Biomarkers In Tumor Cells Research Articles

IL-9 is a Biomarker of BIA-ALCL Detected Rapidly By Lateral Flow Assay.

A delayed seroma around breast implants is the most common clinical presentation of BIA-ALCL. However, most seromas are due to benign causes. Therefore, it is essential to distinguish benign seromas from seromas due to BIA-ALCL. In a prior study mean concentrations of IL-9, IL-10 and IL-13 were found to be significantly higher in BIA-ALCL than in benign seromas. The aim of this research was to test the ability to detect high concentrations of IL-9 rapidly with a lateral flow assay (LFA). Because we previously reported that a LFA for CD30 detected BIA-ALCL in seromas we compared CD30 and IL-9 LFAs in distinguishing BIA-ALCL from benign seromas. Thirty microliter samples of 26 seromas (15 benign, 11 malignant) were tested on in-house prepared strips for IL-9 and CD30. Nanoparticle-conjugated antibodies specific to IL-9 and CD30 were used for detection. IL-9 was analyzed in undiluted samples and CD30 samples were optimized at 1:3 dilution. The dynamic range of detection was determined by spiking recombinant IL-9 into a benign seroma. Image analysis measured intensity of both test line (TL) and control line (CL) and a TL/CL ratio was calculated. IL-9 protein and IL-9 transcription factor PU.1 were stained in BIA-ALCL lines and clinical samples. The IL-9 LFA was reliable in distinguishing BIA-ALCL from benign seromas when the concentration of IL-9 was greater than 10 ng/ml. The CD30 LFA was positive in all 11 malignant cases. In one case with only faint CD30 and IL-10 test lines, the IL-9 LFA was clearly positive. Immunohistochemistry showed IL-9 and its essential transcription factor PU.1 were present in tumor cells in BIA-ALCL lines and clinical samples. IL-9 is a tumor cell biomarker of BIA-ALCL that can be detected by lateral flow assay and immunohistochemistry. Concentrations of IL-9 greater than 10 ng/ml reliably distinguished BIA-ALCL from benign seromas. Moreover, IL-9 LFA could detect BIA-ALCL when CD30 LFA was not definitive and IL-10 was of low concentration with a faint IL-10 TL, suggesting a multiplex LFA including IL-9, CD30 and IL-10 might be more effective in detecting BIA-ALCL in selected cases.

The prognostic value of a combined immune score in tumor and immune cells assessed by immunohistochemistry in triple-negative breast cancer

BackgroundThis study aimed to develop a novel combined immune score (CIS)-based model assessing prognosis in triple-negative breast cancer (TNBC).MethodsThe expression of eight immune markers (PD-1, PD-L1, PD-L2, IDO, TIM3, OX40, OX40L, and H7-H2) was assessed with immunohistochemistry on the tumor cells (TCs) and immune cells (ICs) of 227 TNBC cases, respectively, and subsequently associated with selected clinicopathological parameters and survival. Data retrieved from The Cancer Genome Atlas (TCGA) were further examined to validate our findings.ResultsAll immune markers were often expressed in TCs and ICs, except for PD-1 which was not expressed in TCs. In ICs, the expression of all immune markers was positively correlated between one another, except between PD-L1 and OX40, also TIM3 and OX40. In ICs, PD-1, PD-L1, and OX40L positive expression was associated with a longer progression-free survival (PFS; p = 0.040, p = 0.020, and p = 0.020, respectively). In TCs, OX40 positive expression was associated with a shorter PFS (p = 0.025). Subsequently, the TNBC patients were classified into high and low combined immune score groups (CIS-H and CIS-L), based on the expression levels of a selection of biomarkers in TCs (TCIS-H or TCIS-L) and ICs (ICIS-H or ICIS-L). The TCIS-H group was significantly associated with a longer PFS (p < 0.001). Furthermore, the ICIS-H group was additionally associated with a longer PFS (p < 0.001) and overall survival (OS; p = 0.001), at significant levels. In the multivariate analysis, both TCIS-H and ICIS-H groups were identified as independent predictors of favorable PFS (p = 0.012 and p = 0.001, respectively). ICIS-H was also shown to be an independent predictor of favorable OS (p = 0.003). The analysis of the mRNA expression data from TCGA also validated our findings regarding TNBC.ConclusionOur novel TCIS and ICIS exhibited a significant prognostic value in TNBC. Additional research would be needed to strengthen our findings and identify the most efficient prognostic and predictive biomarkers for TNBC patients.

