Biomarkers Discovery and Validation This Talk is based on Systemic Lupus Erythematosus and Lupus Nephritis

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Systemic Lupus Erythematous
What is Biomarker?
Proteomics
Biomarker Research Design
Lupus Nephritis
Literature
Discussion
Conclusion
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4. • It is often abbreviated to SLE or lupus, It is a systemic
autoimmune disease (or autoimmune connective tissue
disease) that can affect any part of the body.
• As occurs in other autoimmune diseases, the immune
system attacks the body's cells and tissue, resulting in
inflammation and tissue damage.
• It is a Type III hypersensitivity reaction in which
antibody-immune complexes precipitate and cause a
further immune response.
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5. SLE Manifestations
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7. • Not known when Lupus first appeared
– Hippocrates noted similar diseases in Ancient Greece
• Facial rash that resembles the markings of a wolf
• 1851 French-man named Pierre Cazenave
– first clinical records
• More than 1.4 million Americans are affected by SLE
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8. • SLE most often harms the heart, joints, skin, lungs, blood
vessels, liver, kidneys, and nervous system.
• The course of the disease is unpredictable, with periods of illness
(called flares) alternating with remissions.
• The disease occurs nine times more often in women than in
men, especially in women in child-bearing years ages 15 to
35, and is also more common in those of non-European descent
• There is no cure for SLE. It is treated with
immunosuppression, mainly with
cyclophosphamide, corticosteroids and other immunosuppressants.
• SLE can be fatal. The leading cause of death is from
cardiovascular disease due to accelerated atherosclerosis
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9. • SLE is one of several diseases known as "the great imitators"
because it often mimics or is mistaken for other illnesses.
• SLE is a classical item in differential diagnosis because SLE
symptoms vary widely and come and go unpredictably.
• Diagnosis can thus be elusive, with some people suffering
unexplained symptoms of untreated SLE for years.
• Common initial and chronic complaints include
fever, malaise, joint pains, myalgia, fatigue, and temporary loss of
cognitive abilities.
• Because they are so often seen with other diseases, these signs and
symptoms are not part of the diagnostic criteria for SLE. When
occurring in conjunction with other signs and symptoms
however, they are considered suggestive.
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10. There is no one specific cause of SLE. There are, however, a number of
environmental triggers and a number of genetic susceptibilities
@ One manifestation of SLE is abnormalities in apoptosis, a type of programmed cell
death in which aging or damaged cells are neatly disposed of as a part of normal
growth or functioning.
@ In SLE, the body's immune system produces antibodies against itself, particularly
against proteins in the cell nucleus.
@ SLE is triggered by environmental factors that are unknown.
@ In order to preserve homeostasis, the immune system must balance between being
sensitive enough to protect against infection, and becoming sensitized to attack the
body's own proteins (autoimmunity).
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11. • During an immune reaction to a foreign stimulus, such as
bacteria, virus, or allergen, immune cells that would
normally be deactivated due to their affinity for self tissues
can be abnormally activated by signaling sequences of
antigen-presenting cells.
• Thus triggers may include viruses, bacteria, allergens (both
IgE and hypersensitivity), and can be aggravated by
environmental stimulants such as ultraviolet light and
certain drug reactions
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13. • A biomarker, is a measurement, including but not limited to a
genetic, biological, biochemical, molecular or imaging event
whose alternations correlate with disease pathogenesis and/or
manifestations and can be evaluated qualitatively and/or
quantitatively in laboratories.
• Several criteria are required for a laboratory measure to serve as a
reliable biomarker, including:
1. It must be biologically and patho physiologically relevant
2. It must be simple for routine practice
3. It must accurately and sensitively respond to change in disease
activity
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15. Two strategies for biomarker discovery and validation: a “targeted” approach, based on
hypothesis-driven evaluation of specific biomarker candidates, and a “de novo” discovery
approach using different proteomic technologies followed by validation of potential Biomarker
Candidate...
©2008 by American Physiological Society
Matt P et al. Physiol. Genomics 2008;33:12-17
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16. The platform for biomarker discovery in serum and tissue samples at our institution combines
multiple synergistic protein fractionation and separation methods including one- and twodimensional gel electrophoresis (1-DE, 2-DE), differential in-gel electrophoresis...
