Development of a blood-based extracellular vesicle classifier for detection of early-stage pancreatic ductal adenocarcinoma→

Hinestrosa, J.P., Sears, R.C., Dhani, H. et al. https://doi.org/10.1038/s43856-023-00351-4

Abstract

Background

Pancreatic ductal adenocarcinoma (PDAC) has an overall 5-year survival rate of just 12.5% and thus is among the leading causes of cancer deaths. When detected at early stages, PDAC survival rates improve substantially. Testing high-risk patients can increase early-stage cancer detection; however, currently available liquid biopsy approaches lack high sensitivity and may not be easily accessible.

Methods

Extracellular vesicles (EVs) were isolated from blood plasma that was collected from a training set of 650 patients (105 PDAC stages I and II, 545 controls). EV proteins were analyzed using a machine learning approach to determine which were the most informative to develop a classifier for early-stage PDAC. The classifier was tested on a validation cohort of 113 patients (30 PDAC stages I and II, 83 controls).

Results

The training set demonstrates an AUC of 0.971 (95% CI = 0.953–0.986) with 93.3% sensitivity (95% CI: 86.9–96.7) at 91.0% specificity (95% CI: 88.3–93.1). The trained classifier is validated using an independent cohort (30 stage I and II cases, 83 controls) and achieves a sensitivity of 90.0% and a specificity of 92.8%.

Conclusions

Liquid biopsy using EVs may provide unique or complementary information that improves early PDAC and other cancer detection. EV protein determinations herein demonstrate that the AC Electrokinetics (ACE) method of EV enrichment provides early-stage detection of cancer distinct from normal or pancreatitis controls.

Case Report: Early detection of pancreatic pre-cancer lesion in multimodal approach with exosome liquid biopsy→

In this case study, the authors utilized the ExoVita Pancreas assay for a patient undergoing acute pancreatitis. The ExoVita Pancreas assay returned a high likelyhood result. Combined with other results, the patient underwent a Whipple procedure, which revealed the presence of a high-grade lesion. 

Harmeet Dhani, Juan Pablo Hinestrosa, Jesus Izaguirre-Carbonell, Heath I Balcer, Razelle Kurzrock, and Paul R Billings doi: 10.3389/fonc.2023.1170513

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ABSTRACT

Background: The detection of pancreatic ductal adenocarcinoma (PDAC) lesions at pre-cancerous or early-stages is critical to improving patient survival. We have developed a liquid biopsy test (ExoVita®) based on the measurement of protein biomarkers in cancer-derived exosomes. The high sensitivity and specificity of the test for early-stage PDAC has the potential to improve a patient’s diagnostic journey in hopes to impact patient outcomes.

Methods: Exosome isolation was performed using alternating current electric (ACE) field applied to the patient plasma sample. Following a wash to eliminate unbound particles, the exosomes were eluted from the cartridge. A downstream multiplex immunoassay was performed to measure proteins of interest on the exosomes, and a proprietary algorithm provided a score for probability of PDAC.

Results: We describe the case of a 60-year-old healthy non-Hispanic white male with acute pancreatitis who underwent numerous invasive diagnostic procedures that failed to detect radiographic evidence of pancreatic lesions. Following the results of our exosome-based liquid biopsy test showing "High Likelihood of PDAC", in addition to KRAS and TP53 mutations, the patient decided to undergo a robotic pancreaticoduodenectomy (Whipple) procedure. Surgical pathology confirmed the diagnosis of high-grade intraductal papillary mucinous neoplasm (IPMN), which was consistent with the results of our ExoVita® test. The patient’s post-operative course was unremarkable. At five-month follow-up, the patient continued to recover well without complications, in addition to a repeat ExoVita test which demonstrated “Low Likelihood of PDAC”.

Conclusion: This case report highlights how a novel liquid biopsy diagnostic test based on the detection of exosome protein biomarkers allowed early diagnosis of a high-grade precancerous lesion for PDAC and improved patient outcome.

Early-stage multi-cancer detection using an extracellular vesicle protein-based blood test→

In this case-controlled pilot study, the authors explored a cohort of 139 pathologically staged patients with stage 1 and stage 2 pancreatic, ovarian, and bladder cancers. Biological Dynamics’ Verita™ proprietary platform detected 96 percent of stage 1 pancreatic cancers and three-quarters of stage 1 ovarian cancers using isolated exosomes and AI-enabled protein marker analysis. 

Juan Pablo Hinestrosa, Razelle Kurzrock, Jean M. Lewis, Nicholas J. Schork, Gregor Schroeder, Ashish M. Kamat, Andrew M. Lowy, Ramez N. Eskander, Orlando Perrera, David Searson, Kiarash Rastegar, Jake R. Hughes, Victor Ortiz, Iryna Clark, Heath I. Balcer, Larry Arakelyan, Robert Turner, Paul R. Billings, Mark J. Adler, Scott M. Lippman, Rajaram Krishnan. doi.org/10.1038/s43856-022-00088-6

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ABSTRACT

Background: Detecting cancer at early stages significantly increases patient survival rates. Because lethal solid tumors often produce few symptoms before progressing to advanced, metastatic disease, diagnosis frequently occurs when surgical resection is no longer curative. One promising approach to detect early-stage, curable cancers uses biomarkers present in circulating extracellular vesicles (EVs). To explore the feasibility of this approach, we developed an EV-based blood biomarker classifier from EV protein profiles to detect stages I and II pancreatic, ovarian, and bladder cancer.