An imidazole-derived polarity sensitive probe for lipid droplet target and in vivo tumor imaging

Accurate differentiation of tumor cells from normal cells is a fundamental and critical challenge for the early diagnosis of cancer. The intrinsic low-polarity and increased numbers of lipid droplets can be utilized as potential biomarkers for tumor cells. Therefore, we synthesized and screened the polar fluorescent probe (PI-CHO), which exhibiting notable fluorescence color change and spectral shift in solvents of different polarities. PI-CHO has a high sensitivity to polarity changes in the linear range of ET(30) = 37.4–63.1, with a fluorescence enhancement of more than 474-folds. Intracellular imaging experiments showed that PI-CHO has low cytotoxicity, excellent lipid droplet-targeted performance and discernibility between tumor and normal cells. Ex vivo imaging of the tumors and major organs demonstrated the great reliability and sensitive responsiveness of PI-CHO towards tumors. Moreover, the proposed probe was successfully applied to detect tumors in living mice, exhibiting the potential to become a powerful tool in early cancer diagnosis.

Logic-Gated Cell-Derived Nanovesicles via DNA-Based Smart Recognition Module.

Engineering cell-derived nanovesicles with active-targeting ligands is an important strategy to enhance the targeting efficiency. However, the enhanced binding capability to targeting cells also leads to the binding with nontarget cells that share the same biomarkers. DNA-based logic gate is a kind of molecular system that responds to chemical inputs by generating output signals, and the relationship between the input and the output is based on a certain logic. Thus, the DNA-based logic gate could provide a new approach to improve the delivery efficiency of the nanovesicle. In this work, we developed a DNA logic-gated module that coupled two tumor cell-targeting factors (e.g., low pH and a tumor cell biomarker) in a Boolean manner. Immobilization of this module on the surface of the nanovesicle enables the nanovesicle to sense tumor cell-targeting factors and regard these cues as inputs AND logic gate. With the guide of DNA-based logic gate, gold carbon dots (GCDs) encapsulated within nanovesicles were delivered into target cells, and then the intracellular redox status variation was reflected by fluorescence change of GCDs. Overall, we developed DNA logic-gated nanovesicles that contract different targeting factors into a unique tag for target cells. This facile functionalization strategy can pave the way for constructing smart nanovesicles and would broaden their application in the field of precision medicine and personalized treatment.

Highly Sensitive Electrochemical Aptasensor for Detecting the VEGF165 Tumor Marker with PANI/CNT Nanocomposites.

Sensing targeted tumor markers with high sensitivity provides vital information for the fast diagnosis and treatment of cancer patients. A vascular endothelial growth factor (VEGF165) have recently emerged as a promising biomarker of tumor cells. The electrochemical aptasensor is a promising tool for detecting VEGF165 because of its advantages such as a low cost and quantitative analysis. To produce a sensitive and stable sensor electrode, nanocomposites based on polyaniline (PANI) and carbon nanotube (CNT) have potential, as they provide for easy fabrication, simple synthesis, have a large surface area, and are suitable in biological environments. Here, a label-free electrochemical aptasensor based on nanocomposites of CNT and PANI was prepared for detecting VEGF165 as a tumor marker. The nanocomposite was assembled with immobilized VEGF165 aptamer as a highly sensitive VEGF165 sensor. It exhibited stable and wide linear detection ranges from 0.5 pg/mL to 1 μg/mL, with a limit of detection of 0.4 pg/mL because of the complementary effect of PANI/CNT. The fabricated aptasensor also exhibited good stability in biological conditions, selectivity, and reproducibility after several measurement times after the dissociation process. Thus, it could be applied for the non-invasive determination of VEGF, in biological fluid diagnosis kits, or in an aptamer-based biosensor platform in the near future.

