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MyriadRBM.com

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Myriad RBM, inc. Myriad RBM is a leader in the field of biomarker discovery services. With more than a decade of experience developing and validating

multiplexed immunoassays, we provide services for the identification and quantification of important protein biomarkers used in drug and

diagnostic development programs. This is accomplished using our proprietary biomarker discovery platform, operated within a CLIA certified lab, which is based on our customized implementation of the Luminex® xMAP technology, a microsphere-based, multiplexed

immunoassay platform.

technology and platform Our multiplexed assays are based on the familiar “capture-sandwich” format using capture antibodies attached to fluorescently encoded microspheres. After capture of antigen from a biological sample, such as serum, the antigen is then detected using specific detection antibodies coupled to a fluorescent probe (Figure 1A). In the Luminex instrument, micropsheres pass individually through a liquid stream that is interrogated by two lasers (Figure 1B). The fluorescent signals generated from the first laser emanate from within the microsphere providing a unique fluorescent signature. The fluorescent signal generated by the detection antibodies coupled probe are also captured at a distinct wavelength. Using a classic calibration curve, that amount of fluorescence is proportional to the analyte concentration in the sample.

Myriad RBM’s expertise in multiplexed immunoassay development and validation, combined with laboratory automation and a focus on Quality Assurance, create our robust Multi-Analyte Profile (MAP) platform.

Automation of all liquid handling steps

Proprietary blockers to handle most matrix effects

Validation guided by clinical laboratory standards

Neurological disorders affect millions of people annually at a tremendous social and economic cost. As many neurological disorders are chronic conditions resulting in long-term disability or suffering, early and accurate diagnosis is of the utmost importance. The development of clinical diagnostics for many neurodegenerative and neuropsychiatric conditions has been challenging since the molecular bases of these diseases are ill-defined or unknown. Definitive

diagnosis of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, has traditionally relied upon post-mortem neuropathologic tissue analysis, while the accuracy of diagnosis based on established clinical criteria has been estimated at only 70-80%1. Diagnosis of neuropsychiatric disorders has been particularly problematic, as it requires assessment of complex behavioral phenotypes and depends heavily on patient self-reporting. In addition to hindering treatment for affected patients, these limitations have drastically slowed progress towards validating animal models of disease and discovery of novel targets for therapeutic intervention. Recent advances in the identification of biomarkers for neurological disorders are providing researchers and clinicians with powerful new tools to improve

diagnosis and therapeutics.

Our biomarker panels have been featured in over 70 publications covering a wide range of neurodegenerative, neuropsychiatric, and neuromuscular disorders. In a number of these studies, our multiplexed immunoassay panels are the central component of a targeted proteomics approach designed to identify novel biomarkers associated with disease. Myriad RBM has worked with several prominent Alzheimer’s disease research organizations including the Alzheimer’s Disease Neuroimaging Initiative, the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, and the Texas Alzheimer’s Research & Care Consortium (TARCC), in addition to major academic institutions such as the University of Pennsylvania, Cambridge University, and

Figure 1: aURoc analyses ROC analyses assessed the ability of the traditional CSF biomarkers (blue) and 37 Myriad RBM analytes (red) to discriminate patients diagnosed with dementia from controls. Combining the best-performing Myriad RBM analytes with the tau/Aβ42 ratio, improved the area under the curve in many cases (green). Craig-Schapiro et al. 2011

Washington University. The research and collaborations with these groups, among others, underscores our contributions to the field of neurodegenerative biomarkers.

The first example of these biomarker studies identified 37 analytes that were altered in the cerebrospinal fluid (CSF) collected from patients clinically diagnosed with early stage Alzheimer’s disease2. The authors report that using a subset of these markers in combination with the highest performing, previously established Alzheimer’s disease biomarker—the tau/amyloid-β42 ratio—significantly improved diagnostic specificity. This result was expressed as an increase in the area under the Receiver Operating Characteristic (AUROC) curves calculated for a number of these analytes in combination with the tau/amyloid-β42 ratio as compared to tau/amyloid-β42 alone (Figure 1). Other studies have also been successful at identifying novel CSF biomarkers that reflect the rate and severity of cognitive decline (Figure 2)3. These findings not only have the potential to improve diagnosis and prognosis in the clinic, but may also point to novel targets for therapeutic intervention.

