use of endogenous biomarkers to achieve personalized immunosuppression in transplant recipients
TRANSCRIPT
Use of endogenous biomarkers to achieve personalized immunosuppression in transplant recipients
Prof. Dr. med. Dr. h.c. Michael Oellerich
FFPath (RCPI), FRCPathFFPath (RCPI), FRCPath
Department of Clinical Chemistry
George-August-University Goettingen
Germany
I have nothing to disclose.
Disclosure Information
I have nothing to disclose.
PK / PD concepts for monitoring drug therapy
Compliance with intake Prescribed dose Erroneousprescription
Absorption Administered dose Distribution
Biotransformation Excretion
Regional blood flow Drug level in Serum protein binding
Transport mechanisms blood/plasmaTransport mechanisms blood/plasma
Tissue responsiveness Drug concentration other drugs
Diseases at site of action Age
Placebo effects (receptor)
Pharmacodynamic effect
Outcome
Biomarkers
Modified from:Koch-Weser J, N Engl J Med 1972; 287: 277-81
Limitations of immunosuppressive drug level monitoring
• TDM does not precisely predict the effects of immuno-suppressive drugs on immune cells(���� over- or under-immunosuppression)
• Primary value of TDM is to prevent toxicity• Intersubject variability in the sensitivity to suppression • Intersubject variability in the sensitivity to suppression of immune function
• Intersubject variability of intralymphocyte immuno-suppressive drug levels
• Synergistic effects of immunosuppressive drugs• Immunological risk assessment prior to transplantation• Predicting tolerance before drug weaning
Intracellular CsA T-lymphocyte concentration has a potential of predicting rejection
Falck et al, Transplantation 2008; 85: 179-184
Influence of MDR1 3435 genotype on intralymphocyte trough CsA levels
ABCB1 3435C>T
CC CT/TT P value
Number* 22 42
Intracellular concentration (ng/106 cells) 1.5
(1.0-2.2)
2.5
(2.0-3.1)
0.015
Crettol S et al, Pharmacogenet Genom 2008; 18: 307-315
(1.0-2.2) (2.0-3.1)
Blood concentration (ng/ml) 90
(74-111)
111
(101-123)
0.038
Ratio of intracellular to blood concentration 0.017
(0.013-0.022)
0.023
(0.018-0.028)
0.096
Data are given as geometric mean (95% confidence interval).
* transplant recipients (renal, liver, lung)
Biomarkers desirable in addition to TDM?
Inability to measure effects of immunosuppressive
drugs on immune cells in vivo has severely limited:
Dambrin C, Klupp J, Morris RE, Current Opinion in Immunology 2000; 12: 557-562
- preclinical drug development
- design and interpretation of clinical trials
- optimal clinical use in transplantation
Factors limiting long-term outcome in transplantation
• Irreversible chronic rejection� under-immunosuppression
• Side effects of standard immunosuppression(e.g. nephrotoxicity, cardiovascular disease, opportunistic infection, malignancy) � over-immunosuppression
� numerous attempts to develop biomarkers that would complement TDM to achieve personalized immunosuppression
> 50 % of transplanted kidneys fail within 10 years
Sagoo et al, J Clin Invest 2010; 120: 1848-61Wieland et al, Ther Drug Monit 2010; 32: 560-572Schröppel et al, J Clin Invest 2010; 120: 1803-1806
Cyclosporin and Sirolimus Inhibit Different Pathways in the Immune Response
Cyclosporin and Sirolimus Inhibit Different Pathways in the Immune Response
Shaw et al Clinical Therapeutics 2000; 22 (Suppl. B) : B3
Proposed peripheral blood biomarkers
• Drug target enzymes
- Calcineurin phosphatase (CN)���� cyclosporin, tacrolimus
- Inosine monophosphate dehydrogenase (IMPDH)����mycophenolic acid (MPA)
- p70 S6 k phosphorylation ���� sirolimus, everolimus���� sirolimus, everolimus
• Cytokines
- Cytokine mRNA expression (e.g IL-2)
NFAT-regulated gene expression (mRNA expression of IL-2,
IFN-γ, GM-CSF)
- Cytokine production by T-cells (e.g. IL-2, IFN-γ)
T-cell alloreactivity, IFN-γ ELISpot
���� cyclosporin, tacrolimus
Proposed peripheral blood biomarkers
• Markers of lymphocyte proliferation
- PCNA
(proliferating cell nuclear antigen, auxiliaryprotein of DNA polymerase)
• Markers of lymphocyte activation
- CD 25, CD 71, CD 134
(T-cell surface growth factor receptors)
CD 26CD 26
(T-cell signaling, co-stimulation)
CD 28
(co-stimulation of T-cell proliferation)
���� calcineurin-, mTOR inhibitors, MPA
• Marker of Th2 activation
- sCD30
(indicates increased global immunologic responsiveness,
heightened rejection risk)
Proposed peripheral blood biomarkers
• Markers of global immune cell response
- PHA – stimulated ATP production by CD4+ cells
���� calcineurin-, mTOR inhibitors, MPA
• Potential predictors of tolerance
- Natural regulatory T-cells
(CD4+CD25highFOXP3+)
- Signature of B-cell genes
(IGKV4-1, IGLLA, IGKV1D-13)
Calcineurin inhibition in patients after a first single dose of Neoral
120
80
2500
2000
1500
% CN Activity C
sA (µg/L)
PBL
WB
CsA
Halloran et al, Transplantation 1999; 68: 1356
40
0
1000
500
0
0 1 2 4 12 h
post dose #8Post Dose Time (h)
% CN Activity C
sA (µg/L)
Association between pretransplant IMPDH, MMF exposure and acute rejection
7
0
12
4
IMPDH > cut-off */
Dose reduction
- / -
- / +
no rejection
rejectionO %
37 %
IMPDH > cut-off */
Dose reduction
7
0
12
4
no rejection
rejection- / -
- / +
O %
37 %
renal transplant recipients
9
5
7
2
9
0 2 4 6 8 10 12 14
- / +
+ / -
+ / +
Number of patients
36 %
82 %
*8.53 nmol/mg protein/h
Adapted from Glander et al, Am J Transplant 2004; 4: 2045-2051
9
5
7
2
9
0 2 4 6 8 10 12 14
Number of patients
- / +
+ / -
+ / +
36 %
82 %
PK-PD of MMF in pediatric kidney transplant recipients
Stable MMF treatment (n=18)
Fukuda T et al. J Clin Pharmacol 2011; 51: 309-320
Stable MMF treatment (n=18)
Early posttransplant (n=27)
Association between IMPDH inhibition and acute rejection in renal transplant recipients
IMPDH activity (AUECact0-4); EC-MPS therapy
inhibition
Raggi MC et al, Transplantation 2010; 90: 1536-1541
IMPDHinhibition
IMPDH variant allozyme structural analysis
Wu TY et al, Br J Pharmacol 2010; 161: 1584-1598
Allozymes:
Leu275 Phe263
10.2% of wild-type activity decreased activity
Gene sequencing:
73 SNPs 25 SNPs
���� Variation in MPA response may result, in part, from genetic variation in IMPDH1 and IMPDH2
Polymorphisms in type I and II inosine monophosphate dehydrogenase genes and association with clinical outcome
in patients on mycophenolate mofetil
Gensburger O, van Schaik RHN, Picard N, Le Meur Y, Rousseau A, Woillard JB, van Gelder T, Marquet P
IMPDH type I and II as targets of MPA
Study design: DNA and clinical data of 456 renal transplant recipients from clinical trials (Apomygre, FDCC)
Results: IMPDH I rs2278294 SNP was associated with a lower risk of BPAR and a higher risk of leukopenia over the first year post-transplantation.
Conclusion: IMPDH II genotyping may not improve MPA treatment outcome over the first year post-transplantation, in contrast to MPA and calcineurine inhibitor therapeutic drug monitoring and IMPDH I genotyping.