©2008 by American Physiological Society
Matt P et al. Physiol. Genomics 2008;33:12-17
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17. PI
Mr (kDa)
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5
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8 9
10
9: Hsp70 subfamily B suppressor 1-like protein
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97
97
11: Serum albumin
66
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14: Epidermal growth factor
receptor kinase substrate 8like protein 2
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1D- SDS-PAGE GEL
20 Putative serine/threonine-protein
phosphatase 4 regulatory subunit 1-like
2D- Gel Electrophoresis
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18. 2D-DIGE analysis of 2 different serum samples. One serum sample is labeled with Cy3 (green
color in this example), whilst the other is labeled with Cy5 (blue), and equal concentrations of
both samples are labeled with Cy2 (red). All 3 labeled samples are then combined, separated on
the same 2-D gel, and scanned at different emission wavelengths, which allows the differentially
expressed proteins to be viewed as changed in color; see arrows for green or blue spots in
enlarged gel area. Proteins that are equally expressed in both samples appear as white spots.
Matt P et al. Physiol. Genomics 2008;33:12-17
©2008 by American Physiological Society
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19. Mass Software
Mass Machine
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24. • Isolate and identify proteins contained in the LN
Urinary protein signature (PS) of children with
SLE
• Assess the usefulness of the PS-Proteins for
detecting activity of LN over time.
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25. Lupus Nephritis(LN) is among the main
determinants of poor prognosis in Systemic lupus
erythematosus (SLE).
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26. • Systemic Lupus:
– most common and affects major organs
• Discoid Lupus:
– affects only the skin
– not fatal, but can cause severe scarring
• Drug-induced Lupus:
– is systemic Lupus caused by medications
– when the medicine is stopped, the disease goes away
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27. • Lupus nephritis
– one of the most serious manifestations of SLE
– typically arises within 5 years of diagnosis
• commonly within the first 6 to 36 months
• Silent’ lupus nephritis
– normal urinalysis
– no proteinuria
– normal serum creatinine levels
• However, renal biopsy reveals pathological changes
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28. • There is a need for high quality accurate biomarker to
judge Lupus activity and renal damage with SLE.
• By using extensive proteomic approach for the discovery
of novel LN biomarkers and identified a set of PSproteins.
• The set of PS-Proteins identified are
→ Transferrin (Tf)
→ Ceruloplasmin (Cp)
→ α 1-acid-glycoprotein (AGP)
→ lipocalin- type prostaglandin-D-synthetase (L-PGDS)
→ Albumin and Albumin related fragments
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29. Investigation of specific protein
• Comparison of PS-protein plasma concentration
in the three groups of SLE patients (no LN, active
LN, inactive LN) and two groups of controls
(active JIA, inactive JIA (Juvinile Idiopathic
Arthritis)
• Studied the statistically important difference
under a multivariate fixed effect model
framework to analyze the PS-protein effect in
SLE as well as LN.
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30. • To develop the LN protein signature (PS) as a novel
biomarkers, these biomarkers were detected on at least
two different protein chips
• The peak intensity should be > 100 fold increase in
between groups.
• By using SDS-PAGE and 2D-Gel electrophoresis the
significantly expressed bands are identified
• Then the bands are excised and digested with trypsin
• And recovered for Mass spectrometry.
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31.  They measured plasma and urinary transferrin (Tf), plasma ceruloplasmin
(Cp), plasma α-1-acid-glycoprotein (AGP, also: orosomucoid), as well as
plasma and urine lipocalin-type prostaglandin-D synthetase (L-PGDS) by
immunonephelometry (Dade Behring BNII Prospect, Marburg, Germany).
 Urinary Cp was quantified by ELISA (Human Ceruloplasmin ELISA
Quantitation Kit; Genway Biotech, Inc., San Diego, CA, USA); and urinary
AGP by ELISA (Human Orosomucoid ELISA Quantitation Kit; Genway
Biotech, Inc., San Diego, CA, USA).
