Potential Blood-based Biomarkers Research Articles

Abstract OT1-1-09: Phase I/II trial of ruxolitinib in combination with trastuzumab in metastatic HER2 positive breast cancer

Abstract Background Integrated analysis of whole genome RNAi screening with computationally reverse engineered interactome models identified IL6/JAK/STAT as a master regulator pathway essential for growth of ErbB2/HER2 positive breast cancer. Ruxolitinib (R), FDA-approved treatment for myelofibrosis, inhibits JAK1 and JAK2. The combination of R plus Trastuzumab (T) is synergistic in tumor growth inhibition in mouse xenografts of HER2 amplified breast cancer cell lines. These data provide a strong rationale for studying the efficacy of combination R and T in a clinical trial. Trial Design A multi-center, open-label, phase I/II (P1/2) trial of R plus T in HER2+ metastatic breast cancer (MBC) who have progressed on T-based therapy. P1 will be an adaptive design with 10 patients, using the time-to-event continual reassessment method. The recommended P2 dose (RP2D) will be used in a non-randomized, open-label P2 trial with 30 evaluable patients (pts). Given the anticipated limited overlapping toxicities, approximately 36 pts (range: 32-40) are expected for the P1/2. The duration of a treatment cycle will be 21 days. R will be taken orally twice a day continuously. The P1 dosing range will be 10-25 mg BID (dose level 0: 20 mg BID). T will be administered on Day 1 of each cycle at standard dosing. Objective Response Rate (ORR) will be assessed by imaging every 9 weeks. Blood samples will be obtained for biomarker analysis, pre-treatment, on-treatment on C2D1, and then at progression. Pre-treatment biopsies from archival tissue or new biopsy, on treatment biopsy on C2D1, and upon progression of disease will be discussed with pts with accessible disease. Main Eligibility Criteria: 1. HER2 positive MBC 2. Progression on >2 HER2-directed therapy in metastatic setting, including Pertuzumab and T-DM1 3. Measurable or non-measurable disease 4. LVEF >50%. 5. No history of prior JAK2 inhibitor 6. No HIV-positive or active infection 7. No concurrent medications that are potent CYP3A4 inhibitor or inducer Specific Aims 1. Primary: P1: MTD of combined R + T. P2: Progression Free Survival (PFS) 2. Secondary: a) Clinical: ORR, clinical benefit rate (CBR), and tolerability. Pts will be stratified by hormone receptor (HR) status to explore differences in efficacy between HR+ and HR-. b) Explore potential predictive tumor and blood-based predictive biomarkers at baseline, on treatment, and progression: (tumor: pSTAT3 expression); serum: IL-6, IL-8, C-reactive protein; circulating tumor cell pSTAT3 expression; and tumor gene expression. Statistical Methods Assuming a historical PFS of 8 weeks with single-agent agent HER2-targeted therapy in HER2+ MBC after progressing on T-based therapy, we predict that pts receiving the combination of R plus T will have a PFS of at least 13 weeks. With a 2-sided alpha of 0.05, we have 80% power to detect a difference with 30 pts. Target Accrual Sample Size: 32-40 pts; projected over 2 years at 4 sites: Columbia, Einstein, Mount Sinai, and Cornell. Trial will start accruing July 2014. Citation Format: Kevin Kalinsky, Dow-Chung Chi, Shing Lee, Amy Tiersten, Della Makower, Ellen Chuang, Katherine D Crew, Dawn L Hershman, Jose Silva, Andrea Califano, Matthew Maurer. Phase I/II trial of ruxolitinib in combination with trastuzumab in metastatic HER2 positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-09.

