Adam Stark

Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach.

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~1011 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 106 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.

Abstract 3607: Differential protein expression and miR content of sorted subsets of circulating microvesicles from cancer patients and healthy controls

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MicroRNAs (miRs) are small non-coding RNAs that are 20 to 25 nucleotides in length and regulate expression of entire families of genes. A major source of circulating miRs in cancer patients is believed to be circulating microvesicles (cMV) within biologic fluids such as blood. The transfer of these modifiers of RNA translation from diseased cells into the bloodstream can have broad impacts on disease detection, progression and/or prognosis. The goal of these studies was to determine whether there are differences in miR composition within different subpopulations of cMV based on surface protein composition. We used flow cytometry to phenotype and sort plasma-derived cMV from 20 individuals (3 breast cancer, 2 lung cancer, 6 prostate cancer, 1 bladder cancer and 6 non-cancer controls). cMV were stained for proteins associated with membranes such as tetraspanins (CD9, 63, 81) Ago2 and/or GW182 using a Beckman Coulter MoFlo XDP. For phenotypic analysis, events were gated on tetraspanin expression to distinguish cMV from nano-sized irrelevant debris, and co-expression of GW182 and Ago2 was determined. Quadrant-based sorting was performed for single- and double-positive events. miR content was determined using conventional Taqman probes with the ABI 7900 thermal cycler on extracted RNA from the sorted cMV. The results of these studies demonstrate that unfractionated cMV were not able to discriminate cancers from non-cancers using miRs-let-7a, -16, -22, -148a or -451 in this population of patients. When sorted tetraspanin+, Ago2+ and/or GW182+ populations of cMV were compared, miR expression was generally 5-fold higher in cancer patients than in healthy controls. These studies demonstrate that cMV can be consistently phenotyped, analyzed and sorted using a flow cytometer and that subpopulations of cMV contain unique miR profiles which can be useful in distinguishing cancer plasma from non-cancer plasma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3607. doi:1538-7445.AM2012-3607

Poly-ligand profiling differentiates trastuzumab-treated breast cancer patients according to their outcomes

Assessing the phenotypic diversity underlying tumour progression requires the identification of variations in the respective molecular interaction networks. Here we report proof-of-concept for a platform called poly-ligand profiling (PLP) that surveys these system states and distinguishes breast cancer patients who did or did not derive benefit from trastuzumab. We perform tissue-SELEX on breast cancer specimens to enrich single-stranded DNA (ssDNA) libraries that preferentially interact with molecular components associated with the two clinical phenotypes. Testing of independent sample sets verifies the ability of PLP to classify trastuzumab-treated patients according to their clinical outcomes with ROC-AUC of 0.78. Standard HER2 testing of the same patients gives a ROC-AUC of 0.47. Kaplan–Meier analysis reveals a median increase in benefit from trastuzumab-containing treatments of 300 days for PLP-positive compared to PLP-negative patients. If prospectively validated, PLP may increase success rates in precision oncology and clinical trials, thus improving both patient care and drug development.Patients’ selection is particularly important in cancer treatment. Here the authors present a proof-of-principle methodology that could be potentially important in assisting therapeutic decisions in the treatment of breast cancer patients.

KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins

The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular function is yet to be understood. Here we show that silencing KIAA0100 by siRNA in the breast cancer cell line MDA-MB-231 significantly reduced the cancer cells’ aggressive behavior, including cell aggregation, reattachment, cell metastasis and invasion. Most importantly, silencing the expression of KIAA0100 particularly sensitized the quiescent cancer cells in suspension culture to anoikis. Immunoprecipitation, mass spectrometry and immunofluorescence analysis revealed that KIAA0100 may play multiple roles in the cancer cells, including stabilizing microtubule structure as a microtubule binding protein, and contributing to MDA-MB-231 cells Anoikis resistance by the interaction with stress protein HSPA1A. Our study also implies that the interaction between KIAA0100 and HSPA1A may be targeted for new drug development to specifically induce anoikis cell death in the cancer cell.