Methods: Utilizing an alternating current electrokinetics (ACE) platform to purify EVs from plasma, we use multi-marker EV-protein measurements to develop a machine learning algorithm that can discriminate cancer cases from controls. The ACE isolation method requires small sample volumes, and the streamlined process permits integration into high-throughput workflows.

Results: In this case-control pilot study, comparison of 139 pathologically confirmed stage I and II cancer cases representing pancreatic, ovarian, or bladder patients against 184 control subjects yields an area under the curve (AUC) of 0.95 (95% CI: 0.92 to 0.97), with sensitivity of 71.2% (95% CI: 63.2 to 78.1) at 99.5% (97.0 to 99.9) specificity. Sensitivity is similar at both early stages [stage I: 70.5% (60.2 to 79.0) and stage II: 72.5% (59.1 to 82.9)]. Detection of stage I cancer reaches 95.5% in pancreatic, 74.4% in ovarian (73.1% in Stage IA), and 43.8% in bladder cancer.

Conclusion: This work demonstrates that an EV-based, multi-cancer test has potential clinical value for early cancer detection and warrants future expanded studies involving prospective cohorts with multi-year follow-up.

Plasma Biomarker for Post-concussive Syndrome: A Pilot Study Using an Alternating Current Electro-Kinetic Platform→

In this paper, the authors used Biological Dynamics technology to study traumatic brain injury. The work was done in collaboration with Michael Heller's lab at UCSD/OHSU and Dr. Clark Chen, MD, PhD, Head of Neurosurgery at the University of Minnesota Medical School, who would like to continue the work and collaboration due to the exciting results.

Lewis JM, Dhawan S, Obirieze AC, Sarno B, Akers J, Heller MJ, Chen CC. Front. Neurol. 2020 July 14 doi.org/10.3389/fneur.2020.00685

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ABSTRACT

Background: Technology platforms that afford biomarker discovery in patients suffering from traumatic brain injury (TBI) remain an unmet medical need. Here, we describe an observational pilot study to explore the utility of an alternating current electrokinetic (ACE) microchip device in this context.

Methods: Blood samples were collected from participating subjects with and without minor TBI. Plasma levels of glial fibrillary acidic protein (GFAP), Tau, ubiquitin C-terminal hydrolase L1 (UCH-L1), and cell-free DNA (cfDNA) were determined in subjects with and without minor TBI using ACE microchip device followed by on-chip immunofluorescent analysis. Post-concussive symptoms were assessed using the Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) at one-month follow-up.

Results: Highest levels of GFAP, UCH-L1, and Tau were seen in two minor TBI subjects with abnormality on head computed tomography (CT). In patients without abnormal head CT, Tau and GFAP levels discriminated between plasma from minor-TBI and non-TBI patients, with sensitivity and specificity of 64–72 and 50%, respectively. Plasma GFAP, UCH-L1, and Tau strongly correlated with the cumulative RPCSQ score. Plasma UCH-L1 and GFAP exhibited highest correlation to sensitivity to noise and light (r = 0.96 and 0.91, respectively, p < 0.001). Plasma UCH-L1 and Tau showed highest correlation with headache (r = 0.74 and 0.78, respectively, p < 0.001), sleep disturbance (r = 0.69 and 0.84, respectively, p < 0.001), and cognitive symptoms, including forgetfulness (r = 0.76 and 0.74, respectively, p < 0.001), poor concentration (r = 0.68 and 0.76, respectively, p < 0.001), and time required for information processing (r = 0.77 and 0.81, respectively, p < 0.001). cfDNA exhibited a strong correlation with depression (r = 0.79, p < 0.01) and dizziness (r = 0.69, p < 0.01). While cfDNA demonstrated positive correlation with dizziness and depression (r = 0.69 and 0.79, respectively, p < 0.001), no significant correlation was observed between cumulative RPCSQ and cfDNA (r = 0.07, p = 0.81).

Conclusion: We provide proof-of-principle results supporting the utility of ACE microchip for plasma biomarker analysis in patients with minor TBI.

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A Pilot Proof-Of-Principle Analysis Demonstrating Dielectrophoresis (DEP) as a Glioblastoma Biomarker Platform→

In this article, the authors used Biological Dynamic ACE technology to screen plasma samples from brain cancer patients for the presence of both the exosome-associated proteins Tau and GFAP.

Lewis J, Alattar AA, Akers J, Carter BS, Heller MJ, Chen CC. Nature Sci Rep. 2019 Jul 16. doi: 10.1038/s41598-019-46311-8.

