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

1. CNS Cancer-Biomarker
By-Lovnish THAKUR
ASU2014010100099
Integrated Biotech
4th sem

2. 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

3. Protein Biomarker Identification in
the CSF of Patients
With
CNS Lymphoma

4. BACKGROUND
• Brain biopsy is often the test
Brain biopsy is, however, associated with 1.2% to
7% risk of Hemorrhage
• Deep brain structures
• Diagnostic challenge

5. 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 diagnosis
Evaluation of the CSF is markedly less invasive than brain biopsy
and is standard of care

6. 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

7. 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.

8. 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.

9. METHOD
Obtain
CSF
Centrifuged
within 90 minutes
of collection to
remove cellular
debris,
stored at 80°C
2D
LC/MS
analysis

10. Sample Preparation

11. 3 mL of CSF
Albumin
IgG, IgA,
Alpha-1 antitrypsin
Transferrin
Haptoglobin
Were depleted

12. CSF proteome was
Denatured
Reduced
Alkylated
After buffer exchange, digested,
desalted
fractionated into three fractions
on a strong cation-exchange
20 g of processed
peptides
Dissolved in 0.1%
formic acid
injected into the
LC/MS system

13. LC/MS Differential Quantification and
Identification

15. 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.

17. Mostly concentration of Protein metabolite are affected

18. Result
Candidate 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

19. Reference
• Protein Biomarker Identification in the CSF of
Patients With C
• https://www.researchgate.net/.../5790893_Prote
in_Biomarker_Identification
• Heart-cut 2D-LC/MS approach for pharmaceutical
impurity
• https://www.agilent.com/cs/library/applications/
5991-1873EN.pdf

20. Thank YOu