Pharmacogenetics and Genomics 2010; 20: 537-543
Inhibition of lymphocyte proliferation and activation in renal transplant recipients
50
75
100
% PCNA S/G
2M cells
(mean ±SEM)
50
75
100
% surface m
arker p
ositiv
e CD3
(mean ±SEM)
CsA, MMF, prednisone
*p = <0.005
healthy volunteers (n=12) transplant recipients (n=8)
0
25
% PCNA S/G
(mean
0
25
% surface m
arker p
ositiv
e CD3+cells
SEM)
PCNA CD71 CD25
*
**
Adapted from Stalder et al, Ther Drug Monit 2003; 25:22-27
CD28 expression by PBMCs and risk of malignancy
Study design: 134 stable long-term survivors of liver transplantation
CD28 expression by peripheral lymphocytes measured by flow cytometry
Results: Frequency of CD28+CD8+ cells significantly lower in cancer group vs. noncancer group (39 ± 22% vs. 51 ± 21%, P = 0.008)
Negative predictive value: 89.7%
Conclusion: Identification of patients at high risk of developing de novo malignancies
Liver Transpl 2011; 17: 299-305
NFAT-regulated gene expression to assess response to tacrolimus
NFAT-regulated genes:IL-2, IFN-γγγγ, granulocyte-macrophage colony stimulating factor
A B
Patients with CMV disease Patients with acute rejection
* *p = 0.02
p < 0.05
Sommerer C et al, Ther Drug Monit 2011; 33: 373-379
* *
*NFAT RE (%): 1.5 h post Tac dose;stable renal transplant recipients
p = 0.02
No difference in PK data
Percentage of IL-2 producing CD8+ T-cells in liverrecipients with and without acute rejection
Boleslawski et al, Transplantation 2004; 77: 1815 - 1820
Intracellular IL-2 expression in CD8+ T-cells during ISPT withdrawal in stable liver recipients
Millán O et al, Clin Immunol 2010; 137: 337-346
Global CD4+ cellular response measured by iATP synthesis
Sodium heparinate whole blood
Kowalski et al, Clin Transplant 2003; 17: 77-88
Immune Response vs. CD4 Count
Kowalski et al, J Immunotoxicol 2007; 4: 225-32
Comparison of ATP production in mitogen-stimulated CD4+ cells and immunosuppressive drug concentrations
before and 2-hour postdose
Stable renal transplant recipients (n=46)
Akhlaghi F, Gohh RY, Ther Drug Monit 2010; 32: 116-117
ImmuKnow test is a marker for the overall level of immune function
response
ng/mL)
500
600
700
800
900
1000
500
600
700
800
900
1000
r = 0.172p = 0.527
r = 0.024p = 0.918
Ther.range
Correlation between immune response and CNI trough levels in stable pediatric liver transplant recipients
Ther.range
525
strong
525
strong
Immune cell
(iATPng
CsA trough concentration[µg/L]
TCL trough concentration[µg/L]
0
100
200
300
400
500
0 5 10 15 20 25 30 35
0
100
200
300
400
500
0 50 100 150 200 250 300
Brandhorst et al, Clin Chem 2010; 56(suppl.): A241
225
moderate
low
225
moderate
low
Immune response in heart transplant recipients with infection and rejection
Israeli M et al, Transplantation 2010; 89: 968-976
Longitudinal ImmuKnow monitoringin a patient 4 years post-HTx
Are baseline values for individual patients required?
Israeli M et al, Transplantation 2010; 89: 968-976
Immune response in renal transplant recipients with infection or rejection
Single time point immune function assay (ImmuKnowTM)
testing does not aid in the prediction of future
opportunistic infections or acute rejection.
Huskey J, Gralla J, Wiseman AC.
Clin J Am Soc Nephrol 2011; 6: 423-429
Study design:
Retrospective analysis of 1330 ImmuKnow assay values in 583 renal transplant recipients and correlation with OI and AR episodes.
Conclusion:
No association between single time point ImmuKnow test results and adverse event in subsequent 90 days.
Association between pretransplant iATP levels (ImmuKnow, Cylex) with kidney graft outcome
iATP ng/m
l
p = 0.04
600
500
400
Adapted from Reinsmoen et al, Transplantation 2008; 85: 462-470
iATP ng/m
l
No clinical reason Negative Ab-mediated AR
Positive AR
n = 57 n = 8 n = 18 n = 4
Biopsies
300
200
100
ImmuKnow (iATP) values in lung transplant recipients with infections
CMV: cytomegalovirus; FC: fungal colonization;
FD: fungal disease; PNEU: bacterial pneumonia
TB: tracheobronchitis; VIRAL: viral infection
STABLE: control group
Husain et al, Transplantation 2009; 87: 1852-1857
Progression of Aspergillus colonization to invasive pulmonary aspergillosis (iA)
moderate
strong
low
Husain et al, Transplantation 2009; 87: 1852-1857
Immune response in adult liver transplant recipients classified with biopsy findings
Hashimoto et al, Clin Transplant 2010; 24: 701-708
Acyclovir treatmentTCL discontinued
EBV not detectable< 103 copies
400
500
EBV infection
1.4 x 105 copies
TCL
EBV relapse
5.0 x 103 copies
EBV not detectable< 103 copies
600 CsA treatment
CsA
Acyclovir treatment
700
800
response(iATP
mL)
Immune cell response in a liver transplant recipient (2 y, boy) with EBV infection
525
strong
moderate
27.05.09
279
200
400
300
100
0
13.03.09
181
TCL19.8 ng/mL
25.08.09
143
407
28.09.09
CsA124 ng/mL
Immune cellresponse
ng/mL
Brandhorst et al, Clin Chem 2010; 56(suppl.): A241
225
low
iATPng/mL)
AST 97 U/LALT 143 U/LγGT 449 U/L
MPA 2.7 mg/L
AST 49 U/LALT 67 U/LγGT 328 U/L
MPA 3.3 mg/L
AST 257 U/LALT 379 U/LγGT 690 U/L
MPA 0.45 mg/L
AST 41 U/LALT 55 U/LγGT 465 U/L
MPA 2.7 mg/LTCL <2.4 µg/L
AST 51 U/LALT 46 U/LγGT n.d.
MPA 1.9 mg/LTCL 8.1 µg/L
AST 104 U/L ALT 131 U/L γGT 757 U/L
MPA 1.0 mg/LTCL 6.1 µg/L
>1000
800
900
1000
>1000
838
>1000
Immune cell response in a liver transplant recipient (15 y, girl) with acute rejection
17.06.0927.02.09 20.07.09 31.08.09 09.11.0928.09.09
Immune cellresponse(iATP
200
400
600
700
500
300
100
0
325
Acute rejection
838
348
strong
moderate
low
525
225
Brandhorst et al, Clin Chem 2010; 56(suppl.): A241
Immune profiles of liver transplant recipients who developed rejection when IST was withdrawn
Millán et al, Transplantation 2009; 88: S78-S84
Relationship between immune cell response (Cylex)
and acute rejection (AR)
Significant results (p < 0.05) Author
Renal transplantation
↑↑↑↑AR risk with higher iATP levels Kowalski 2006; Reinsmoen 2008
No significant relationship Huskey 2010; Serban 2009
Cardiac transplantationCardiac transplantation
↑↑↑↑AR risk with higher iATP levels Israeli 2010; Kowalski 2006
No significant relationship Gupta 2008; Kobashigawa 2010
Hepatic transplantation
↑↑↑↑AR risk with higher iATP levels Kowalski 2006; Cabrera 2009;Brandhorst 2010; Hashimoto 2010
No significant relationship Millán 2009
Relationship between immune cell response (Cylex)
and infection
Significant results (p < 0.05) Author
Renal transplantation
↑↑↑↑ Incidence of infection with lower iATP levels Kowalski 2006; Serban 2009; Cadillo-Chávez 2006; De Paolis 2011
No significant relationship Huskey 2010
Cardiac transplantation
↑↑↑↑ Incidence of infection with lower iATP levels Israeli 2010; Kowalski 2006; ↑↑↑↑ Incidence of infection with lower iATP levels Israeli 2010; Kowalski 2006; Kobashigawa 2010
No significant relationship Gupta 2008
Hepatic transplantation
↑↑↑↑ Incidence of infection with lower iATP levels Xue 2010; Hashimoto 2010;Kowalski 2006; Cabrera 2009; Lee 2006
No significant relationship --------
Lung transplantation
↑↑↑↑ Incidence of infection with lower iATP levels Bhorade 2008; Husain 2009
No significant relationship --------
Association between immune response and mortality risk
Screening of mortality in transplant patients
using an assay for immune function.