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ABSTRACT

Extracellular vesicles (EVs) are small, membrane-bound particles released by all cells that have emerged as an attractive biomarker platform. We study the utility of a dielectrophoretic (DEP) micro-chip device for isolation and characterization of EVs derived from plasma specimens from patients with brain tumors. EVs were isolated by DEP chip and subjected to on-chip immunofluorescence (IF) staining to determine the concentration of glial fibrillary acidic protein (GFAP) and Tau. EVs were analyzed from the plasma samples isolated from independent patient cohorts. Glioblastoma cell lines secrete EVs enriched for GFAP and Tau. These EVs can be efficiently isolated using the DEP platform. Application of DEP to clinical plasma samples afforded discrimination of plasma derived from brain tumor patients relative to those derived from patients without history of brain cancer. Sixty-five percent (11/17) of brain tumor patients showed higher EV-GFAP than the maximum observed in controls. Ninety-four percent (16/17) of tumor patients showed higher EV-Tau than the maximum observed in controls. These discrimination thresholds were applied to plasma isolated from a second, independent cohort of 15 glioblastoma patients and 8 controls. For EV-GFAP, we observed 93% sensitivity, 38% specificity, 74% PPV, 75% NPV, and AUC of 0.65; for EV-Tau, we found 67% sensitivity, 75% specificity 83% PPV, 55% NPV, and AUC of 0.71 for glioblastoma diagnosis. This proof-of-principle study provides support for DEP-IF of plasma EVs for diagnosis of glioblastoma.

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Integrated Analysis of Exosomal Protein Biomarkers on Alternating Current Electrokinetic Chips Enables Rapid Detection of Pancreatic Cancer in Patient Blood→

In this article, the authors used Biological Dynamic ACE technology to screen whole blood and plasma samples from pancreatic cancer patients for the presence of both the exosome-associated protein CD63 and glypican-1 (GPC-1).

 Lewis JM, Vyas AD, Qiu Y, Messer KS, White R, Heller MJ. ACS Nano. 2018 Mar 28. doi: 10.1021/acsnano.7b08199.

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ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult. Here, we present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless "sample-to-answer" liquid biopsy screening and improve early stage cancer diagnostics.

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Dielectrophoretic recovery of DNA from plasma for the identification of chronic lymphocytic leukemia point mutations→

In this article,  the authors demonstrate feasibility of detecting cancer mutations in eluted cell-free DNA that was isolated from the plasma of chronic lymphocytic leukemia (CLL) patients using Biological Dynamics platform.

Sareh Manouchehri, Stuart Ibsen, Jennifer Wright, Laura Rassenti, Emanuela M Ghia, George F Widhopf, Thomas J Kipp & Michael J Heller

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ABSTRACT

Aim: Circulating cell free (ccf) DNA contains information about mutations affecting chronic lymphocytic leukemia (CLL). The complexity of isolating DNA from plasma inhibits the development of point-of-care diagnostics. Here, we introduce an electrokinetic method that enables rapid recovery of DNA from plasma. Materials & methods: ccf-DNA was isolated from 25 µl of CLL plasma using dielectrophoresis. The DNA was used for PCR amplification, sequencing and analysis. Results: The ccf-DNA collected from plasma of 5 CLL patients revealed identical mutations to those previously identified by extracting DNA from CLL cells from the same patients. Conclusion: Rapid dielectrophoresis isolation of ccf-DNA directly from plasma provides sufficient amounts of DNA to use for identification of point mutations in genes associated with CLL progression.

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids→

In this review, the authors discuss emerging cell-free nanoparticles isolation technologies that demonstrate the potential for being translated into point-of-care application. The simplicity of sample preparation used in Biological Dynamics’ AC dielectrophoretic separation enables future applications of molecular diagnostics at point-of-care.

Lewis JM, Heineck DP, Heller MJ. Expert Rev Mol Diagn. 2015 Jul 18. doi: 10.1586/14737159.2015.1069709. 

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Abstract

As we move into the era of individualized cancer treatment, the need for more sophisticated cancer diagnostics has emerged. Cell-free (cf) nucleic acids (cf-DNA, cf-RNA) and other cellular nanoparticulates are now considered important and selective biomarkers. There is great hope that blood-borne cf-nucleic acids can be used for ‘liquid biopsies,’ replacing more invasive tissue biopsies to analyze cancer mutations and monitor therapy. Conventional techniques for cf-nucleic acid biomarker isolation from blood are generally time-consuming, complicated and expensive. They require relatively large blood samples, which must be processed to serum or plasma before isolation of biomarkers can proceed. Such cumbersome  sample preparation also limits the widespread use of powerful, downstream genomic analyses, including PCR and DNA sequencing. These limitations also preclude rapid, point-of-care diagnostic applications. Thus, new technologies that allow rapid isolation of biomarkers directly from blood will permit seamless sample-to-answer solutions that enable next-generation point-of-care molecular diagnostics.

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