CD44-Associated Tn Antigen as a New Biomarker of Tumor Cells with Aberrant Glycosylation.

Tn antigen is a tumor-associated antigen that appears on cancer cells as a result of aberrant O-glycosylation. The most studied form of Tn antigen is found in mucins, in particular, in mucin 1 (MUC1). Antibodies against this form of Tn antigen are used to diagnose tumors, as well as to generate T-killers with a chimeric receptor. Some carcinomas do not carry MUC1 and antibodies of a different specificity are required to detect Tn antigen on these cells. In our work, we searched for anti-Tn antibodies without preliminary assumptions about the proteins that may be carriers of the Tn antigen. For this purpose, we obtained several pairs of isogenic cell lines with the wild type and knockout of the Cosmc gene, which is essential for correct protein O-glycosylation. Using the created lines as immunogens, we generated a monoclonal antibody AKC3, which reacted with the Cosmc-deficient A549 lung adenocarcinoma cells and did not bind to the wild-type cells. Using mass spectrometry, as well as co-immunoprecipitation, it was shown that the AKC3 antibody recognized the Tn antigen in the context of CD44 protein - a protein important for tumor growth. The AKC3 antibody can be used for tumor diagnosis, and to generate T cells with a chimeric receptor for treatment of tumors that do not express mucins.

Abstract 2795: Ex vivo modelling of vascularized pdx-derived ovarian tumors

Abstract Predicting clinical response to anticancer drugs remains a major challenge in cancer therapy research. The current treatment of patients with ovarian cancer involves surgery and platinum-based chemotherapy, which still result in recurrence on advanced stages of the disease. During tumor progression, cancerous cells continuously accumulate mutations giving rise to a heterogeneous population of cells. Additionally, spatial distribution of stromal, endothelial and immune cells in the microenvironment affects tumor progression, cell morphology, and ultimately drug response. In order to predict clinical outcome of chemotherapy in vitro, tumor heterogeneity and microenvironment constituents must be conserved. Current models of tumor biology and microenvironment consist in xenografts of human tumors implanted in immunodeficient mice. These models allow studying systemic treatment response, but its application is limited to small developmental studies. Here, we present a high throughput in vitro 'grafting' platform where we co-culture blood vessels with tumor explants. Each unit in this platform is composed of two parallel microfluidic channels and a central chamber. Two endothelial tubules are generated in the microfluidic channels and cultured in presence of a gradient of angiogenic factors (S1P, VEGF, bFGF and PMA) added to the central chamber of the culture unit. Angiogenic tubules form vascular beds within 3-5 days, after which tumor explants are loaded to the central chamber on top of the vascular beds. The model shown in this study consists of ovarian serous papillary adenocarcinoma collected after xenograft growth in immunodeficient mice. This ovarian cancer explant was previously characterized as resistant to Paclitaxel. In this study, we observe how the vascular bed remodels in the presence of the explant and closely interacts with ovarian tumor tissue. Vessel perfusion and stabilization of vascular bed was monitored by real time imaging of 150 kDa FITC-Dextran. Cultures were evaluated by assessment of morphology and presence of endothelial and tumor cell biomarkers. Moreover, co-culture response to Sorafenib (anti-angiogenic), Palbociclib and Paclitaxel was detected by distinctive proliferation rates as compared to control conditions. The established ovary cancer-on-a-chip platform enables the study of fundamental aspects of tumor disease and progression. In addition, these co-cultures serve as a platform for understanding tumor-endothelial cell crosstalk and its consequences for tumor aggressiveness. Moreover, these models constitute a suitable platform for drug screenings of anti-cancer and anti-angiogenic compounds, making them a powerful translational tool for drug selection in personalized medicine applications. Citation Format: Silvia Bonilla, Jean-François Mirjolet, Pauline Berger, Elodie Rajon, Erik Walinga, Remko van Vught, Henriëtte Lanz, Jos Joore, Paul Vulto, Fabrice Viviani, Karla Queiroz. Ex vivo modelling of vascularized pdx-derived ovarian tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2795.