Berglund D, Bengtsson M, Biglarnia A, Berlund E, Yamamoto S, von Zur-Mühlen B,
Lorant T, Tufveson G.
Transpl Immunol 2011, in press
Study design:1031 ImmuKnow assays (iATP) in 362 patients (pts)(kidney, pancreas, islet cells, liver allografts)
Results:Mortality: 14.4 % iATP < 175 ng/ml (at least once)
5.2 % iATP > 175 ng/ml
Conclusion:Potential usefulness of ImmuKnow assay for identification of pts with increased short-term mortality risk.
Allograft tolerance in solid organ transplantation
Spontaneous operational tolerance:
- long-term maintenance of stable graft function without a clinically significant, detrimental response or immune deficit following discontinuation of conventional immunosuppression
- stable renal transplant recipients without immunosuppression ≥ 1 year (serum creatinine < 10 % increase)
"Almost tolerance":"Almost tolerance":
- stable graft function in minimally immunosuppressed recipients (low dose monotherapy)
Roussey-Kesler et al, Am J Transplant 2006; 6: 736-746Newell et al, J Clin Invest 2010; 120: 836-847
Sánchez-Fueyo A et al, Gastroenterology 2011; 140: 51-64Sagoo P et al, J Clin Invest 2010; 120: 1848-1861
Estimated incidence of operational tolerance:
- liver transplantation: ≤≤≤≤ 20%
- renal transplantation: low frequency
Biomarker signatures related to tolerance in transplantation
Goal: Identification of recipients who would benefit from immunosuppression withdrawal or minimization
Indices of tolerance:Indices of tolerance:
- Signature of B-cell differentiation genes (IGKV4-1, IGLLA, IGKV1D-13) highly predictive of renal transplant tolerance
- Number of regulatory T-cells (Treg) FOX P3 demethylation as Treg signature useful for monitoring Treg in human peripheral blood
Newell et al, J Clin Invest 2010; 120: 1836-47Wieczorek et al, Cancer Res 2009; 69: 599-608
Sagoo et al, J Clin Invest 2010; 120: 1848-61
Gene expression signatures
Identification of a B cell signature associated with renal transplant tolerance in humans
Kenneth A. Newell et al
Study design:
Identification of recipients who would benefit from IS withdrawal or minimization
J Clin Invest 2010; 120: 1836-47
minimizationCohort of 25 tolerant renal transplant recipients (> 1 y, no IS)
Results:
Tolerant patients exhibited increased numbers of total and naive B cells and showed increased expression of multiple B cell differentiation genes. Signature of 3 genes (IGKV4-1, IGLLA, IGKV1D-13) was highly predictive of tolerance.
Conclusion:
Transitioning or maturing B cells involved in tolerance induction and/or maintenance
FOXP3 demethylation as Treg cell signature
Baron et al, Eur J Immunol 2007; 37: 2378-89
T-cell subsets in peripheral blood from liver transplant patients with renal dysfunction
CD3+CD8
+
cells/nLCD3
+CD4+
cells/nLCD4
+CD25highFOXP3
+ cells/nL
MMF group (n=22)*
Baseline 0.613 ± 0.092 1.068 ± 0.099 0.062 ± 0.016
Month 12 0.424 ± 0.055+ 0.756 ± 0.085+ 0.056 ± 0.010
Adapted from Cicinnati et al, Aliment Pharmacol Ther 2007; 26: 1195-1208
Control group (n=14)**
Baseline 0.474 ± 0.085 0.645 ± 0.096 0.051 ± 0.037
Month 12 0.530 ± 0.091 0.728 ± 0.089 0.034 ± 0.008†
+ P < 0.001 vs. baseline; † P < 0.05 vs. baseline; mean ± S.E.M.
* MMF 2x1 g; CsA 25-50 µg/l or TAC 2-4 µg/l
** CsA 80-120 µg/l or TAC 5-7 µg/l
Biomarker signature to detect renal transplant tolerance
- a set of 10 genes with significantly altered expression
- elevated numbers of peripheral blood B and NK cells
Tolerance signature comprising:
Sagoo et al, J Clin Invest 2010; 120: 1848-1861
- diminished numbers of recently activated CD4+
T cells
- donor-specific hyporesponsiveness of CD4+ T cells (IFN-γ ELISpot)
- high ratio of FoxP3/α-1,2-mannosidase gene expression in peripheral blood
Bio-markers for immunosuppressive drug
effects
Biomarker combinations for immune monitoring
Overall immune function(Over-immunosuppression)
• CD4+ cellular response measured by iATP synthesis (mitochondrial metabolic competence)
Risk of rejectionRisk of rejection
• IL-2 expression by CD8+ T-lymphocytes (cytotoxic properties)
Indices of tolerance
• Regulatory T-cells (FOXP3 demethylation signature)• Signature of B-cell differentiation genes
Biomarkers for personalized
immunosuppression
Potential complementary tools in addition to TDM
- may identify candidates for minimization of immunosuppressive therapy
- may identify patients at risk for acute rejection or infection- may be useful to guide immunosuppressant weaning
PD-monitoring using bio-markers is in its early stages
- optimal combinations of biomarkers may be necessary- baseline values for individual patients may be required- no prospectively validated target ranges available
Development of "tolerance permissive" immunosuppressive regimens would be desirable
Personalized immunosuppression –
outlook
- Shift emphasis from reaction to prevention
- Make immunosuppressive drugs safer
Reduce cost of health care- Reduce cost of health care
Adapted from R. Valdes, IATDMCT 2011
Update on Monitoring of Antiretroviral Update on Monitoring of Antiretroviral
Drugs in HIV TherapyDrugs in HIV Therapy
Natella Rakhmanina, MD, PhD, FAAP, AAHIVSAssociate Professor of Pediatrics
2011 ASCP Annual Meeting
Associate Professor of PediatricsDirector, Special Immunology ProgramChildren’s National Medical CenterThe George Washington University
Disclosure InformationDisclosure Information
Dr. Rakhmanina has been supported by
Department of Health and Human Services
NICHD K231K23HD060452-01A1, MO1-RR-
2011 ASCP Annual Meeting
NICHD K231K23HD060452-01A1, MO1-RR-
020359 and NICHD 1U10 HD45993 grants
Learning GoalsLearning Goals
� Understand the principles of the antiretroviral
therapy
� Review current application of the therapeutic
drug monitoring in the management of HIV
2011 ASCP Annual Meeting
drug monitoring in the management of HIV
� Identify therapeutic targets of antiretroviral
drugs
� Review the role of biomarkers in predicting
the response and monitoring of HIV therapy
HIV HIV Life CycleLife Cycle
2011 ASCP Annual Meeting
Goals of Antiretroviral TherapyGoals of Antiretroviral Therapy
�Maximal suppression of HIV replication
�Maximal recovery and preservation of
immune function
� Suppression of HIV-associated inflammation
ARV=Antiretroviral
2011 ASCP Annual Meeting
� Suppression of HIV-associated inflammation
� Prevention of opportunistic and other forms
of infection
� Preservation of the quality and normal
expectancy of life
Classes of Antiretroviral DrugsClasses of Antiretroviral Drugs
� Nucleoside Reverse Transcriptase Inhibitors
(NRTIs)
� Non-Nucleoside Reverse Transcriptase
Inhibitors (NNRTIs)
ARV=Antiretroviral
2011 ASCP Annual Meeting
Inhibitors (NNRTIs)
� Protease Inhibitors (PIs)
� Entry and Fusion Inhibitors (EIs and FIs)
� Integrase Inhibitors (IIs)
Measuring Therapeutic Effect of ARTMeasuring Therapeutic Effect of ART
Laboratory Efficacy:
� HIV RNA viral load
� CD4+ T lymphocyte count/percentage
Laboratory Toxicity:
ART=Antiretroviral Therapy
2011 ASCP Annual Meeting
Laboratory Toxicity:
� Liver enzymes(AST, ALT, bilirubin)
� Lipids (cholesterol, triglycerides) and glucose