While CSF markers of neurodegenerative diseases have been useful as part of clinical trial enrollment criteria, blood-based biomarkers may be even more valuable to the clinic. A study focusing on plasma biomarkers by Doecke et al., identified a panel that could distinguish between cognitively normal subjects and Alzheimer’s disease patients with a high degree of sensitivity and specificity4. The authors, using the Human DiscoveryMAP®, combined clinical assessments with blood-based analysis of samples from the AIBL study.

Following multiple statistical analyses, 18 biomarkers were selected in a disease prediction model that achieved 85% sensitivity and specificity when combined with demographic variables. A reduced panel consisting of eight biomarkers had only slightly decreased accuracy of disease prediction (Figure 3).

Figure 2: novel biomarkers reflect the rate of cognitive decline in Mci. Partial residual plots of MAP analytes versus rates of subsequent cognitive decline in patients with mild cognitive impairment. X-axis represents “fold change” of protein. Linear fit and 95% confidence interval for fit are shown for each graph. MMSE = mini-mental status examination . Hu et al. 2010

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Because blood-based analysis is simpler and less invasive than CSF, continued identification and validation of serum and plasma biomarkers for disease is of critical importance in the development of clinical diagnostic tools.

The Myriad RBM biomarker services have also been successfully applied to the field of neuropsychiatry. Domenici et al., used a targeted proteomic discovery strategy in an attempt to identify clusters of biomarkers that correlate with depression and schizophrenia5. Until recently, there has been limited success in achieving diagnostic specificity using any molecular approach across the wide range of patients and presentations of these disorders. To address this problem, the authors used Myriad RBM’s HumanMAP® panel to measure biomarkers in plasma samples collected from patients diagnosed with major depressive disorder or schizophrenia. Samples were evaluated for levels of 79 proteins, including cytokines, chemokines, neurotrophins and hormones. This analysis yielded several markers that appear to be modulated specifically in patients with either depression or schizophrenia as compared to controls. Figure 4 shows the relative difference in the levels of the analytes exhibiting the highest statistically significant changes. The authors then applied multivariate analysis to select a panel of the 10 biomarkers that most significantly improved discrimination of cases from controls, constituting a novel set of candidate biomarkers of schizophrenia and depression. Several similar studies by Schwarz et al. have also been successful in identifying and distinguishing biological signatures for schizophrenia and other neuropsychiatric conditions such as major depression and bipolar disorder6-8.

Finally, recent work searching for biomarkers of the neuromuscular disease Spinal Muscular Atrophy (SMA) demonstrates the versatility of Myriad RBM’s multiplex immunoassay approach for neurological disorders. Researchers from the SMA Foundation used a combination of the Discovery- and OncologyMAP® panels to measure 267 biomarkers in plasma samples from confirmed SMA patients and matched controls. This analysis identified 72 novel biomarker candidates that correlate with clinical measures of disease severity.

Figure 3: Receiver operating characteristic curves for comparison of biomarker set performance. Data sets used (1) age, sex and presence of apolipoprotein (APOE) ε4 allele (red line); (2) age, sex, APOE ε4 allele and 18 biomarker set A (blue line); and (3) age, sex, APOE ε4 allele and reduced 8 biomarker set B (black line). Doecke et al. 2012

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Figure 5: Differential early response to treatment of leptin, insulin and c-peptide between the short- and long-term relapse groups. The figure shows the difference in the change of levels during treatment in serum from patients in the two different relapse groups. Schwarz et al. 2012

Figure 4: plasma protein markers with the highest significance in schizophrenia and Major Depressive Disorder. Box plots of individual analytes with high significance. The bracketed values in the titles refer to the data transformation. The white line corresponds to the median and the full box represents the central 50%. Domenici et al. 2010

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Following this initial research, a collaborative effort was undertaken with Myriad RBM to develop immunoassays for 8 other potential biomarkers. The top 25 protein targets were then used to generate predicted scores of disease severity that correlated highly with actual patient scores (Figure 6)9. As a result of this collaboration, Myriad RBM has now developed a custom SMA MAP consisting of 27 plasma proteins designed for use in evaluating the severity of SMA and assessing drug efficacy in clinical trials.