Abstract P4-02-09: RUNX3 hypermethylation in circulating tumor DNA is a biomarker of breast cancer distant metastasis

Abstract Breast cancer detection has not resulted in the expected decrease in mortality suggesting that current screening strategies are not finding metastasis-prone lesions before metastasis occurs and instead are detecting many lesions that will not progress to metastatic disease. Although several improvements in the management of breast cancer have been made, an estimated 90% of breast cancer related deaths are due to the development of distant metastasis to organs such as lung, bone, liver, and brain. Metastatic breast cancer affects 10-15% of breast cancer patients, is often refractory to therapy and associated with poor outcomes. However, a blood test indicating a high-risk population at the time of breast cancer diagnosis might shift the current strategy and improve outcomes. Efforts for identifying predictive markers of breast cancer metastasis have been impeded by a lack of data defining the genetic and epigenetic determinants of the disease. Numerous studies suggest that DNA methylation could be a useful biomarker for improving the clinical management. Recently circulating tumor (ct)DNA has attracted attention for clinical use in the context of risk prediction, prognostication and prediction in human cancer. Various types of DNA alterations have been reported in ctDNA but methylated DNAs are chemically stable and are among the most sensitive and specific ctDNA cancer markers. The objective of our study was to identify and validate a blood-based DNA methylation biomarker for breast cancer patients at high risk of distant metastasis. Using the HumanMethylation27K bead array, we recently identified RUNX3 hypermethylation involving 3 CpG probes in 32 breast brain metastasis tumor tissue compared to a series of 50 unmatched early stage breast cancer samples. We calculated the area under the curve (AUC) to exceed 0.8, with a higher AUC achieved in Her2-positive samples. In an independent, first of its kind analysis, we performed whole genome bisulfite sequencing (WGBS) of ctDNA from plasma samples representing three pools of 40 women with distant metastatic breast cancer to various organs, 40 women with metastasis-free breast cancer, and 40 non-breast cancer controls. In this unbiased analysis, these data demonstrated RUNX3 hypermethylation in the plasma of patients with metastatic breast cancer in the same region corresponding to the CpG probes of the methylation array. We are now performing bisulfite pyrosequencing of each individual plasma sample, and in an additional cohort of 50 archival primary/brain metastasis tumor pairs with matching serum and clinical outcomes data. To determine regional specific differences we have also selected two additional regions of RUNX3 not differentially methylated for validation by bisulfite pyrosequencing. Determining the extent to which RUNX3 hypermethylation occur in the context of brain metastasis versus other distant sites are ongoing studies. Together these studies point towards RUNX3-regional specific methylation as a potential blood-based biomarker of breast cancer metastasis. Such a biomarker will help establish surveillance methods and eventually preventative regimens that will increase the likelihood of long-term progression-free survival for breast cancer patients at risk of distant relapse. Citation Format: Bodour Salhia, Gerald C Gooden, Kyle N Johnson, Waibhav Tembe, Garrick Wallstrom, Winnie Liang, Manmeet Ahluwalia, Steven N Kalkanis, Tom Mikkelsen, Steven A Toms. RUNX3 hypermethylation in circulating tumor DNA is a biomarker of breast cancer distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-09.

FIRST STEPS IN THE DEVELOPMENT AND PRODUCTION OF CLINICAL REFERENCE MATERIAL FOR AMYLOID-B1-42 IN HUMAN CEREBROSPINAL FLUID

were determined using a Taq-Man assay. A total of 35 PFAA profiles were measured by liquid chromatography-mass spectrometry (LCMS). The PFAA profiles were also compared with ageand gender-matched healthy control subjects (HC, n 1⁄4 36) who had undergone a comprehensive health screening.Results: Several PFAAs have shown significant changes between patients with AD and cognitively normal subjects as well as ageand gendermatched healthy subjects. Neither age at onset, MMSE score, nor APOE genotype affects the PFAA profiles significantly in patients with AD. The area under the curve (AUC) of receiver operating characteristic (ROC) curve by a certain PFAA was found to be 0.86, suggesting a high discriminating power of PFAA to make a diagnosis of AD. Conclusions: Our results suggest that PFAA profile could be a potential blood-based biomarker for AD. We will discuss the implications of our findings for the development of screening tool and companion diagnostic test for patients with AD.