� Hematologic parameters (WBC, RBC, Hb/Hct)
� Renal function parameters (BUN, Creatinine, UA)
Principles of TDM and ARV TherapyPrinciples of TDM and ARV Therapy
TDM=Therapeutic Drug Monitoring
Narrow
therapeutic index
or significant
consequences of
therapy failure
Evidence that an
optimized drug Rapid, accurate
and affordable
NNRTIs
or all ARVs
Most ARVs
2011 ASCP Annual Meeting
Therapeutic Drug
Monitoring
optimized drug
exposure
improves clinical
outcome
Wide inter-
subject and low
intra-subject
variability in PK
Concentration-
effect
relationship for
efficacy and/or
toxicity
and affordable
assay of relevant
moiety
NNRTIs, PIsNNRTIs, PIs
All and few ARVs
Major Barriers to TDM of ARV DrugsMajor Barriers to TDM of ARV Drugs
� Lack of the data on therapeutic range of
concentrations for all ARV drugs
� Limited number of clinical laboratories with
quality assurance/quality control standards
TDM=Therapeutic Drug Monitoring
2011 ASCP Annual Meeting
quality assurance/quality control standards
� Shortage of experts with ARV drugs clinical
pharmacological and virological expertise
� Lack of large prospective studies on ARV
drugs TDM
Current Application of TDM in HIV InfectionCurrent Application of TDM in HIV Infection
� Evaluation of virologic failure with established adherence
�Optimization of the dose in treatment experienced patents
TDM=Therapeutic Drug Monitoring
2011 ASCP Annual Meeting
experienced patents
� Prevention of ART associated toxicity
�Management of drug-drug interactions
�Management of patients with significant physiologic changes (hepatic/renal impairment, pregnancy, pediatrics)
Pharmacokinetic Considerations for ARTPharmacokinetic Considerations for ART
�Maximal efficacy concentrations (EC)
required to reduce viral replication of the wild
type virus by minimum of 50% (EC50)
� The area under the time-concentration (AUC)
EC=Efficacy Concentration
2011 ASCP Annual Meeting
� The area under the time-concentration (AUC)
and plasma trough concentration (Cmin) are
linked to the exposure/efficacy and sustained
virologic suppression
� AUC and peak plasma concentration (Cmax)
are associated with the exposure/toxicity
�
Therapeutic Targets of ARTTherapeutic Targets of ART
Efficacy trough
� NNRTIs: Efavirenz, Nevirapine
� PIs: Fosamprenavir, Atazanavir, Darunavir,
Indinavir, Nelfinavir, Lopinavir, Saquinavir,
ART=Antiretroviral Therapy
2011 ASCP Annual Meeting
Indinavir, Nelfinavir, Lopinavir, Saquinavir,
Tipranavir
Toxicity trough
� NNRTIs: Efavirenz
� PIs: Indinavir
TDM of NRTIsTDM of NRTIs
� NRTIs are metabolized inside the cell to
active triphosphate metabolites
�Only a few studies established relationship
between NRTIs plasma concentrations and
NRTIs=Nucleoside Reverse Transcriptase Inhibitors
2011 ASCP Annual Meeting
between NRTIs plasma concentrations and
virologic and immunologic outcomes
� It is not known whether the plasma
concentrations reflect the real time NRTIs
exposure
TDM and NNRTIsTDM and NNRTIs
� NNRTIs have a low plasma concentration
resistance threshold
� Resistance to first generation NRTIs is
unlikely to overcome with increased drug
NNRTIs=Non-Nucleoside Reverse Transcriptase Inhibitors
2011 ASCP Annual Meeting
unlikely to overcome with increased drug
exposure
� Second generation NNRTIs have a different
mechanism of viral resistance and may
benefit from increase drug exposure
TDM of PIsTDM of PIs
� PIs have high plasma concentration
threshold for HIV resistance
� Increasing plasma PIs exposure has been
shown to overcome resistance and improve
PIs=Protease Inhibitors
2011 ASCP Annual Meeting
shown to overcome resistance and improve
virologic outcome
� Combined use of virologic resistance test
with TDM results provide mechanism for
optimizing the PIs pharmacodynamics
Therapeutic Targets of PIsTherapeutic Targets of PIs
� Inhibitory quotient (IQ)=the ratio of real time
plasma trough concentration (Cmin) to the
parameters of viral resistance/ susceptibility
to ARV drugs
PIs=Protease Inhibitors
2011 ASCP Annual Meeting
to ARV drugs
� Phenotypic viral resistance is used to
calculate phenotypic IQ(pIQ), virtual IQ(vIQ),
or normalized IQ (nIQ)
Therapeutic Targets of PIsTherapeutic Targets of PIs
� pIQ=Cmin/in vitro IC50
� vIQ=Cmin/fold change in virtual IC50 from genotype
x matched reference wild-type protein adjusted IC50
� nIQ=patient specific IQ/reference IQ*
PIs=Protease Inhibitors
2011 ASCP Annual Meeting
� nIQ=patient specific IQ/reference IQ*
* - calculated as the ratio of typical Cmin for a given
dose and wild type viral IC50 which normalizes the
IQ target across ARV to the ratio of >1
Therapeutic Targets of PIsTherapeutic Targets of PIs
�Genotypic tests are used to calculate a
genotypic IQ (gIQ)
� gIQ=Cmin/number of ARV specific resistance-
associated mutations
PI=Protease Inhibitors
2011 ASCP Annual Meeting
associated mutations
� gIQ for PIs: Fosamprenavir, Atazanavir,
Darunavir, Indinavir, Lopinavir, Saquinavir,
Tipranavir
The Role of Biomarkers in HIV TherapyThe Role of Biomarkers in HIV Therapy
� Prediction of the progression of HIV disease
and response to treatment
� Evaluation of chronic inflammation
� Prediction and evaluation of the immune
IRIS=Immune Reconstitution Infalmmatory Syndrome
2011 ASCP Annual Meeting
� Prediction and evaluation of the immune
reconstitution inflammatory syndrome (IRIS)
� Prediction of HIV-associated co-morbidity
� Prediction and evaluation of drug-associated
toxicity
Chronic Inflammation in Patients on ARTChronic Inflammation in Patients on ART
� Higher levels of soluble inflammatory and
endothelial dysfunction makers (plasminogen
activator inhibitor type 1, soluble tumor
necrosis factor (TNF) receptor type 1 and
Guzmán-Fulgencio M., et al. J Infect 2011
2011 ASCP Annual Meeting
necrosis factor (TNF) receptor type 1 and
intercellular and vascular adhesion
molecules)
� Two-fold increase in Framingham scores for
cardiovascular disease (coronary heart
diseases and stroke)
IRIS and HIVIRIS and HIV
� Restoration of immune system resulting in an
exuberant host response to pathogens
and/or antigens
� Frequently observed with Mycobacterium
IRIS=Immune Reconstitution Inflammatory Syndrome
2011 ASCP Annual Meeting
� Frequently observed with Mycobacterium
tuberculosis and Mycobacterium avium co-
infections
� Significant challenge to initiation and
continuation of ART and treatment of
infections
Prediction and Evaluation of IRIS in HIVPrediction and Evaluation of IRIS in HIV
� Higher levels of the interleukin (IL)-6 and
soluble IL-6 in patients with IRIS Stone SF, et al. HIV Med 2001
AIDS=Acquired Immunodeficiency Syndrome
2011 ASCP Annual Meeting
� Higher levels of the C-reactive protein (CRP),
D-dimer,IL-6, IL-8, TNF-α, and interferon-γ
associated with IRIS, AIDS and death Boulware DR, et al. J Infect Dis 2011
Prediction and Evaluation of the Prediction and Evaluation of the
Progression of HIV DiseaseProgression of HIV Disease
� Significant decline in D-dimer (not IL-6 and
high sensitivity CPR) in patients with
immediate vs. delayed ART initiation Baker JV, et al. J Acquir Immune Defic Syndr 2011