 Then they determine the concentration of plasma and the identified specific
important proteins concentration by Tukey-Post hoc Testing technique.
 An AUCROC of 1.0 represents a perfect biomarker whereas a value of 0.5 is
no better than expected by chance. Statistical computations were conducted
using SAS version 9.1 (SAS, Cary, NC, USA) software. P-values < 0.05
were considered statistically significant.
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32. Sample
Collection
Fractionation
1D-SDS-PAGE
Mass
Spectrometry
In gel Digestion
2DElectrophoresis
Statistical
Analysis
Conformational
Assay
Validation
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34. Values are means and SE. Significant differences are based on Tukey post-hoc testing.
The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS
(D) for the groups defined as Figure 1. Uncorrected PS-protein levels (per mL or dL
of urine) are depicted. Significant differences between groups are indicated as
follows: * = P<0.004; **= P<0.002; ¶= P<0.00001.
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35. Values are means and SE. Significant differences are based on Tukey post-hoc testing.
The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS
(D) for the groups defined as Figure 1. PS-protein excretion standardized by urine
creatinine (mg/mL urine) is shown. Significant differences between groups are
indicated as follows: *= P<0.0005; **= P<0.0001; = P<0.05; ¶ = P<0.001.
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36. Values are means and SE. Significant differences are based on Tukey post-hoc
testing. The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C)
and L-PGDS (D) for the groups defined as Figure 1. PS-protein excretion
standardized by nonselective proteinuria is depicted with significant differences
between groups indicated as follows: * = P<0.05; ** = P<0.005; = P<0.02; ¶ =
P<0.009.
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37. Values are geometric means of uncorrected urinary levels of Tf (A), Cp (B), AGP (C) and L-PGDS
(D) at months -6, -3 and 0, respectively. Month 0 is the time point when the clinical diagnosis of
the course of LN is made and months -3 corresponds to the time point of 3 months prior to the
clinical diagnosis of the LN flare. ‘Improved LN’ describes the course of LN with decreasing renal
SLEDAI scores; ‘worse LN’ describes the course of LN associated with an increase of the renal
SLEDAI scores; ‘stable active LN’ describes patients with stable renal SLEDAI scores > 0; and
‘inactive LN’ describes the course of continuously inactive LN (renal SLEDAI = 0). Significant
differences in the levels between two consecutive visits are indicated in the plots as follows. = P<
0.009; ¶ = P<0.0001; * = P<0.001. The above defined LN courses are depicted as follows:
Improved LN, squares; Worsened LN, circles; Stable active LN, triangles; Inactive LN, diamonds.
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38. • They found high and increasing urinary levels of Tf
associated with active LN and impending worsening of LN
flares.
• Tf is co-regulated by interferon-α, involved in iron
delivery, and the innate immune system.
• Plasma Tf levels were correlated to global SLE disease
activity in the past (Yilmaz.A et al 2005).
• Thus, this study confirms these earlier findings in SLE, and
new evidences provided that urinary Tf excretion may
represent a predictive biomarker for LN.
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39. • Cp plays a critical physiological role in controlling the rate of iron
efflux from cells with mobilizable iron (Hellman et al 2003) .
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Like Tf, plasma Cp has been recommended as a marker of global
SLE disease activity (Yilmaz et al 2005, Hrycek.A et al 2007) .
• Conversely, the results support that urinary Cp concentrations only
differ with LN activity rather than extra renal disease activity.
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Possibly, because urinary Cp levels vary widely in SLE, they were
unable to detect a meaningful relationship to the course of LN.
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40. α1-acid-glycoprotein)
• AGP is a predictive biomarker for diabetic renal disease (Gomes et
al 2004) , and we provide initial evidence that this is also the case
for LN.
• More importantly, urine concentrations of AGP (similar to Tf and
L-PGDS) appear useful to anticipate LN flares, i.e. these markers
may allow clinicians to preemptively adjust therapy prior to the
appearance of overt worsening of LN.
• Previous studies proposed plasma AGP to be a biomarker of SLE
global disease activity (Lacki JK et al 1997, Meijer C et al 1993) .