Abstract P4-12-08: Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay

Introduction: Previous attempts to use individual aptamers as diagnostic reagents have failed to consistently achieve performance comparable to antibodies. Here we report a novel systems biology approach using poly-ligand aptamer libraries to identify responders and non-responders to traztuzumab-based regimens in metastatic breast cancer. Methods: To overcome the fundamental limitation of the individual aptamer binding affinities, large libraries (106 species) were created so that potentially thousands of aptamers could bind to each of a multitude of targets related to the whole cellular changes in response to trastuzumab therapy. A set of breast cancer patients, which received trastuzumab mono- or combined therapy for at least 7 months were classified as “Responders” (R); cases with particular regimen discontinued in the period not exceeding 5 months were classified as “Non-Responders”(NR). A library of 2x1012 unique 90-mer ssDNA oligodeoxynucleotides (ssODN) was trained on FFPE tissue of both R and NR patients. Partitioning of aptamer libraries was done by microdissection of the tumor tissue, after incubation of aptamer library with the entire tissue section, to drive selection pressure toward cancer cells. A total of 10 cases of R and NR, 6 Her2+ cases each, were used to train separate aptamer libraries, with 1 positive and 2 counter selection cases per enrichment. Enriched libraries were screened on 20 R and 20 NR cases (11 Her2+ cases each) by adopting modified immunohistochemistry protocol. Each library was used as an independent reagent (similar to an antibody in IHC) across all 40 cases to evaluate the efficacy of the aptamer library to distinguish differences between the R and NR groups. Staining (DAB chromogen) profiles were scored from 0 to 3+ (nuclear and cytoplasmic staining) by a pathologist without any knowledge of the clinical outcomes. Initial validation was done by t-test using raw histological scores. Four libraries showed significant p-values between groups of responders and non-responders, a classification algorithm was constructed and evaluated using area under the receiver-operator characteristic curve (AUC). The datasets of two best-performing libraries were combined into one model using logistic regression to further improved the classifier performance. Results: Of seventeen trained libraries, eight were evaluated and four showed significant correlation to clinical benefit with a minimum accuracy of 75% for each library when evaluated independently. Furthermore, two libraries showed exceptional performance (ROC curve AUC of 0.86 and 0.77). Combination of the profiling data from these two libraries using logistic regression resulted in an AUC of 0.985. A prospective validation of aptamer histochemical theranostic testing has been initiated. Summary: Enriched aptamer libraries appear to distinguish trastuzumab responsiveness in metastatic breast cancer. This technology could be used as an additional technique beyond FISH testing to determine sensitivity to anti-HER2 agents. The demonstrated platform is applicable to virtually any disease where the safe and effective use of corresponding drug is yet to be improved. Citation Format: Spetzler D, Domenyuk V, Santhanam R, Wei X, Stark A, Wang J, Gatalica Z, Miglarese M, Vidal G, Schwartzberg LS. Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-08.

Abstract 2754: A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients

Improved technologies capable of characterizing system-wide changes associated with complex diseases will be required to be able to detect millions of proteins and their isoforms as well as multi-molecular complexes. We present a method for developing aptamer libraries using blood plasma exosomes that provides unprecedented system-wide coverage of native exosomal complexes. To train a naive aptamer library toward cancer samples (positive selection), the library (~1013 biotinylated ssODN species) was incubated with plasma from individual cancer patients and aptamer-bound exosomes were isolated using polymer-based precipitation. Negative selection was performed by contacting the aptamer library with exosomes from donors without breast cancer and recovering unbound aptamers from the supernatant. In all, 12 libraries trained toward 12 individual breast cancer patients were used to probe additional samples. Exosome-bound aptamers were identified and quantified by Next Generation Sequencing (NGS) to build highly accurate signatures for cancer/healthy donor classification. Using these signatures, cancer patients’ binding profiles were easily distinguishable from controls. Interestingly, cancer-trained libraries did not distinguish any of the negative control samples from each other, indicating that the selection pressure for cancer was high and noise due to inherent inter-healthy donor heterogeneity was minimal. Full validation studies are ongoing. Aptamer libraries may ultimately be deployed as a minimally-invasive diagnostic adjunct in breast and other cancers. Citation Format: Valeriy Domenyuk, Symon Levenberg, Adam Stark, Mark Miglarese, David Spetzler. A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2017-2754

Abstract LB-135: Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility

Introduction: Single stranded DNA (ssDNA) libraries consisting of several trillion oligodeoxynucleotides (ODNs) can adopt a nearly infinite number of three-dimensional structures. These structures can potentially bind any biomolecule and can be screened for specificity toward important biomarkers by employing suitable enrichment schemes. Since no prior knowledge on the binding partner is required, massively parallel biomarker identification is possible even on complex matrices like biological fluids and across a wide range of biological conditions. Here we present Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) as a platform for biomarker and target discovery. We employed ADAPT for the molecular profiling of exosome-associated proteins in small volume plasma samples from women with breast cancer and healthy donors. Results: Random ssDNA-libraries of 10 11 unique ODNs were subjected to a number of selection and counter-selection steps on pooled blood plasma of breast cancer and healthy women. Several positive and negative enrichment schemes were employed, and exosome isolation and ODNs library partitioning were performed by ultracentrifugation and/or PEG precipitation. After library enrichment reduction of complexity to 10 6 -10 7 ), ODN libraries were used to probe an independent set of individual plasma samples from women with or without breast cancer. Two thousand differentially-binding aptamers with significant p-values were re-synthesized and combined in equimolar amounts to create a profiling library (L2000). The L2000 library was used to probe plasma samples from 323 individuals (206 from breast cancer patients and 117 from healthy donors) in triplicate. Using Next Generation Sequencing, we quantitated bound ODN from each plasma sample. ANOVA revealed 350 aptamers with significant p-values in distinguishing plasma samples from cancer patients and healthy donors, far in excess of the number of ODNs that would have achieved statistical significance by random sampling of the 2000 ODNs. Generalized linear model showed an AUC in a ROC curve of 0.94 for the training set. Random forest modelling was used to assess classification performance and revealed an AUC of 0.73 (p Conclusions: We have demonstrated the feasibility of aptamer library enrichment directly on blood plasma and have identified a set of 2000 DNA aptamers that distinguish plasma from women with breast cancer from women without breast cancer. This liquid biopsy approach requires only 200 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve ROC AUC values >0.6. Further optimization of the aptamer library and testing on additional samples is ongoing. Upon complete validation, an ADAPT TM - derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Valeriy Domenyuk, Zhenyu Zhong, Jie Wang, Adam Stark, Nianqing Xiao, Mark Miglarese, George Poste, Michael Famulok, Gunter Mayer, David Spetzler. Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility. [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 LB-135.