CRP=C-reactive Protein
2011 ASCP Annual Meeting
� Resumption of HIV replication following ART
interruption is associated with increase in
plasma cytokines and chemokines (TNF-α,
IL-10, and CXCL10) Cozzi-Lepti A, et al. AIDS 2011
IRIS and HIVIRIS and HIV
� Restoration of immune system resulting in an
exuberant host response to pathogens
and/or antigens
� Frequently observed with Mycobacterium
IRIS=Immune Reconstitution Inflammatory Syndrome
2011 ASCP Annual Meeting
� Frequently observed with Mycobacterium
tuberculosis and Mycobacterium avium co-
infections
� Significant challenge to initiation and
continuation of ART and treatment of
infections
Biomarkers of the Biomarkers of the NevirapineNevirapine ToxicityToxicity
� Phase II activation of the NVP metabolite 12-
hydroxy-NVP mediates NVP binding to
bionucleophiles NVP toxicity
� In vitro model using the synthetic electrophile
Antunes AM, et al. Chem Res Toxicol 2010
2011 ASCP Annual Meeting
� In vitro model using the synthetic electrophile
12-mesyloxy-NVP as a surrogate of the 12-
sulfoxy-NVP
� LC-ESI-MS/MD and MALDI-TOF-TOF-MS
� Identification of cysteine, lysine, tryptophan,
histidine, serine and N-terminal valine of Hb
Inflammatory and Thrombotic Markers in Inflammatory and Thrombotic Markers in
ILIL--2 Therapy2 Therapy
� IL-2 cycling in patients on ART has been
shown to increase the long-term CD4 cell
counts
� The clinical benefit is unclear
Porter BO, et al. AIDS 2009
2011 ASCP Annual Meeting
� The clinical benefit is unclear
� The use of IL-2 cycling increased high-
sensitivity C-reactive protein and D-dimer
regardless of HIV RNA suppression
� Possible increased risk of thrombotic events
ConclusionsConclusions
� Limited data from small prospective studies
support role of TDM in improving virologic
response and/or decreasing the ARV drugs
concentration-related drug toxicities
2011 ASCP Annual Meeting
concentration-related drug toxicities
� Studies of cytokines and chemokines support
the use of biomarkers in predicting and
evaluating response to ART and ART
associated inflammation and/or toxicity
ConclusionsConclusions
� Large prospective studies on TDM of ARV
drugs are necessary to further investigate the
application in therapy of HIV
� Earlier initiation of ART and growing
2011 ASCP Annual Meeting
� Earlier initiation of ART and growing
evidence supporting universal ART in HIV-
positive patient prompt further development
of biomarkers evaluating the suppression of
HIV-associated inflammation and ARV drugs
response and/or toxicities
Early detection of Alzheimer’s Disease: are
CSF Aβ1-42 and tau biomarker tests ready for
the challenge?the challenge?
Leslie M Shaw
Department of Pathology and Laboratory Medicine
Perelman School of Medicine
University of Pennsylvania
Early detection of Alzheimer’s Disease: are
and tau biomarker tests ready for
the challenge?the challenge?
Leslie M Shaw
Department of Pathology and Laboratory Medicine
Perelman School of Medicine
University of Pennsylvania
Disclosures
Grant support: ADNI 1, ADNI GO, ADNI 2, NIH/NIA;
Pfizer/UPenn rbm studies
Consultant to: Innogenetics/Fujirebio; Janssen Research & Development
Disclosures
Grant support: ADNI 1, ADNI GO, ADNI 2, NIH/NIA;
; Janssen Research & Development
Alzheimer’s disease
• AD is one of the most disabling & burdensome health conditions worldwide
• An estimated 5.3 million people in the US and 35 million people have dementia today
• 4.6 million new cases diagnosed each year
• Number of people affected is expected to • Number of people affected is expected to double every 20 yrs to reach ~81 million by 2040
• Dementia prevalence <1% at age 60increases exponentially, thus by age >85 prevalence is 24
Lancet 2005;366:2112; 2006;368:387; 2009 Alzheimer’s Facts & Figures and 2009 World Alzheimer
Report from the Alzheimer’s Association and Alzheimer’s Disease
http://www.alz.co.uk
Science 2006 vol 314, Nov 3.
Alzheimer’s disease
AD is one of the most disabling & burdensome health conditions worldwide
An estimated 5.3 million people in the US and 35 million people have dementia today
4.6 million new cases diagnosed each year
Number of people affected is expected to Number of people affected is expected to double every 20 yrs to reach ~81 million by
Dementia prevalence <1% at age 60-64, increases exponentially, thus by age >85 prevalence is 24-33%
Lancet 2005;366:2112; 2006;368:387; 2009 Alzheimer’s Facts & Figures and 2009 World Alzheimer
Report from the Alzheimer’s Association and Alzheimer’s Disease International;http://www.alz.org/national;
Senile plaques&neurofibrillary tangles are characteristic lesions in the Senile plaques&neurofibrillary tangles are characteristic lesions in the
medial temporal lobe structures & cortical areas of AD brain.medial temporal lobe structures & cortical areas of AD brain.
•• Plaques are extracellular depositsPlaques are extracellular deposits
of amyloidof amyloid--ββ surrounded bysurrounded by
dystrophic neurites, reactivedystrophic neurites, reactive
astrocytes, & microgliaastrocytes, & microglia
•• Tangles are intracellular aggregatesTangles are intracellular aggregates
composed of a hyperphosphorylatedcomposed of a hyperphosphorylated
form of the microtubular stabilizingform of the microtubular stabilizing
Senile plaques&neurofibrillary tangles are characteristic lesions in the Senile plaques&neurofibrillary tangles are characteristic lesions in the
medial temporal lobe structures & cortical areas of AD brain.medial temporal lobe structures & cortical areas of AD brain.
form of the microtubular stabilizingform of the microtubular stabilizing
protein, Tauprotein, Tau
•• Degeneration of neurons and synapsesDegeneration of neurons and synapses
is a characteristic finding associated is a characteristic finding associated
with plaques & tangleswith plaques & tangles
•• Oxidative stress & neuronal/neuriticOxidative stress & neuronal/neuritic
dysfunction (eg, impairment of acetyldysfunction (eg, impairment of acetyl
choline transmitter activity) accompanycholine transmitter activity) accompany
these lesionsthese lesions
The Most Promising Biochemical Biomarkers
Detection: CSF Aβ1
• Changes in CSF: lower Aβ1-42, elevated t
• Attributes
– Most widely studied so far
– Linked to AD pathology
– May detect pathology before memory dysfunction or dementia appear clinically
– May detect pathology before memory dysfunction or dementia appear clinically
• Challenges
– Can be abnormal to varying degrees in non
– Analytical methods for measurement need
– May detect pathology before memory dysfunction or dementia appear clinically
Biochemical Biomarkers for AD
1-42, t-tau, pTau181
, elevated t-tau, p-tau181
May detect pathology before memory dysfunction or dementia appear May detect pathology before memory dysfunction or dementia appear
Can be abnormal to varying degrees in non-AD neurodegenerative diseases
Analytical methods for measurement need standardization
May detect pathology before memory dysfunction or dementia appear
• Why do we need biomarkers for Alzheimer’s Disease (AD)?
