Name: Biomarkers from Discovery to Clinical Diagnostics Applications of Single-Molecule Array Technology
Uploaded: 2015-02-17T00:00:00
Duration: 39 min 21 s
Description: Speaking at Diagnostics & Biomarkers 2015

SELECTBIO

Diagnostics & Biomarkers 2015

Speaking at Diagnostics & Biomarkers 2015

Biomarkers from Discovery to Clinical Diagnostics Applications of Single-Molecule Array Technology

Targeted mass spectrometry, called MRM or SRM, allows for the rapid quantification of hundreds of proteins in a single assay.  This tool is very specific, highly quantitative and requires very little sample (e.g. < 5 uL plasma).  Recently, we evaluated the performance of 190 candidate protein biomarkers to discriminate between patients with benign or malignant lung nodules.  The candidate markers came from the substantial literature on cancer-associated proteins as well as from mass spectrometry studies, conducted in-house to identify proteins up-regulated either on the surface of tumors or in the secretome of tumors, as compared to adjacent normal lung tissue.  Plasma samples were obtained retrospectively for analysis from those with either benign or malignant nodules.  A 13 protein panel was identified with a high negative predictive value, able to remove approximately half of those with benign nodules from further consideration.  The test has been commercialized, using MRM mass spectrometry, by Integrated Diagnostics.

Creating a Clinical Diagnostic Test from Discovery Through Launch with Mass Spectrometry

Biomarkers offer potential to accelerate drug development, present a reliable diagnostic readout of human disease or be predictive of therapeutic outcome during clinical trials. However, they can fail to deliver their promise not because of the underlying science but due to poor choice of assay and lack of validation. The fit-for-purpose (F-P) approach provides a robust solution to biomarker method validation. The approach is conducted in multi-stages but at its core it is based on defining the purpose of the biomarker, choosing an appropriate assay and then demonstrating by experimentation that the assay does indeed achieve its purpose. Comparing technical parameters in isolation of purpose – conventional bioanalytical method validation –does not guarantee that the assays will be successful in the clinical. Technical parameters, although essential, tell you that you are doing the test correctly, but not that you are using the correct test.  F-P takes account of the position of the biomarker in the spectrum between research tool and diagnostic test, the nature of the analytical technique from qualitative assay to absolute quantitation and all relevant regulatory requirements. To illustrate these principles, a worked example is presented of circulating tumour cell enumeration as a prognostic/predictive biomarker in cancer research.

Fit-for-Purpose Biomarker Assay Validation - From Research Tool to Diagnostic Test

Present diagnostic procedures and therapeutic strategies solid tumours rely on the detection and targeting of specific marker molecules to classify the tumour and to design an efficient therapy. Examples for such markers/targets, in the case of breast cancer, are the oestrogen receptor (ER), progesterone receptor (PgR), and the Erb2/Her2 molecule, a receptor tyrosine protein kinase that is overexpressed in a subset of breast cancers. There are number of treatment options for tumours that express the ER, PgR or Her2 receptors, however up to 20 percent of the newly diagnosed cases belong to a subset, which is negative for all three of the receptor molecules. This presentation will discuss applications of high-resolution hybrid mass spectrometry for quantitative genome-scale studies of protein abundance and post-translational modifications. The current state-of-the-art technology for large-scale proteome analysis of individual solid tumours is discussed. Case studies from experiments with triple-negative breast and colorectal tumours illustrating the capability of the proteome-based strategies are presented, as well as mechanistic investigations in cultured cells that have been informed by tumour proteomics and focus on specific phosphorylation hotspots and protein interactions of the tumour suppressor Scribble. 

Genome-Scale Protein Expression Analysis ofIndividual Solid Tumors

Imaging has been at the heart of Medicine for many decades. However, its influence is changing as the need increases not only for diagnosis of disease, but also for quantification of disease extent, identification of suitable candidates for (often expensive) treatment and for testing of both treatment response or assessment of potential side effects for new treatments. The modalities are largely driven by large data generation, either using CT, MRI or PET methods. This puts an emphasis on data handling, development of software tools and particularly (with the advent of multicentre trials) the requirement for rigorous quality assurance of all participating sites. Not all imaging is currently ready for use as a biomarkers, yet many man hours are being invested into creating the tools required to further both more advanced treatments as well as for more personalised healthcare delivery. This presentation will discuss the potential role of imaging-based biomarkers, the requirements and some examples of how this can be applied towards clinical trials.


Imaging Biomarkers - Future or Reality

"Metabolomics has been successfully applied to identify and validate biomarkers for congestive heart failure. 
Diagnosis and risk stratification of heart failure (HF) is challenging. Yet, the clinical utility of established biomarkers is limited in particular by their lack of sensitivity for the early detection of HF. Metabolomics of plasma samples was performed by both untargeted and targeted GC-MS and LC-MS/MS. In the initial single center identification phase metabolites were nominated for multimarker panels and their performance was verified in an independent validation cohort from three clinical centers (in total >800 HF patients and >330 healthy controls). The metabolite multimarkers not only showed a similar diagnostic performance compared to NT-proBNP, but also demonstrated a significant added diagnostic value when combining with NT-proBNP for several HF subgroups. We conclude that metabolomic biomarkers may be useful in the early detection of HF. 