A pilot study of cdc6 as a biomarker for circulating tumor cells in patients with lung cancer.

BackgroundCell division cycle 6 (cdc6) is a key factor of DNA replication initiation license system and a proto‐oncogene. It has been detected in some tumor tissues to aid cancer diagnosis in many research projects. However, it remains unclear that if cdc6 could be detected in the peripheral blood, just like liquid biopsy, in solid tumor patients. The aim of this study is to investigate the possibility of cdc6 as a biomarker for circulating tumor cells in patients with lung cancer.MethodsWe first detected the expression of cdc6 in peripheral blood mononuclear cells (PBMCs) and tumor cells by in situ hybridization with cdc6 RNA probe. Then, we examined the expression of cdc6 in PBMCs from health individual, mononuclear cells from cord blood, or A549 cell line by RT‐qPCR. Furthermore, we used RT‐qPCR to test the cdc6 expression in PBMCs from tumor patients (test group) and non‐tumor individuals as a control group. Chi‐square test with Fisher's exact test was used to analyze the statistical significance of the difference. P < .05 is considered as statistically significant difference.ResultsWhen compared the cdc6 expression in cells from difference sources, we found that A549 tumor cell line had the strongest expression of cdc6, samples from cord blood showed the least expression level, indicating the detection strategy of RT‐qPCR is reasonable. Using this method, we studied whether cdc6 in Peripheral blood could be detected as a biomarker by examining cdc6 expression from PBMCs of patients with lung cancer. We found 20% of patients with lung cancer were cdc6 positive in PBMCs, whereas only 4.26% was found positive in the control group (P = .039, P < .05).ConclusionCell division cycle 6 has a potential to be used as a circulating tumor cell biomarker for lung cancer. Further study in clinical application is still broad needed.

Single-arm, phase II study to evaluate the safety and efficacy of sacituzumab govitecan in patients with metastatic castration-resistant prostate cancer who have progressed on second generation AR-directed therapy.

TPS251 Background: In patients with mCRPC, androgen receptor (AR) pathway inhibitors such as enzalutamide or abiraterone acetate are often used as first-line systemic therapy. After progression on either of these inhibitors, the likelihood of response to the other is significantly lower than in the first-line setting. Multiple resistance mechanisms have been identified, including the emergence of AR splice variants. Trop2 is a cell-surface antigen that is expressed in &gt; 70% of prostate cancers and is enriched in mCRPC expressing AR splice variants. Sacituzumab govitecan (SG; IMMU-132) is a novel antibody drug conjugate composed of humanized anti-Trop2 antibody conjugated to SN-38 payload by hydrolyzable linker. SG has potential to be an effective therapy after progression on initial AR pathway inhibitors to meet this significant unmet clinical need. Methods: This is a single-arm, open-label, multicenter phase 2 trial (NCT03725761) to determine the efficacy and safety of SG. The primary endpoint is PSA response rate (≥50% PSA decline at or within 9 weeks of starting treatment); secondary endpoints include rPFS and overall survival. 55 subjects with mCRPC who have progressed on second-generation AR-directed therapy in either the hormone sensitive or CRPC setting will be included. Fresh or archived metastatic site biopsy is required. Subjects will be treated with SG IV at 10 mg/kg on Day 1 and Day 8 of 21-day cycles until disease progression. Subjects who receive ≥1 SG dose and have ≥1 PSA post-dose assessment will be included in final efficacy analysis. Circulating tumor cell biomarker studies will be performed throughout treatment and at progression to investigate potential mechanisms of response/resistance to SG. Tumor biopsies at baseline and after 3 therapy cycles will evaluate genomic alterations that predict treatment response. Subjects who remain progression free will undergo repeated evaluations of extent of disease every 9 weeks. The trial is actively accruing. Managed by: Prostate Cancer Clinical Trials Consortium; Funding: Immunomedics; Support: Prostate Cancer Foundation. Clinical trial information: NCT03725761.