Proteomics Approaches to Understand Linkage Between Alzheimer’s Disease and Type 2 Diabetes Mellitus

Alzheimer's disease (AD) is a progressive neurological disease of the brain leading to the irreversible loss of neurons and intellectual abilities. Diabetes mellitus type 2 (T2DM) is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. The prevalence of AD and T2DM is increasing at an alarming rate and has become a major public health concern worldwide. The clinico-pathological relationship between AD and T2DM has been debated for more than a decade. Recent epidemiological studies have provided direct evidence that T2DM is a strong risk factor for AD and numerous studies have demonstrated that patients with diabetes have an increased risk of developing AD as compared with healthy individuals. The underlying biological mechanisms that link the development of diabetes with AD are not fully understood and therefore are worth intensive research. The existence of proteomic links between AD and diabetes is an important topic currently under active debate. An understanding of the complex association between diabetes and AD is necessary for the development of novel drug therapies and lifestyle guidelines aimed at the treatment and/or prevention of these diseases. This review aims to summarize what is currently known about the biological and especially proteomic relationships and similarities between these two age-related devastating diseases of modern day life. This study may also aid in future for the identification of a single or a panel of potential blood-based protein biomarkers for early diagnosis of AD and T2DM with high sensitivity and specificity.

Dermcidin expression is associated with disease progression and survival among breast cancer patients

Improved diagnostic screening has led to earlier detection of many tumors, but screening may still miss many aggressive tumor types. Proteomic and genomic profiling studies of breast cancer samples have identified tumor markers that may help improve screening for more aggressive, rapidly growing breast cancers. To identify potential blood-based biomarkers for the early detection of breast cancer, we assayed serum samples via matrix-assisted laser desorption ionization-time of flight mass spectrometry from a rat model of mammary carcinogenesis. We found elevated levels of a fragment of the protein dermcidin (DCD) to be associated with early progression of N-methylnitrosourea-induced breast cancer, demonstrating significance at weeks 4 (p = 0.045) and 5 (p = 0.004), a time period during which mammary pathologies rapidly progress from ductal hyperplasia to adenocarcinoma. The highest serum concentrations were observed in rats bearing palpable mammary carcinomas. Increased DCD was also detected with immunoblotting methods in 102 serum samples taken from women just prior to breast cancer diagnosis. To validate these findings in a larger population, we applied a 32-gene in vitro DCD response signature to a dataset of 295 breast tumors and assessed correlation with intrinsic breast cancer subtypes and overall survival. The DCD-derived gene signature was significantly associated with subtype (p < 0.001) and poorer overall survival [HR (95 % CI) = 1.60 (1.01-2.51), p = 0.044]. In conclusion, these results present novel evidence that DCD levels may increase in early carcinogenesis, particularly among more aggressive forms of breast cancer.

Abstract OT2-6-05: Phase I/II trial of ruxolitinib in combination with trastuzumab in metastatic HER2 positive breast cancer

