing positive or borderline ALK FISH and negative IHC using ALK1 antibody. Of these, 21 cases with sufficient tissue remaining for selected reaction monitoring (SRM) and an additional case within normal range FISH split (4%) were sent to confirm ALK IHC using other antibodies (5A4 and D5F3). Target protein levels measured by multiplexed SRM were correlated with records of clinical outcomes after treatment with crizotinib or cytotoxic chemotherapy. Results: Of 22 cases with various ALK FISH results and negative IHC (ALK1), IHC using other antibodies (5A4 and D5F3) identified 4 positive cases, whereas the SRM assay identified 7 cases with tumor-specific ALK protein. IHC showed lower sensitivity in specimen with delayed fixation but SRM showed superior sensitivity in those samples as SRM is independent to modified epitope. Of 8 crizotinib-treated cases, 4 cases with detectable levels of either ALK or cMET proteins had complete or partial responses; cases without either of these proteins (n = 4) did not respond to crizotinib regardless of ALK FISH results. Cases with 3 or more predictive proteins specific to chemotherapeutic agents were found to be more responded to those agents and had a higher probability of progression-free survival. Conclusions: The SRM assay showed superior sensitivity in poorly fixed specimens and can inform therapeutic decision-making in NCSLC patients with ALK screening discordances. Citation Format: TAE-JUNG KIM, HIDEKI TERAI. Utility of multiplexed biomarker analysis in non-small-cell lung cancer patients with discrepancies between FISH and immunohistochemical results of ALK testing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A33.

Application of Efficient Nanoparticles for Early Diagnosis and Treatment of Cancer.

Cancer is considered as a prevalent cause of human deaths and undoubtedly, is the most complex disease with multiple cellular physiological systems involved. During the last decade, the application of nanotechnological products for cancer treatment has received considerable attention. These sophisticated tools and materials treat cancer though the early diagnosis, the prediction, the prevention and the personalized therapy. This technology enabled the development of nanoscale particles that can be conjugated with one or multiple functional molecules simultaneously. Nanoparticles have the capability to be delivered directly through blood vessels to the tumor site and interact with targeted tumor-specific proteins located inside or on the surface of cancer cells, since their size is a hundred to thousand times smaller than cancer cells. In this review, comprehensive outline of all the latest scientific and technological applications such as quantum dots and gold nanoparticles alongside with their applications in cancer diagnosis and treatment have been presented.

Protein Identification of Blood Nucleoprotein Complexes

Circulating nucleoprotein complexes were isolated-from blood plasma by affinity chromatography using immobilized polyclonal anti-histone antibodies. It was found, that the main part of DNA from histone-contained nucleoprotein complexes have size 170-180 b.p., in blood of breast cancer patients DNA with size 170-180 b.p. and DNA more then 6 k.b.p. are presented in equal quantity. Proteins from circulating nucleoprotein complexes were identified using MALDI-TOF mass-spectrometry. It was shown that nucleoprotein complexes from blood of breast cancer patients contain tumor-specific proteins, such as LDOC1L, ADP/ATP translocase 3 and Lamellipodin. These data indicate, that a part of circulating extracellular DNA have tumor origin.

Suppressor of Ty homolog-5, a novel tumor-specific human telomerase reverse transcriptase promoter-binding protein and activator in colon cancer cells.

The human telomerase reverse transcriptase (hTERT) promoter promotes differential hTERT gene expression in tumor cells and normal cells. However, information on the mechanisms underlying the differential hTERT transcription and induction of telomerase activity in tumor cells is limited. In the present study, suppressor of Ty homolog-5 (SPT5), a protein encoded by the SUPT5H gene, was identified as a novel tumor-specific hTERT promoter-binding protein and activator in colon cancer cells. We verified the tumor-specific binding activity of SPT5 to the hTERT promoter in vitro and in vivo and detected high expression levels of SUPT5H in colorectal cancer cell lines and primary human colorectal cancer tissues. SUPT5H was more highly expressed in colorectal cancer cases with distant metastasis than in cases without distant metastasis. Inhibition of endogenous SUPT5H expression by SUPT5H gene-specific short hairpin RNAs effectively attenuated hTERT promoter-driven green fluorescent protein (GFP) expression, whereas no detectable effects on CMV promoter-driven GFP expression in the same cells were observed. In addition, inhibition of SUPT5H expression not only effectively repressed telomerase activity, accelerated telomere shortening, and promoted cell senescence in colon cancer cells, but also suppressed cancer cell growth and migration. Our results demonstrated that SPT5 contributes to the up-regulation of hTERT expression and tumor development, and SUPT5H may potentially be used as a novel tumor biomarker and/or cancer therapeutic target.