CtcRbase: the gene expression database of circulating tumor cells and microemboli.

Circulating tumor cells/microemboli (CTCs/CTMs) are malignant cells that depart from cancerous lesions and shed into the bloodstream. Analysis of CTCs can allow the investigation of tumor cell biomarker expression from a non-invasive liquid biopsy. To date, high-throughput technologies have become a powerful tool to provide a genome-wide view of transcriptomic changes associated with CTCs/CTMs. These data provided us much information to understand the tumor heterogeneity, and the underlying molecular mechanism of tumor metastases. Unfortunately, these data have been deposited into various repositories, and a uniform resource for the cancer metastasis is still unavailable. To this end, we integrated previously published transcriptome datasets of CTCs/CTMs and constructed a web-accessible database. The first release of ctcRbase contains 526 CTCs/CTM samples across seven cancer types. The expression of 14 631 mRNAs and 3642 long non-coding RNAs of CTCs/CTMs were included. Experimental validations from the published literature are also included. Since CTCs/CTMs are considered to be precursors of metastases, ctcRbase also collected the expression data of primary tumors and metastases, which allows user to discover a unique 'circulating tumor cell gene signature' that is distinct from primary tumor and metastases. An easy-to-use database was constructed to query and browse CTCs/CTMs genes. ctcRbase can be freely accessible at http://www.origin-gene.cn/database/ctcRbase/.

Porous carbon supported nanoceria derived from one step in situ pyrolysis of Jerusalem artichoke stalk for functionalization of solution-gated graphene transistors for real-time detection of lactic acid from cancer cell metabolism

Effective detection of biomarkers for tumor cells has been the focus of attention. In this work, we have successfully fabricated a highly sensitive sensor based on solution-gated graphene transistors (SGGT) for detecting lactic acid content accumulated in tumor cells through their glycolysis metabolism. The sensing mechanism of the lactic acid sensor is attributed to electrochemical catalysis of H2O2 produced by the oxidation of lactic acid by lactate oxidase near the gate electrode. The key component of the sensor is the functionalization of porous carbon loaded with ceria nanoparticles derived from a novel one step in situ pyrolysis of pretreated Jerusalem artichoke stalk, which significantly improved the sensor sensitivity, i.e. a detection limit as low as 300 nM and linear range from 3 μM to 300 μM. The optimized lactic acid sensor has successfully applied to the detection of lactic acid in practical cell culture samples with high credibility. The SGGT-based lactic acid biosensor shows great potential for the application in tumor microenvironment due to its superior biocompatibility and accuracy.

Clinical Significance of the PRAME Gene Expression in Oncohematological Diseases

Although the PRAME activity was first discovered in solid tumors, this gene is very frequently expressed in oncohematological diseases. PRAME can be regarded as a reliable biomarker of tumor cells. Determination of PRAME transcripts is used in residual disease monitoring and molecular relapse diagnostics. Experimentation with PRAME expressing lines of leukemia cells yielded controversial results. Therefore, it is hardly possible to estimate the prognostic value of PRAME activity in oncohematological diseases. In chronic myeloproliferative disease and chronic myeloid leukemia, however, PRAME activity proves to be a predictor of negative prognosis, and on the contrary, it can be regarded as a positive prognostic factor in acute myeloid or lymphoid leukemia. Despite many clinical studies prognostic value of PRAME expression in some diseases requires further investigation. The present literature review contains the data concerning PRAME expression in oncohematological diseases.

Regulation of DNA replication-coupled histone gene expression.

The expression of core histone genes is cell cycle regulated. Large amounts of histones are required to restore duplicated chromatin during S phase when DNA replication occurs. Over-expression and excess accumulation of histones outside S phase are toxic to cells and therefore cells need to restrict histone expression to S phase. Misregulation of histone gene expression leads to defects in cell cycle progression, genome stability, DNA damage response and transcriptional regulation. Here, we discussed the factors involved in histone gene regulation as well as the underlying mechanism. Understanding the histone regulation mechanism will shed lights on elucidating the side effects of certain cancer chemotherapeutic drugs and developing potential biomarkers for tumor cells.