Abstract Background Integrated analysis of whole genome RNAi screening with computationally reverse engineered interactome models identified IL6/JAK/STAT as a master regulator pathway essential for growth of ErbB2/HER2 positive breast cancer. Ruxolitinib (R), FDA-approved treatment for myelofibrosis, inhibits JAK1 and JAK2. The combination of R plus Trastuzumab (T) is synergistic in tumor growth inhibition in mouse xenografts of HER2 amplified breast cancer cell lines. These data provide a strong rationale for studying the efficacy of combination R and T in a clinical trial. Trial Design A multi-center, open-label, phase I/II (P1/2) trial of R plus T in HER2+ metastatic breast cancer (MBC) who have progressed on T-based therapy. P1 will be a dose-escalation study with a 3 + 3 design to determine maximum tolerable dose (MTD) for the combination. The recommended P2 dose (RP2D) will be used in a non-randomized, open-label P2 trial with 30 evaluable patients (pts). Given the anticipated limited overlapping toxicities, 33 pts are expected for the P1/2 (range: 33-42 pts). The duration of a treatment cycle will be 21 days. R will be taken orally twice a day continuously. The P1 dosing range will be 10-25 mg BID (dose level 0: 20 mg BID). T will be administered on Day 1 of each cycle at standard dosing. Objective Response Rate (ORR) will be assessed by imaging every 9 weeks. Blood samples will be obtained for biomarker analysis, pre-treatment, on-treatment on C1D8, and then every 9 weeks. Pre-treatment biopsies from archival tissue or new biopsy, on treatment biopsy after C1, and upon progression of disease will be discussed with pts with accessible disease. Main Eligibility Criteria: 1. HER2 positive MBC 2. Progression on at least one T-containing regimen in the metastatic setting 3. Measurable or un-measurable disease 4. LVEF &amp;gt;50% 5. No history of prior JAK2 inhibitor 6. No HIV-positive or active infection 7. No concurrent medications that are potent CYP3A4 inhibitor or inducer Specific Aims 1. Primary: P1: MTD of combined R + T. P2: Progression Free Survival (PFS) 2. Secondary: a) Clinical: ORR, clinical benefit rate (CBR), and tolerability. Pts will be stratified by hormone receptor (HR) status to explore differences in efficacy between HR+ and HR-. b) Explore potential predictive tumor and blood-based predictive biomarkers at baseline, on treatment, and progression: (tumor: pSTAT3 expression); serum: IL-6, IL-8, C-reactive protein; circulating tumor cell pSTAT3 expression; and tumor gene expression. Statistical Methods Assuming a historical PFS of 8 weeks with single-agent agent HER2-targeted therapy in HER2+ MBC after progressing on T-based therapy, we predict that pts receiving the combination of R plus T will have a PFS of at least 13 weeks. With a 2-sided alpha of 0.05, we have 80% power to detect a difference with 30 pts. Target Accrual Sample Size: 33-42 pts; projected over 2 years at 4 sites: Columbia, Einstein, Mount Sinai, and Cornell. Contact Kevin Kalinsky/ Matt Maurer kk2693@columbia.edu/mm2058@columbia.edu. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-05.

Zwitterionic polymer-coated immunobeads for blood-based cancer diagnostics

Both total plasma and tumor-derived microvesicle (TMV)-associated miRNAs have been proposed as potential blood-based biomarkers for cancer diagnosis. However, there has been no comparison of the two types of miRNAs for biomarker discovery because of technological challenges of isolating TMVs from human plasma. The effective isolation of TMVs can be hardly achieved with conventional immunobead-based methods due to the high content of plasma proteins. In the current study, zwitterionic sulfobetaine-conjugated immunobeads are prepared using cluster of differentiation 83 (CD83) as a candidate protein marker for breast cancer-derived microvesicles. The zwitterionic immunobeads are more than 10-fold efficient for isolating TMVs from clinical plasma samples by suppressing nonspecific protein binding than conventional immunobeads. Early-stage breast cancer can be distinguished from benign breast disease by using the sulfobetaine-modified immunobeads, whereas conventional immunobeads show poor discriminatory performance. Further, we demonstrate that miRNAs in the form of TMVs offer a major improvement over total plasma miRNAs for early cancer detection. The analyses of miRNA expression levels show that in total, 6 miRNAs are significantly upregulated in the CD83-positive microvesicles of breast cancer patients, whereas differential miRNA expression is not detected on using total plasma RNA. The results indicate that our zwitterionic immunobead platform may constitute a powerful tool to identify circulating biomarkers and open a new avenue for highly sensitive blood-based cancer diagnostics.

Candidate Serum Biomarkers for Early Intestinal Cancer Using 15N Metabolic Labeling and Quantitative Proteomics in the ApcMin/+ Mouse