• The results support this (data not shown) but they also provide
evidence that urinary AGP constitutes a biomarker of LN rather
than extra renal disease activity.
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41. • Lipocalins play a role in many biological processes, among them
immune responses and prostaglandin synthesis.
• L-PGDS, a lipocalin, is involved in nitric oxide regulation and the
induction of apoptosis in the kidney.
• L-PGDS has not been previously found to be a LN biomarker.
Urine and plasma L-PGDS are considered sensitive indicators of
chemotherapy- induced renal damage and diabetes-associated
hypertension (Hirawa N 2002, Ogawa et al 2002).
• They found urinary L- PGDS unrelated to the creatinine clearance
in both JIA and SLE; L-PGDS also did not significantly change
with cyclophosphamide exposure in SLE patients.
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42. • There is a need for high-quality accurate biomarkers to judge LN
activity and renal damage with SLE.
• By using a proteomic approach for the discovery of novel LN
biomarkers and identified a set of PS-proteins (i.e. Tf, Cp, AGP
and L-PGDS).
• In quantitative analysis, particularly urinary rather than plasma
levels of the PS-proteins increased significantly with the presence
of active LN.
• Tf, CP, AGP and L-PGDS to be promising LN biomarkers, as their
levels do not seem to change with the use of angiotensin inhibiting
medications and even help discriminate patients who are at risk of
a future LN flare.
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43. • Tf, and AGP are part of LN protein signature (Varghese et al 2007)
• At present there is no universally accepted gold standard for the
measurement of LN activity.
• The relevance of this findings is strengthened by the fact that the
PS-Proteins performed similarly well to capture and anticipate the
course of LN.
• The sensitivity of moderately elevated protein :creatinine ratio to
angiotensin blocking medications and its unproven ability for
predicting LN flares .
• They consider Tf, Cp, AGP, and L-PGDS to be promising LN
biomarkers as their levels do not seem to change with the use of
angiotensin inhibiting medications and risk of LN flare.
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44. 
Tf, Cp, AGP and L-PGDS are promising LN biomarkers.
 Their initial validation suggests superior measurement
properties compared to most traditional LN biomarkers and
that Tf, AGP and L-PGDS are candidates of a novel set of
predictive LN biomarkers.
 Additional validation studies are mandatory to evaluate the
usefulness of such a LN Renal Panel to predict the course of
LN, the severity of kidney pathology, and the future
development of renal damage with SLE.
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47. The AUCROC was calculated to assess the concurrent validity of the PSproteins and the traditional renal biomarkers to diagnose the presence of
active LN as measured by the SLEDAI and the BILAG, respectively (Table
3). Individual urinary PS-Proteins in performed all in the fair to good range
according to current ROC interpretation standards (12), they were all better
diagnostic markers of active LN than traditional renal biomarkers (all
AUCROC <0.63) with the exception was the urine protein:creatinine ratio
with an AUCROC at 0.91 (SLEDAI) and 0.85 (BILAG), respectively.
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48.  In Mass Spectrometry the Proteins are analyzes on a normal binding Protein chip to
confirm the aimed mass spectrum.
 Peptides recovered from the in-gel digest were identified either by peptide mass
fingerprints (PMF) on the SELDI-TOF platform or MALDI-TOF/MS and MS/MS
fragmentation with sequencing individual peptides.
 These two methods are necessary as albumin or albumin fractions are often were present.
 Due to the unsuccessful removing of albumin by various methods there is a need to
perform two methods to identify the specific novel proteins.
 PMFs and MS/MS-fragmentation data were collected for each sample.
 Both MALDI-TOF and TOF/TOF approaches were used, since the extreme abundance of
albumin fragments interfered with the PMF identification for many of the bands.
 The acquired peptide data from SELDI-TOF MS were searched via Mascot database
search engine and the International Protein Index (IPI) human protein database.
 For the MALDI-TOF MS/MS spectra, data were processed using an integrated
GPSExplorer interface from Applied Bio systems coupled to a local Mascot Server with
database searches against the entire NCBI database.
 In either case, standard Mascot statistical criteria were used to indicate positive protein
identification.
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