Elevation of YAP promotes the epithelial-mesenchymal transition and tumor aggressiveness in colorectal cancer

Tumor metastasis is the leading cause of death in cancer patients. Identifying metastatic biomarkers in tumor cells would help cancer diagnoses and the development of therapeutic targets. Yes-associated protein (YAP) plays an important role in organ development and has gained much attention in tumorigenesis. However, the role of YAP and the underlying mechanism in tumor metastasis of colorectal cancer (CRC) is still unclear. In this study, we generated metastatic 116-LM cells from the HCT116 CRC cell line. We found that the capacity for tumor aggressiveness was elevated in 116-LM cells and identified that YAP and its mRNA level were upregulated in 116-LM cells. Moreover, expression of YAP was found to correlate with epithelial-mesenchymal transition (EMT) marker expressions, whereas suppression of YAP decreased EMT marker expressions and impeded tumor migration and invasion. Additionally, upregulation of YAP was identified in colon cancer patients, and it was correlated with EMT gene expressions. Furthermore, we identified LBH589, a histone deacetylase inhibitor, that was capable of inhibiting tumor growth and aggressiveness in both HCT116 and 116-LM cells. LBH589 potentially inhibited YAP and its mRNA expression, accompanied by diminished expressions of YAP downstream genes and EMT markers. Together, YAP plays a crucial role in aggressiveness and metastasis of CRC, and YAP may be an attractive therapeutic target.

Prognostic and Predictive Biomarkers in Colorectal Cancer. From the Preclinical Setting to Clinical Practice.

Colorectal cancer (CRC) is the second largest cause of cancer mortality in Western countries, mostly due to metastasis. Understanding the natural history and prognostic factors in patients with metastatic CRC (mCRC) is essential for the optimal design of clinical trials. The main prognostic factors currently used in clinical practice are related to tumor behavior (e.g., white blood counts, levels of lactate dehydrogenase, levels of alkaline phosphatase) disease extension (e.g., presence of extrahepatic spread, number of organs affected) and general functional status (e.g., performance status as defined by the Eastern Cooperative Oncology Group). However, these parameters are not always sufficient to establish appropriate therapeutic strategies. First-line therapy in mCRC combines conventional chemotherapy (CHT) (e.g., FOLFOX, FOLFIRI, CAPOX) with a number of agents targeted to specific signaling pathways (TA) (e.g., panitumumab and cetuximab for cases KRAS/NRAS WT, and bevacizumab). Although the response rate to this combination regime exceeds 50%, progression of the disease is almost universal and only less than 10% of patients are free of disease at 2 years. Current clinical trials with second and third line therapy include new TA, such as tyrosin-kinase receptors inhibitors (MET, HER2, IGF-1R), inhibitors of BRAF, MEK, PI3K, AKT, mTORC, NOTCH and JAK1/JAK2, immunotherapy modulators and check point inhibitors (anti-PD-L1 and anti- PD1). Despite the identification of multiple prognostic and predictive biomarkers and signatures, it is still unclear how expression of many of these biomarkers is modulated by CHT and/or TA, thus potentially affecting response to treatment. In this review we analyzed how certain biomarkers in tumor cells and microenvironment influence the response to new TA and immune-therapies strategies in mCRC pre-treated patients.

Development and Characterization of New Monoclonal Antibodies against Human Recombinant CA XII

Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.