Current screening procedures for colorectal cancer are imperfect and highly invasive and result in increased mortality rates due to low compliance. The goal of the experiments reported herein is to identify potential blood-based biomarkers indicative of early stage intestinal cancers using the ApcMin/+ mouse model of intestinal cancer as an experimental system. Serum proteins from tumor-bearing ApcMin/+ mice were quantitatively compared to tumor-free Apc+/+ wild-type mice via in anima metabolic labeling with 14N/15N-labeled Spirulina algae and an LTQ Orbitrap mass spectrometer. Out of 1116 total serum proteins quantified, this study identified 40 that were differentially expressed and correlated with the increase in intestinal neoplasms. A subset of these differentially expressed proteins underwent a secondary quantitative screen using selected reaction monitoring-mass spectrometry with stable isotope-labeled peptides. Using both quantitative techniques, we identified MGAM and COL1A1 as downregulated and ITIH3 and F5 as upregulated in serum. All but COL1A1 were similarly differentially expressed in the mRNA of neoplastic colonic tissues of ApcMin/+ mice compared to normal wild-type tissue. These differentially expressed proteins identified in the ApcMin/+ mouse model have provided a set of candidate biomarkers for future validation screens in humans.

Peripheral signatures of the clusterin-DKK neurotoxicity pathway as potential blood-based biomarkers

Peripheral signatures of the clusterin-DKK neurotoxicity pathway as potential blood-based biomarkers

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

BackgroundMicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.MethodsThe pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.ResultsMore than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.ConclusionsOur study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis.

Inflammatory Bowel Disease – comprised of Crohn's Disease and Ulcerative Colitis (UC) - is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.

Abstract A9: De novo sequencing of circulating microRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

Abstract Purpose: MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. Patients and Methods: The pre-treatment sera of 42 stage II–III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II–III BC patients was used to assess the power of identified miRNA markers. Results: More than 800 miRNA species were detected in the circulation, and exhibited patterns associated with the histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundances determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II–III BC patients. Conclusion: Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications. Citation Format: Xiwei Wu, George Somlo, Melanie Palomares, Yun Yen, John Rossi, Harry Gao, Shizhen Emily Wang. De novo sequencing of circulating microRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A9.

Abstract 4157: Identification and validation of galectin-7 as a novel blood biomarker for NSCLC diagnosis

Abstract Lung cancer is the leading cause of cancer-related moralities in the US and worldwide. Non small cell lung cancer (NSCLC) accounts for 80 % of all diagnosed lung cancer cases. The lack of reliable, non-invasive diagnostic tests prompted the search for viable diagnostic protein markers. We report here the identification and validation of galectin7 as a novel blood-based biomarker for diagnosis of NSCLC. To identify potential blood protein markers that could be used for diagnosis of NSCLC, we implemented a multiphase validation scheme exploiting a large pool of novel protein markers shown to be over expressed in NSCLC tissues by our group by shotgun- mass spectrometry. Top candidates were first selected using bioinformatics tools including Ingenuity Pathway Analysis (IPA), Web gestalt and DAVID by applying a combination of statistical and biological filters including expression levels in cancer tissues, reported presence in plasma and other biofluids, and statistical significance. The resulting list of candidates was further refined by cross examination against other publically available data bases such as Plasma Proteome project of the Human Proteome Organization (PPP-HUPO) and Oncomine. We decided to focus our efforts on galectin 7, one of our top candidates that has not been reported previously as a biomarker for lung cancer. A case-control pilot study was designed to evaluate the relative levels of galectin 7 in plasma samples of peripheral blood from a small cohort of 30 NSCLC and 15 control nonmalignant lung disease patients using ELISAs. The results of this study were subjected to a second round of validation in an independent set of plasma samples from controls and NSCLC patients. Finally, we verified the identity of our protein candidates by means of immunoprecipitation (IP) and Western blot analyses. Statistical analysis of our ELISA data on galectin7 using the Wilcoxon cox nonparametric test revealed that galectin7 was detected at significantly higher levels in NSCLC patient plasma compared to controls. To further validate our findings, a second round of interrogation was performed using a separate sample set of 30 plasma samples and their matched tissues from nonmalignant and NSCLC patients. To verify the identity of our protein markers detected by ELISA, we successfully detected galectin 7 from both tissue lysates and plasma from NSCLC patients by Western blot analysis. Based on our preliminary results, we believe that we present the first evidence that galcetin 7 is a potential diagnostic blood-based biomarker for NSCLC. Retrospective clinical studies are currently underway to test the utility of galectin7 for diagnosis of early stage NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2011-4157