Abstract P2-01-08: Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue

Introduction: Breast cancer screening relies upon mammography, but for women with dense breast tissue this method is often uninformative. Routine screening identifies suspicious breast lesions in some women, but the pain and risk associated with follow-up biopsies along with the poor accuracy of traditional histopathology urgently call for improved approaches to breast cancer screening. This is especially important for those high-risk patients for whom mammography is of limited value. We describe a non-invasive liquid biopsy method of profiling plasma exosome preps designed to improve the accuracy and safety of breast cancer screening for women with dense breast tissue. Results: We incubated plasma samples (300 microliters per sample) from breast cancer patients (n=60) and a control cohort (n=60) with a high-complexity DNA aptamer library using a modified SELEX scheme, termed “adaptive dynamic artificial poly-ligand targeting (ADAPTTM)”. Differentially bound (cancer vs. non-cancer) aptamers were recovered from precipitated exosomes and were identified by deep sequencing. Two thousand aptamer sequences were resynthesized and used to probe a larger set of 500 plasma samples from a patient cohort (n=206) and a control cohort comprised of self-reported healthy volunteers (n=117) and patients whose biopsies led to a diagnosis of non-cancer (n=177). We employed several statistical models to build a cancer/non-cancer predictor, including a Random Generalized Linear Model (RGLM) and a Random Forest Model (RFM). Both models yielded an equivalent classification performance with areas under the receiver-operator characteristic curve (ROC AUC) of 0.7. Testing the prediction performance by 100 Out-of-Bag permutations or by pre-filtered (read cutoff and estimated sample size) cross-validation (CV) resulted in ROC AUC values of 0.66 and 0.62, respectively. When samples were randomly assigned to groups, the aptamers were no longer able to distinguish the groups (ROC AUC = 0.54), indicating that the underlying information driving the model is truly specific to cancer. Importantly, incorporation of BIRAD results as a clinical covariate did not influence model performance, signifying that predictions by ADAPTTM were independent of breast tissue density. Conclusions: We have identified a set of 2000 DNA aptamers that distinguish women with breast cancer from women without breast cancer. Our liquid biopsy approach requires only 300 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve cross validation ROC AUC values approaching 0.7. The performance of the predictor was not affected by BIRAD scores, supporting its potential utility in difficult cases where imaging is insufficient, such as in women with dense breast tissue. Further optimization of the aptamer library and testing on additional samples should improve performance. Upon complete validation, an ADAPTTM – derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Domenyuk V, Zhong Z, Wang J, Stark A, Chen W, Xiao N, Miglarese MR, Famulok M, Mayer G, Spetzler DB. Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-08.

Abstract 3610: GW182 associates with circulating microvesicles and microRNA in human plasma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL To date, microRNAs (miR) in human plasma have been discovered within circulating microvesicles (cMV), bound to Argonaute 2 and associated with HDL and LDL microvesicles. The protein GW182 shares an association with both multivesicular bodies and the Argonaute family of proteins. GW182 has the capacity to bind all human Argonaute proteins (1-4) and their associated miRs. In the cell, GW182 is associated with the membrane of multivesicular bodies and has the ability to congregate Argonaute-loaded RISC complexes. In addition, GW182 has been observed on the surface of purified exosomes. Here we investigated the relationship of GW182 with Argonaute and cMV in human plasma. A monoclonal antibody directed toward GW182 was used to capture the protein. This isolate also contained Argonaute proteins as determined by Western analysis. The co-precipitation of GW182 and Argonaute suggests that these two proteins retain their functional relationship in plasma. RNA was then isolated from precipitates for miR detection and analysis. The GW182-associated miR profile from human plasma contained individual miRs whose abundance either equaled or surpassed that of their matched Argonaute 2 immuno-precipitated miRs. This implies that GW182 maintains an association with the family of Argonaute proteins and a subset of cMV in human plasma. Phenotypic analysis of GW182-associated human plasma microparticles was performed using flow cytometry with a Beckman Coulter MoFlo XDP. A subpopulation of plasma-derived cMV was observed that co-expressed tetraspanins, GW182 and Argonaute 2. Since tetraspanins are transmembrane proteins highly associated with cMV, these results suggest that both GW182 and Argonaute can associate with cMV in human plasma. Thus, precipitation of GW182 enables the isolation and purification of miRs from human plasma including those bound to Argonaute 1-4 and a subset of cMV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3610. doi:1538-7445.AM2012-3610