CNS Cancer-Biomarker

By-Lovnish THAKURASU2014010100099Integrated Biotech4th sem

Introduction

• Central nervous system (CNS) lymphoma is a disease in which malignant (cancer) cells form in the lymph tissue of the brain and/or spinal cord

• Fatal Condition• x-rays, MRI and computed tomography (CT)

scans• Lack technique for early diagnosis

Protein Biomarker Identification in the CSF of PatientsWith

CNS Lymphoma

BACKGROUND

• Brain biopsy is often the testBrain biopsy is, however, associated with 1.2% to

7% risk of Hemorrhage

• Deep brain structures

• Diagnostic challenge

PURPOSE

• Elucidation of the CSF proteome may yield insights into the pathogenesis of CNS disease.

• Tested the hypothesis that individual CSF proteins distinguish CNS lymphoma from benign focal brain lesions.

• Noninvasive diagnosisEvaluation of the CSF is markedly less invasive than brain biopsy

and is standard of care

Technique Used

• Liquid chromatography/mass spectrometry• Identify several hundred CSF proteins in CNS

lymphoma • Used enzyme-linked immunosorbent assay

(ELISA) to confirm one of these markers • Use potential of mass spectrometry(MS)to

identify biofluid peptide profiles that might facilitate early diagnosis of cancer

Properties of CSF

• Volume of CSF -150 mL, compared with the 5-L blood volume.

• CSF compartment is specialized to CNS and is not exposed to the systemic circulation and to multiple organs.

• Both features favor the relative over representation of brain and brain tumor related proteins in the CSF.

Common Method

High-resolution two-dimensional (2D) polyacrylamidegel electrophoresis with MS for identification of excised gel spots.

• This process is limited by low sensitivity

In this an alternate strategy for differential proteomic analysis that also includes large-scale identification of more than 500 proteins to identify the major CSF proteins which distinguish B-cell CNS lymphoma from benign conditions.

METHOD

Obtain CSF

Centrifuged within 90 minutes

of collection to remove cellular

debris,stored at 80°C

2D LC/MS

analysis

Sample Preparation

3 mL of CSFAlbuminIgG, IgA,

Alpha-1 antitrypsinTransferrin

HaptoglobinWere depleted

CSF proteome wasDenatured Reduced Alkylated

After buffer exchange, digested, desalted

fractionated into three fractions on a strong cation-exchange 20 g of processed

peptidesDissolved in 0.1% formic acid injected into theLC/MS system

LC/MS Differential Quantification and Identification

Uses high-resolution LC/MS data for profiling differential quantification without isotopic labeling, combined with extensive protein identification by LC/MS/MS.Molecular Ion signal intensities were normalized

Approximately 500 proteins were identified by tandem MS using control CSF samples

All profiled molecular ions were additionally constrained when matched against the library via accurate m/z and chromatographic retention times.

Mostly concentration of Protein metabolite are affected

ResultCandidate biomarker that they pursued is the serine protease

inhibitor ATIII

Function-: ATIII is normally synthesized by hepatocytes and has an established role in the regulation of blood coagulation.

Demonstrated endogenous expression of ATIII RNA transcripts within diagnostic biopsy specimens of primary CNS lymphoma tumors.

Used immunoblot to confirm the high relative expression of ATIII protein in the CSF of CNS lymphoma patients

Reference

• Protein Biomarker Identification in the CSF of Patients With C

• https://www.researchgate.net/.../5790893_Protein_Biomarker_Identification

• Heart-cut 2D-LC/MS approach for pharmaceutical impurity

• https://www.agilent.com/cs/library/applications/5991-1873EN.pdf

Thank YOu