Plasma microRNAs as potential biomarkers for non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Developing minimally invasive techniques that can diagnose NSCLC, particularly at an early stage, may improve its outcome. Using microarray platforms, we previously identified 12 microRNAs (miRNAs) the aberrant expressions of which in primary lung tumors are associated with early-stage NSCLC. Here, we extend our previous research by investigating whether the miRNAs could be used as potential plasma biomarkers for NSCLC. We initially validated expressions of the miRNAs in paired lung tumor tissues and plasma specimens from 28 stage I NSCLC patients by real-time quantitative reverse transcription PCR, and then evaluated diagnostic value of the plasma miRNAs in a cohort of 58 NSCLC patients and 29 healthy individuals. The altered miRNA expressions were reproducibly confirmed in the tumor tissues. The miRNAs were stably present and reliably measurable in plasma. Of the 12 miRNAs, five displayed significant concordance of the expression levels in plasma and the corresponding tumor tissues (all r>0.850, all P<0.05). A logistic regression model with the best prediction was defined on the basis of the four genes (miRNA-21, -126, -210, and 486-5p), yielding 86.22% sensitivity and 96.55% specificity in distinguishing NSCLC patients from the healthy controls. Furthermore, the panel of miRNAs produced 73.33% sensitivity and 96.55% specificity in identifying stage I NSCLC patients. In addition, the genes have higher sensitivity (91.67%) in diagnosis of lung adenocarcinomas compared with squamous cell carcinomas (82.35%) (P<0.05). Altered expressions of the miRNAs in plasma would provide potential blood-based biomarkers for NSCLC.

Correlation and quantitation of microRNA aberrant expression in tissues and sera from patients with breast tumor

Objectives MicroRNAs (miRNAs) have been underlined as a promising potential biomarker for breast cancer but are limited to tissue specimens. Clinical specimens of sera are more abundant and more conveniently collected than tissues. This work was designed to investigate the expression and correlation of a selected panel of miRNAs associated with breast tumor in tissues and matching serum samples. Methods Tumor tissues, adjacent non-tumor tissues and matching serum samples were collected from 68 patients with newly diagnosed breast tumors. Normal control sera were collected from 40 healthy subjects. A panel of 6 miRNAs (miRNA-21, 106a, 126, 155, 199a and 335) were selected and their aberrant expression levels were quantified by using real-time PCR technique. Results A high correlation of miRNA expression level was found between breast tumor tissues and sera. MiR-21, miR-106a and miR-155 were significantly over-expressed in the tumor specimens compared with those in normal controls ( P < 0.05), whereas miR-126, miR-199a and miR-335 were significantly under-expressed ( P < 0.05). Furthermore, the relative expression of miR-21, miR-126, miR-155, miR-199a and miR-335 was closely associated with clinicopathologic features of breast cancer ( P < 0.05), such as histological tumor grades and sex hormone receptor expression. Conclusions Selective expression and modulation of miRNAs could be potential blood-based biomarkers for breast cancer diagnosis, grading and prognosis. Our results should encourage further studies on the use of miRNAs in serum samples as an easy and convenient method of breast cancer screening.

Circulating microRNAs as potential blood-based biomarkers for detection of colorectal cancer

e15040 Background: Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. While there is a strong correlation between stage and prognosis in this disease, current screening methods for CRC have significant limitations, and newer technological approaches are desired. Circulating nucleic acids in body fluids have been studied as a source for diagnostic information and for cancer screening, yet the potential of microRNAs, a family of small non-coding regulatory RNAs, has not yet been thoroughly explored. Here we investigated the utility of microRNAs as potential serum biomarkers for early detection of CRC. Methods: We developed protocols for extracting and quantifying microRNA levels in serum. Serum levels of more than 350 microRNAs were measured using qRT-PCR on samples from 10 healthy controls and 10 CRC patients. Most microRNAs showed consistent levels across different individuals. A subset of microRNAs had significant differences in abundance between the two groups and was studied on a larger cohort of 118 patients and controls. Results: We initially identified a subset of microRNAs that showed significant differential abundance between sera of CRC patients and controls. Measuring the serum levels of 22 microRNAs on a cohort of 118 patients and controls, we showed that levels of circulating microRNAs can be very informative in the identification of CRC. Conclusions: The results demonstrate the potential of the microRNA processing and analysis methods that we developed. Certain microRNAs were found in different amounts in sera of CRC patients compared to healthy controls. Thus, circulating microRNAs represent promising candidates for diagnostic biomarkers in CRC. [Table: see text]