• What characteristics define a good biomarker?
• Standardization of biomarker measurements is a key need
Objectives
• Standardization of biomarker measurements is a key need
• What are the most promising recent findings about AD
biomarkers?
• What does the future hold for AD biomarker science and
practice?
Why do we need biomarkers for Alzheimer’s Disease (AD)?
What characteristics define a good biomarker?
Standardization of biomarker measurements is a key needStandardization of biomarker measurements is a key need
What are the most promising recent findings about AD
What does the future hold for AD biomarker science and
• Early diagnosis of AD is a high priority need
– Definitive diagnosis requires autopsy confirmation
• Diagnostic accuracy rate of ~90% using consensus criteria for probable
AD
– Diagnosis is especially difficult at early pre
Clinical Diagnosis of AD is Imprecise
– Diagnosis is especially difficult at early pre
AD
• Confusion with other dementias is common
• Hypotheses: CSF biochemical biomarkers can improve clinical diagnostic accuracy and predict risk of progression to AD from preclinical disease
Early diagnosis of AD is a high priority need
Definitive diagnosis requires autopsy confirmation
Diagnostic accuracy rate of ~90% using consensus criteria for probable
Diagnosis is especially difficult at early pre-clinical stages of
Clinical Diagnosis of AD is Imprecise
Diagnosis is especially difficult at early pre-clinical stages of
Confusion with other dementias is common
Hypotheses: CSF biochemical biomarkers can improve clinical diagnostic accuracy and predict risk of progression to AD from pre-
Overview• More than 30 studies (mostly single center small studies) have shown the diagnostic utility of
CSF Aβ1-42 and tau measurement for AD detectionincrease in tau in comparison to age-matched normals
• ELISA and Luminex xMAP multiplex immunoassays and used methods; Mesoscale Diagnostics immunoassay and others in development
• Several studies show the predictive performance for Aprogression from Cog Norm→MCI or MCI→AD (Hansson, 2006; Fagan, 2007; Shaw, 2009; Mattson, 2010).
• Studies from UWash, WashU, ADNI show that about a third of normal elderly have these changes but requires many years of time to observe conversion to MCI or early AD; relationship
• Studies from UWash, WashU, ADNI show that about a third of normal elderly have these changes but requires many years of time to observe conversion to MCI or early AD; relationship to changes in certain neuropsych. tests are significant.
• It is possible to obtain reproducible results within one laboratory, using one lot of manufacturer’s reagents, such that batched samples can be assayed with confidence in the results, provided that appropriate qc materials are used to check performance continuously.
• A primary reference material in CSF matrix is needed for accuracy assessments.
• High level standardization requirements for sample collection, storage, handling, reagent manufacture, lab performance has been reported.
• An international CSF qc program sponsored by the provides feedback to participating labs and should lead to improved practice worldwide (> 35 participating labs)
OverviewMore than 30 studies (mostly single center small studies) have shown the diagnostic utility of
and tau measurement for AD detection-50% or more decrease in Aβ1-42 and ~2-3 fold normals.
multiplex immunoassays and Innogenetics reagents most commonly Diagnostics immunoassay and others in development
Several studies show the predictive performance for Aβ1-42 and t-tau/ Aβ1-42 as predictors of or MCI→AD (Hansson, 2006; Fagan, 2007; Shaw, 2009;
, ADNI show that about a third of normal elderly have these changes but requires many years of time to observe conversion to MCI or early AD; relationship
, ADNI show that about a third of normal elderly have these changes but requires many years of time to observe conversion to MCI or early AD; relationship
. tests are significant.
It is possible to obtain reproducible results within one laboratory, using one lot of manufacturer’s reagents, such that batched samples can be assayed with confidence in the results, provided that appropriate qc materials are used to check performance continuously.
A primary reference material in CSF matrix is needed for accuracy assessments.
High level standardization requirements for sample collection, storage, handling, reagent manufacture, lab performance has been reported.
An international CSF qc program sponsored by the Alz Association was established in 2010 that provides feedback to participating labs and should lead to improved practice worldwide (> 35
Efforts underway to improve standardization
• ADNI
• Alz Assn: International qc program; Global Consortium for Standardization of Biomarkers
• CAMD
• PPMI (Parkinson’s Progression Markers Initiative)• PPMI (Parkinson’s Progression Markers Initiative)
• UPenn/Wash U collaboration on ELISA/
• Upenn ADNI/Japanese ADNI collaboration on immunoassay
• ABSI: Innogenetics-sponsored workgroup on standardization guidelines
Efforts underway to improve standardization
: International qc program; Global Consortium for Standardization of Biomarkers
PPMI (Parkinson’s Progression Markers Initiative)PPMI (Parkinson’s Progression Markers Initiative)
/Wash U collaboration on ELISA/xMAP relationships
ADNI/Japanese ADNI collaboration on xMAP
sponsored workgroup on
GOALS OF ADNI(10/2004 To 9/2009 + 1 Year No Cost Extension To 9/2010)
• Public/private sponsorship by NIH(NIA)/industry, private foundations
• Optimize and standardize biomarkers (imaging & biochemical)for clinical trials
• Validate biomarkers as measures of change• Validate biomarkers as measures of change
• Validate biomarkers as diagnostics or predictors
• All ADNI data posted on the website
• Establish a world-wide network for clinical AD studies and treatment trials
• ADNI GO underway, sponsored by NIA: 9/2009
• ADNI 2 funded by NIA + private sponsorship: 9/2010
GOALS OF ADNI-1 (10/2004 To 9/2009 + 1 Year No Cost Extension To 9/2010)
Public/private sponsorship by NIH(NIA)/industry, private
Optimize and standardize biomarkers (imaging & biochemical)for
Validate biomarkers as measures of changeValidate biomarkers as measures of change
Validate biomarkers as diagnostics or predictors
All ADNI data posted on the website
wide network for clinical AD studies and
ADNI GO underway, sponsored by NIA: 9/2009-9/2011
ADNI 2 funded by NIA + private sponsorship: 9/2010-9/2015
Naturalistic study of AD progression
•200 normal 3 yrs
•400 MCI 3 yrs
•200 AD 2 yrs
•Visits every 6 months
•57 sites
•Clinical, blood, LP
•Cognitive tests
•1.5T MRI
Some also have
•3.0T MRI
•FDG-PET (50%)•FDG-PET (50%)
•PiB-PET (approx.100)
ADNI cores:
•Administrative
•Clinical
•Imaging•MRI
•PET
•Biochemical biomarker
•Neuropathology
•Bioinformatics
•Biostatistics
•Genetics
ADNI-1
ADNI CoresThere are 9 ADNI cores (MWeiner is overall PI)
Core PIs
Administrative Mike Weiner
Clinical Ron Peterson
Paul Aisen
MRI Cliff Jack
PET Wm JagustPET Wm Jagust
Biomarker John Trojanowski
Les Shaw
Neuropathology John Morris
Informatics Art Toga
Genetics Andy Saykin
Biostatistics Laurel Beckett
ADNI Coresis overall PI):
Institution
Mike Weiner UCSF
Ron Peterson
Aisen
Mayo Clinic
UCSD
Cliff Jack Mayo Clinic
Jagust UC BerkeleyJagust UC Berkeley
Trojanowski
Les Shaw
UPenn