Rapid translation of circulating tumor cell biomarkers into clinical practice: technology development, clinical needs and regulatory requirements

The great hope in circulating tumor cell (CTC) research lies in the use of these rare cells as an accessible "fluid biopsy" that would permit frequent, minimally invasive sampling of tumor cells for similar molecular assays that are performed on traditional biopsies. Given the rarity of CTCs in peripheral circulation, microscale methods show great promise and superiority to capture and analyze these cells from patients with solid tumors. Novel technologies that produce validated CTC biomarkers may finally provide medical oncologists the tools needed to provide precise, personalized medical care for patients with advanced cancer. However, few CTC technologies demonstrate both experimental and clinical evidence of an accurate, reliable and reproducible assay that also meets the regulatory requirements to enter routine clinical practice. Many opportunities exist to incorporate clinical needs and regulatory benchmarks into technology development to more quickly garner FDA approval to direct decisions on patient care. This review will address: 1) device development tailored to address predictive, prognostic and/or therapeutic needs across the multitude of malignancies and disease stages; 2) validation benchmarks for clinical assay development; 3) early establishment of standard operating procedures for sample acquisition and analysis; 4) demonstration of clinical utility; 5) clinical qualification of a novel biomarker; and 6) integration of a newly validated and qualified technology into routine clinical practice. Early understanding and incorporation of these regulatory requirements into assay development can simplify and speed the integration of these novel technologies into patient care. Meeting these benchmarks will lead to the true personalization of cancer therapies, directing initial and subsequent treatments for each individual based on initial tumor characteristics while monitoring for emerging mechanisms of resistance in these continually evolving tumors.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Targeted metabolic analysis of nucleotides and identification of biomarkers associated with cancer in cultured cell models

Cancer, like other diseases accompanied by metabolic changes, shows characteristic DNA/RNA modifications and activities of modifying enzymes, resulting in fluctuations in nucleoside levels. In this study, we undertook targeted metabolomic analyses of nucleotides in different cancer cell culture models using a sensitive and reproducible ion-pair HPLC method. The experimental data were analyzed by principal component analysis (PCA) to identify potential biomarkers in cancer cells, and statistical significance was determined by one-way analysis of variance. As a result, a clear differentiation of normal and tumor cells into two clusters was shown, indicating abnormal metabolism of nucleotides in tumor cells. Six variables (AMP, UDP, CTP levels with a significance of P<0.05; ATP, UTP and GMP levels with a significance of P<0.01) were considered as potential biomarkers; the content of AMP, UTP, GMP and ATP was significantly higher in cancer cells. The receiver operating characteristic (ROC) curve analysis allowed us to discriminate normal cells from tumor cells based on area under the curve (AUC). The sequence of their AUC values were: ATP (0.979)>UTP (0.938)>CTP=GMP (0.896)>AMP (0.812)>UDP (0.792), so we conclude that ATP and UTP are the best potential biomarkers in tumor cells. This study may provide a valuable tool for studying minute alterations of intracellular nucleotide pools induced by anticancer/antiviral drugs, diseases or environmental factors.

Hormonally responsive breast cancer cells in a microfluidic co-culture model as a sensor of microenvironmental activity.

Breast cancer cell growth and therapeutic response are manipulated extrinsically by microenvironment signals. Despite recognition of the importance of the microenvironment in a variety of tumor processes, predictive measures that incorporate the activity of the surrounding cellular environment are lacking. In contrast, tumor cell biomarkers are well established in the clinic. Expression of Estrogen Receptor-alpha (ERα) is the primary defining feature of hormonally responsive tumors and is the molecular target of therapy in the most commonly diagnosed molecular subtype of breast cancer. While a number of soluble factors have been implicated in ERα activation, the complexity of signaling between the cellular microenvironment and the cancer cell implies multivariate control. The cumulative impact of the microenvironment signaling, which we define as microenvironmental activity, is more difficult to predict than the sum of its parts. Here we tested the impact of an array of microenvironments on ERα signaling utilizing a microfluidic co-culture model. Quantitative immunofluorescence was employed to assess changes in ERα protein levels, combined with gene expression and phosphorylation status, as measures of activation. Analysis of microenvironment-induced growth under the same conditions revealed a previously undescribed correlation between growth and ERα protein down-regulation. These data suggest an expanded utility for the tumor biomarker ERα, in which the combination of dynamic regulation of ERα protein and growth in a breast cancer biosensor cell become a read-out of the microenvironmental activity.