Assessing the potential usefulness of IGF-related peptides and adiponectin for predicting disease risk

ObjectiveInsulin-like growth factors (IGF), their binding proteins and adiponectin have been investigated as potential blood-based biomarkers for a variety of diseases. Before these circulating proteins can be considered as biomarkers, their variation within and between individuals and between published studies must be critically assessed. The purpose of this study was to use the D-value to predict the potential usefulness of IGF-related peptides and adiponectin as biomarkers for the diagnosis of colorectal cancer (CRC). DesignIntra- and inter-individual variation of total IGF-I and -II, IGF binding protein 1 (IGFBP-1), -2 and -3 and adiponectin, was examined in 10 healthy subjects over a 5 week period. This data was analysed in conjunction with previous publications to provide a D-value, which is a theoretical value that identifies the usefulness of the analyte individually and as a panel, as a biomarker for CRC. ResultsA single measurement of total IGF-I and -II, and adiponectin provided a reproducible representation of their circulating concentrations. The D-value for total IGF-II and IGFBP-3 were 0.5 and 0.47, respectively, which corresponded to area under the curve (AUC) values of 64 and 63%. Combining these analytes into a panel only slightly improved the D-value to 0.63 (AUC was 67%). ConclusionsAlthough serum levels of total IGF-I, total IGF-II and IGFBP-3 are stable and reproducible, the D-value calculations indicate that they have limited importance when used as biomarkers of CRC.

Blood-Based Biomarkers of SU11248 Activity and Clinical Outcome in Patients with Metastatic Imatinib-Resistant Gastrointestinal Stromal Tumor

There is an unmet need for noninvasive markers to measure the biological effects of targeted agents, particularly those inhibiting the vascular endothelial growth factor (VEGF) receptor (VEGFR) pathway, and identify patients most likely to benefit from treatment. In this study, we investigated potential blood-based biomarkers for SU11248 (sunitinib malate), a multitargeted tyrosine kinase inhibitor, in patients with metastatic imatinib-refractory gastrointestinal stromal tumors. Patients (n=73) enrolled in a phase I/II trial received SU11248 daily for 14 or 28 days followed by 14 days without treatment per cycle. Clinical benefit was defined as progression-free survival of >6 months. We assessed plasma markers, including VEGF and soluble VEGFR-2 (sVEGFR-2), and two cellular populations bearing VEGF receptors: monocytes and, in a subset of patients, mature circulating endothelial cells (CEC). Compared to patients with progressive disease, patients with clinical benefit had significantly greater increases in CECs (0.52 versus -0.01 CEC/microL/d, P=0.03) and smaller decreases in monocyte levels (47% versus 60%, P=0.007) during cycle 1. VEGF increased by 2.2-fold and sVEGFR-2 decreased 25% during the first 2 weeks of treatment. Neither plasma marker correlated with clinical outcome although a modest inverse correlation was observed between sVEGFR-2 changes and plasma drug levels. Monocytes, VEGF, and sVEGFR-2 all rebounded towards baseline off treatment. Monocytes, VEGF, and sVEGFR-2 were consistently modulated by treatment, suggesting that they may serve as pharmacodynamic markers for SU11248. Changes in CECs and monocytes, but not the plasma markers, differed between the patients with clinical benefit and those with progressive disease. These end points merit further investigation in future trials to determine their utility as markers of SU11248 activity and clinical benefit in gastrointestinal stromal tumors and other tumor types.