John Morris Wash U
Art Toga UCLA
Andy Saykin Indiana Univ
Laurel Beckett UC Davis
Qualification of the analytical and clinical performance of CSF A
tau181p in the ADNI study1. Selection of CSF Aβ1-42, tau, p-tau181p based on prior studies that showed their promise for AD
detection & a consensus among experts in this field
2. Pre-analytical factors for the lp & CSF handlingIdentify and control for pre-analytical variables
• Time of day for lp -morning following overnight fast
• Use of narrow gauge blunt (Sprotte) needle
• Collection & aliquot tube type -avoid PS and glass tubes & use PP tubes
• Transport temperature -avoid storage at refrigerator temp
• # of freeze-thaw cycles -minimize
• Time from collection to freezing -minimize
• Sample id & annotation of details on each sample collection/processing history
3. Analytical performance3. Analytical performanceAssure stability of reproducibility of test performance
• Follow consistently detailed method protocol
• Within each run
• Day to day
• Among expert laboratories
• From batch to batch of immunoassay reagents
• AA-sponsored international CSF external blinded quality control program
4. Clinical diagnostic performanceEstablish diagnostic and predictive performance using the qualified test method
• Establish sensitivity & specificity in ADNI-independent CSF samples from autopsy
• Use these diagnostic cutpoints to characterize AD CSF pathologic biomarker signatures in ADNI subjects
• Evaluate predictive performance for MCI→AD converters
• Characterize the longitudinal changes in CSF biomarker changes in a subset of ADNI CSF donors
• Study multiple biomarker types in combination for optimal disease detection and progression
Qualification of the analytical and clinical performance of CSF Aβ1-42, tau and p-
in the ADNI studybased on prior studies that showed their promise for AD
detection & a consensus among experts in this field
morning following overnight fast
avoid PS and glass tubes & use PP tubes
avoid storage at refrigerator temp
minimize
minimize
Sample id & annotation of details on each sample collection/processing history
sponsored international CSF external blinded quality control program
Establish diagnostic and predictive performance using the qualified test method
independent CSF samples from autopsy-confirmed AD subjects
to characterize AD CSF pathologic biomarker signatures in ADNI subjects
Evaluate predictive performance for MCI→AD converters
Characterize the longitudinal changes in CSF biomarker changes in a subset of ADNI CSF donors
Study multiple biomarker types in combination for optimal disease detection and progression
ADNI Interlaboratory Study of the Luminex Multiplex Immunoassay Using
Innogenetics INNO-BIA AlzBio3 Reagents for Tau, pA Study of the Repeatability and Reproducibility of Measuring Concentrations of Tau,
p-tau181p and Aββββ1-42 in pooled CSF samples and aqueous buffered qualitySamples
Report Prepared by:
Prof. Dr. Les Shaw and Dr. Hugo Vanderstichele
ADNIADNI AALZHEIMER’S LZHEIMER’S
Prof. Dr. Les Shaw and Dr. Hugo Vanderstichele
Dept of Pathology and Laboratory Medicine
University of Pennsylvania Medical Center, US
and Innogenetics, Belgium
October 24, 2007
ADNI Interlaboratory Study of the Luminex Multiplex Immunoassay Using
BIA AlzBio3 Reagents for Tau, p-tau181p and Aββββ1-42:A Study of the Repeatability and Reproducibility of Measuring Concentrations of Tau,
in pooled CSF samples and aqueous buffered quality-assessment
Samples
Report Prepared by:
Prof. Dr. Les Shaw and Dr. Hugo Vanderstichele
LZHEIMER’S LZHEIMER’S DDISEASE ISEASE NNEUROIMAGING EUROIMAGING IINITIATIVENITIATIVE
Prof. Dr. Les Shaw and Dr. Hugo Vanderstichele
Dept of Pathology and Laboratory Medicine
University of Pennsylvania Medical Center, US
and Innogenetics, Belgium
October 24, 2007
ADNI CSF Biochemical Biomarker
Study (All Data Are o
Participating Centers & Investigators
University of Pennsylvania: Leslie M Shaw, John Trojanowski, Virginia MLee, Margaret Knapik-Czajka
Innogenetics: Hugo Vanderstichele
Sahlgrenska University Hospital: Sahlgrenska University Hospital: Kaj
Friedrich-Alexander-Universitat ErlangenLewczuk
Pfizer Global Research & Development:
Eli Lilly & Company: Robert A Dean, Eric Salfen, (Linco)
Merck Research Laboratories: Adam Simon, William Potter
ADNI CSF Biochemical Biomarker Interlaboratory
Study (All Data Are on ADNI Website)
Participating Centers & Investigators
Leslie M Shaw, John Trojanowski, Virginia M-Y
Kaj Blennow
Erlangen-Nurnberg: Jens Wiltfang, Piotr
Pfizer Global Research & Development: Holly Soares, Nancy Raha
Robert A Dean, Eric Siemers, Richard Lachno, Brent
Adam Simon, William Potter
Reproducibility of xMAP Immunoassay (
the ADNI biomarker core lab
Avg test/re-test %CV:Avg test/re-test %CV:
Aβ42, 5.7%
t-tau, 5.6%
p-tau181, 11.5%
Immunoassay (Innogenetics reagents/Luminex) in
the ADNI biomarker core lab
test %CV:test %CV:
, 5.7%
tau, 5.6%
, 11.5%
Established CSF Aβ1-42 and t-tau biomarker threshold concentrations in an ADNI
population with autopsy-proven AD and applied these
tau biomarker threshold concentrations in an ADNI-independent
proven AD and applied these cutpoint concentrations to the ADNI subjects.
AADD
N 100 196
Male/Female 59/43 (42% female) 134/66
Age, y
Median 76 75
Mean+SD 75±8 75
Range 73 - 77 74
MMSE
Characteristics of 410 ADNI subjects who provided a BASELINE CSF sampleCharacteristics of 410 ADNI subjects who provided a BASELINE CSF sample
MMSE
Median 24 27
Mean+SD 23.5±1.9 26
95% CI 23.2-23.9 2
ADAS Cog 11
Median 17.2 11.3
Mean+SD 18.2±6.2 11.6±4.5
95% CI 16.9-19.4 11
APOE ε ε ε ε4+/εεεε4- 71/31 (70% ε4+) 108/92 (54%
Shaw LM, et al, Ann Neurol 2009
MMCCII NNCC
196 114
134/66 (33% female) 58/56 (49% female)
75 76
75±7 76±5
74 - 76 75 - 77
Characteristics of 410 ADNI subjects who provided a BASELINE CSF sampleCharacteristics of 410 ADNI subjects who provided a BASELINE CSF sample
27 29
26.9±1.8 29.1±1.0
26.7-27.2 28.9-29.3
11.3 6.3
11.6±4.5 6.4±2.9
11-12.3 5.9-6.9
108/92 (54% ε4+) 27/87 (24% ε4+)
Tau Aββββ1-42p-Tau
ROC AUC 0.831 0.913 0.753
Threshold values 93 pg/mL 192 pg/mL 23 pg
Sensitivity (%) 69.6 96.4 67.9
CSF Biomarker Cutpoints Established Using
Independent Autopsy-Based AD and
Normal Subjects
Specificity (%) 92.3 76.9 73.1
Test accuracy (%) 80.6 87.0 73.1
Positive predictive
value (%)90.7 81.8 67.9
Negative
predictive value
(%)
73.8 95.2 70.4
Shaw LM, et al, Ann Neurol 2009
Tau181p Tau/Aββββ1-42 p-tau181p/Aββββ1-42LR TAA
0.753 0.917 0.856 0.938
pg/mL 0.39 0.10 0.22
67.9 85.7 91.1 100
Established Using CSFs Collected from ADNI-
AD and Age-Matched Cognitively
Normal Subjects
73.1 84.6 71.2 76.9
73.1 85.2 81.5 88.9
67.9 85.7 77.3 82.4
70.4 84.6 88.1 100
A diagnosis-independent analysis of ADNI data reveals an AD
biomarker signature in cognitively normal elderly people• “decision boundary” at 188 pg/mL for A
• Best mixture model obtained with combination
of Aβ42 & pTau181 or t-tau
• When applied to an independent group of 71 Belgian
autopsy-based AD premortem CSFs 67 (94%) were classified as AD
• When applied to ADNI cohorts, AD, MCI & NC: 91%, 73% & 38% were classified as having the AD biomarker signatureclassified as having the AD biomarker signature
• This independent statistical analysis confirms that there is an AD biomarker signature in more than 1/3rd of the elderly cognitively normal control group at BASELINE.