Together these studies highlight the unique advantages of using Myriad RBM’s powerful multiplexed immunoassays to quantify a large number of analytes within a single study. The continued identification and characterization of novel biomarker candidates for neurodegenerative, neuropsychiatric, and neuromuscular diseases promises to make significant contributions towards early detection and the development of therapeutic intervention. Myriad RBM strives to be a leading provider of biomarker assays for neurological disorders.

Figure 6: preliminary predictive power of a subset of sMa plasma protein analytes.A group of 25 MAP plasma protein analytes were used to generate predicted Modified Hammersmith Functional Motor Scale (MHFMS) scores for the samples from SMA patients. The subpanel analytes were able to account for up to 95% of the variance in the MHFMS scores. Kobayashi et al. 2011

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1. Gearing M, Mirra SS, Hedreen JC, Sumi SM, Hansen LA, Heyman A. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part X. Neuropathology confirmation of the clinical diagnosis of Alzheimer’s disease. Neurology. 995 Mar;45(3 Pt 1):461-6.

2. Craig-Schapiro R, Kuhn M, Xiong C, Pickering EH, Liu J, Misko TP, Perrin RJ, Bales KR, Soares H, Fagan AM, Holtzman DM. Multiplexed immunoassay panel identifies novel CSF biomarkers for Alzheimer’s disease diagnosis and prognosis. PLoS One. 2011 Apr 19;6(4):e18850.

3. Hu WT, Chen-Plotkin A, Arnold SE, Grossman M, Clark CM, Shaw LM, Pickering E, Kuhn M, Chen Y, McCluskey L, Elman L, Karlawish J, Hurtig HI, Siderowf A, Lee VM, Soares H, Trojanowski JQ. Novel CSF biomarkers for Alzheimer’s disease and mild cognitive impairment. Acta Neuropathology. 2010 June; 119(6): 669-678.

4. Doecke JD, Laws SM, Faux NG, Wilson W, Burnham SC, Lam CP, Mondal A, Bedo J, Bush AI, Brown B, De Ruyck K, Ellis KA, Fowler C, Gupta VB, Head R, Macaulay SL, Pertile K, Rowe CC, Rembach A, Rodrigues M, Rumble R, Szoeke C, Taddei K, Taddei T, Trounson B, Ames D, Masters CL, Martins RN; Alzheimer’s Disease Neuroimaging Initiative; Australian Imaging Biomarker and Lifestyle Research Group. Blood-based protein biomarkers for diagnosis of Alzheimer Disease. Arch Neurology. 2012; 69(10); 1318-25.

5. Domenici, E, Wille DR, Tozzi F, Prokopenko I, Miller S, McKeown A, Brittain C, Rujescu D, Giegling I, Turck CW, Holsboer F, Bullmore ET, Lefkos M, Merlo-Pich E, Alexander RC, Muglia P. Plasma protein biomarkers for depression and schizophrenia by multi analyte profiling of case-control collections. PLoS One. 2010; 5(2): e9166.

6. Schwarz E, Guest PC, Rahmoune H, Harris LW, Wang L, Leweke FM, Rothermundt M, Bogerts B, Koethe D, Kranaster L, Ohrmann P, Suslow T, McAllister G, Spain M, Barnes A, van Beveren NJ, Baron-Cohen S, Steiner J, Torrey FE, Yolken RH, Bahn S. Identification of a biological signature for schizophrenia in serum. Mol Psychiatry. 2012 May;17(5):494-502.

7. Schwarz E, Guest PC, Rahmoune H, Martins-de-Souza D, Niebuhr DW, Weber NS, Cowan DN, Yolken RH, Spain M, Barnes A, Bahn S. Identification of a blood-based biological signature in subjects with psychiatric disorders. World J Biol Psychiatry. 2012 Dec;13(8):627-32.

8. Schwarz E, Guest PC, Steiner J, Bogerts B, Bahn S. Identification of blood-based molecular signatures for prediction of response and relapse in schizophrenia patients. Translational Psychiatry (2012) 2, e82.

9. Kobyashi DT, Chung B, Stephen L, Ballarad KL, Dewey R, Shi J, Walker M, McCarthy K, Paushkin S, Joyce C, Mapes J, Chen KS. Development of a multiplex immunoassay biomarker panel for Spinal Muscular Atrophy. 15th International SMA Research Group Meeting. Orlando, FL. July 2011.

Myriad RBM, Inc.3300 Duval Road · Austin, Texas 78759

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