BENEFITS
- Learn how metabolite data enables discovery of new biomarker for diagnostics
- Outline how to leverage metabolomics results to validated biomarkers
- Understand the current limitations of established heart failure diagnosis and how to overcome these
- Comprehend how established clinical markers (e.g. NT proBNP) can be combined with the new metabolic signature applying cutting-edge biostatistics for an improved diagnosis

New Metabolomics Biomarker for Heart Failure

We have developed an approach to study intratumoral heterogeneity, and to uncover the molecular basis of aggressive tumor cell phenotypes.  Some tumor cell subsets in an individual tumor are more competent than others in forming tumors, or to metastasize, or to resist therapy.  Collectively, these properties have been attributed to 'cancer stem cells' or CSC.  We have developed novel processes and methods to extract CSC from clinical biospecimens, primarily culture them in vitro, and live-sort candidate CSC-subsets in order to jointly validate distinctive phenotypes and directly associate these properties with molecular signatures using OMICs.  This strategy will enable to both discover novel 'behavior-specific' therapeutic targets, as well as rational combinatorial approaches to treat cancer.

Novel Vertically-Integrated Platform for Discovery and Characterization of Lung Cancer Stem Cells

Molecular biomarkers, a cornerstone of precision oncology, are critical for management of a number of cancers. Testing practices are varied and intensely debated, impacting diagnostic quality and affecting pathologists, oncologists and patients. However, little is known about testing approaches used in the US clinical practice. This presentation will outline key issues in oncology biomarker testing and describe findings from a study of practices used by National Cancer Institute (NCI) designated cancer centers, the leading U.S. oncology institutions, for testing key biomarkers in three solid tumors - breast, gastroesophageal and non-small cell lung cancers. Taken together, these three cancers represent an epitome of molecular profiling practices. We will discuss these practices as the groundwork for a U.S. practice baseline, in the context of current guidelines, testing challenges and implications for community-based oncology. Finally, we will outline emerging trends in biomarker testing, namely next-generation tumour sequencing, and related adoption and reimbursement implications. 

Oncology Biomarker Testing Current US Practice Baseline and Emerging Trends

Molecular imaging is increasingly being used to support decision making in early-stage drug development, particularly in the CNS. Providing invaluable information on the so-called ‘three pillars of drug survival’, namely tissue delivery, target engagement and pharmacological effect, it can be seen as a kind of precision pharmacology However, a lack of appreciation of when and how to use imaging and a lack of suitable tools means imaging is often an afterthought and employed in a post mortem of drug failure. Timely development of imaging biomarkers will ensure the full translational value of this technology can be realised This talk will illustrate, with case studies, how appropriate and timely use of imaging biomarkers can significantly reduce the risk, cost and time of drug development. Key benefits:


Parallel Tracking i-biomarkerT Development with Drug Development for Maximum Benefit

Despite extensive research effort there are no accepted clinical biomarkers that predict for response to systemic combination chemotherapy in advanced CRC. Current assessment is based on computed tomography scans, approximately 6-8 weeks after treatment. Earlier molecular predictors of response would be highly beneficial for both patients and clinicians. We have investigated whether useful protein biomarkers could be found in patient plasma, through the use of an advanced MS technique known as SRM-MS. Here, we will discuss the discovery-through-validation pipeline of identifying and validating clinically-relevant biomarkers that can be rapidly translated into the clinic. Briefly, we examined whether the type of blood collection tube would impact on SRM measurement of plasma proteins, in addition to examining the stability of the selected plasma proteome over a 19 week time-course. We then applied SRM to monitor 31 plasma proteins in a trial cohort of advanced CRC patients receiving FOLFOX chemotherapy. Our results indicated that the baseline levels of plasma vitronectin were associated with chemotherapy response, which was successfully validated in a larger patient cohort using SRM and ELISA (n=29 patients). These findings pave the way for further testing in larger cohorts and could lead to more cost-effective and rational uses of systemic chemotherapy.

Plasma Vitronectin Predicts Response to FOLFOX Chemotherapy in Metastatic Colorectal Cancer

Discernment of rare alleles in circulating free DNA (cfDNA) may be a useful method for detecting the presence of new or recurring cancer long before it becomes symptomatic and, perhaps, when it is more treatable. Unfortunately the false-positive rate of current methods reduce specificity to an unacceptable level at allele frequencies of less than ~0.5%.
I will discuss both bench and informatic methods used at Washington University to increase specificity in the apprehension of rare cfDNA alleles.

Tissue and cfDNA Rare Alleles How Low Can We Go

This presentation will cover how bottlenecks in the validation of candidate tumour progression-related biomarkers can be overcome. In particular, the use of tissue microarray technology and digital pathology will be highlighted. In addition, a novel mechanistically anchored biomarker panel that outcompetes current prognostic classifers in breast cancer will be introduced for the first time.

Biomarkers from Discovery to Clinical Diagnostics Applications of Single-Molecule Array Technology

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February 17, 2015

Diagnostics & Biomarkers 2015

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