Aβ1-42 concentration, pg/
Normal MCI
independent analysis of ADNI data reveals an AD
biomarker signature in cognitively normal elderly peoplefor Aβ1-42
Best mixture model obtained with combination
When applied to an independent group of 71 Belgian
CSFs 67 (94%) were classified as AD
When applied to ADNI cohorts, AD, MCI & NC: 91%, 73% & 38% were classified as having the AD biomarker signatureclassified as having the AD biomarker signature
This independent statistical analysis confirms that there is an AD biomarker signature in more than 1/3rd of the elderly cognitively normal control group
concentration, pg/mL
MCI AD
The five most widely studied biomarkers of AD pathology
• Decreased CSF Aβ1-42
• Increased CSF t-tau
• Decreased fluorodeoxyglucose uptake on PET
• PET amyloid imaging
• Structural MRI measures of cerebral atrophy
The five most widely studied biomarkers of AD pathology
uptake on PET
Structural MRI measures of cerebral atrophy
• Revise 1984 criteria for Alzheimer’s disease dementia
• Intended for:
—General healthcare providers (little access to
measures
—Specialized investigators
• Now biomarker evidence is integrated into the diagnosis of probable and possible AD for use in
research settings
• Clinical criteria remain cornerstone of diagnosis in clinical practice
• Biomarker evidence expected to enhance pathophysiological specificity of AD dementia diagnosis
• Validation of the diagnostic utility of biomarkers for AD diagnosis is ongoing
Revise 1984 criteria for Alzheimer’s disease dementia
General healthcare providers (little access to neuropsych testing, advanced imaging and CSF
Now biomarker evidence is integrated into the diagnosis of probable and possible AD for use in
Clinical criteria remain cornerstone of diagnosis in clinical practice
Biomarker evidence expected to enhance pathophysiological specificity of AD dementia diagnosis
diagnostic utility of biomarkers for AD diagnosis is ongoing
New diagnostic criteria for AD
• Preclinical stage of the disease, mild cognitive impairment (MCI),
precedes dementia
• In 1984 AD was defined by NINCDS
• Redefinition of research criteria for AD diagnosis to include the earlier,
pre-dementia stage of the disease (pre-dementia stage of the disease (
• Inclusion of mildly impaired subjects requires the use of biomarkers
(including MRI, CSF, FDG PET) in addition to clinical criteria to improve
reliability of diagnosis
• This year (2011) in a series of papers the new criteria for AD diagnosis in
the research setting were further described including the role and use
of biomarkers
New diagnostic criteria for AD
Preclinical stage of the disease, mild cognitive impairment (MCI),
In 1984 AD was defined by NINCDS-ADRDA as a dementia disorder
Redefinition of research criteria for AD diagnosis to include the earlier,
dementia stage of the disease (DuBois, 2007)dementia stage of the disease (DuBois, 2007)
Inclusion of mildly impaired subjects requires the use of biomarkers
(including MRI, CSF, FDG PET) in addition to clinical criteria to improve
This year (2011) in a series of papers the new criteria for AD diagnosis in
the research setting were further described including the role and use
% of ADNI patients in whom biomarker signature was detected using ROC
AD
Aβ1-42 91
UPenn Autopsy-Based CSF Biomarker
AD Signature in the ADNI Study Cohorts
Aβ1-42 91
Tau/Aβ1-42 ratio 89
LRTAAmodel 89
LRLRTAATAA = a logistic regression model that includes Tau, A= a logistic regression model that includes Tau, A
allele number; ROC = receiver operating characteristic curveallele number; ROC = receiver operating characteristic curve
% of ADNI patients in whom biomarker signature was detected using ROC
cutpoints
AD MCI NC
91 78 34
Based CSF Biomarker
AD Signature in the ADNI Study Cohorts
91 78 34
89 69 34
89 70 31
= a logistic regression model that includes Tau, A= a logistic regression model that includes Tau, Aββ4242, and ApoE , and ApoE εε4 4
allele number; ROC = receiver operating characteristic curveallele number; ROC = receiver operating characteristic curve
Survival analyses for ADNI MCI subjects:
progression to AD for BASELINE CSF biomarkers > or <
Aβ42<192 pg/mL
As of June 28, 2010
Survival analyses for ADNI MCI subjects:
progression to AD for BASELINE CSF biomarkers > or < cutpoints
t-tau/Aβ42>0.39
t-tau/Aβ42<0.39
Tau and p-Tau Increase With Decreasing Normalized
Whole Brain Volume in Very
DAT = dementia of Alzheimer’s type
Fagan AM, et al. Ann Neurol. 2009;65:176-183.
- CSF Aβ1-42 plateaus
- tau and p-Tau increase
as brain volume decreases
Tau Increase With Decreasing Normalized
in Very Mild-Mild DAT
CSF Aββββ1-42 vs plaque counts (neocortex and
hippocampus)
60
80
Non-demented
Demented
42
155 autopsy cases
CSF Aβ1-42 is Strongly Correlated to Plaque
autopsied brains and Plaque Burden
CSF A
Strozyk D, et al. Neurology 2003;60:652-656; Jagust W, et al,
0 5 10 15 200
20
40
60 Non-demented
Plaque count
CSF-Aß1-42
(pg/mL)
Pittsburgh compound-B labeled positron emission tomography; SUVR = standard uptake value ratio
is Strongly Correlated to Plaque Counts in
and Plaque Burden by PiB testing
CSF Aββββ1-42 vs mean cortical PiB SUVR
Jagust W, et al, Neurology 2009; 73:1193-1199
B labeled positron emission tomography; SUVR = standard uptake value ratio
The message in this Perspective paper is that early knowledge is very helpful to the affected
Individual and to family to plan for best response to the disease before the dementia phase.
In increasing numbers the early detection of AD is not seen so negatively as it once was and can
be beneficial. Nevertheless some are sceptical that early diagnosis will be beneficial.
The message in this Perspective paper is that early knowledge is very helpful to the affected
Individual and to family to plan for best response to the disease before the dementia phase.
In increasing numbers the early detection of AD is not seen so negatively as it once was and can
that early diagnosis will be beneficial.
Proposed staging framework for preclinical AD
Sperling R, et al, Alz & Dementia, 2011. Report of the NIA &
Proposed staging framework for preclinical AD
& Dementia, 2011. Report of the NIA & Alz Assn Workgroup
Figure 25. Worldwide ADNI sites. NA-ADNI, North American ADNI;
Argentinean ADNI; E-ADNI, European ADNI; C-ADNI, Chinese ADNI; K
ADNI; J-ADNI, Japanese ADNI; T-ADNI, Taiwanese ADNI; A
Weiner M, 2011, Alz & Dementia, in press
ADNI, North American ADNI; Arg-ADNI,
ADNI, Chinese ADNI; K-ADNI, Korean
ADNI, Taiwanese ADNI; A-ADNI, Australian ADNI.
It takes a great team effort!
John Q Trojanowski
Virginia M-Y Lee
Chris Clark
Steve Arnold
Hugo Vanderstichele
Magdalena Korecka
Margaret Knapik-Czajka
Sarah Pan
William Hu
Ju Hee Kang
Jon Toledo
Uwe Christians
Kaj Blennow
Holly Soares
Adam SimonMargaret Knapik-Czajka
Magdalena Brylska
Teresa Waligorska
Michal Figurski
Ravi Patel
Leona Fields
Adam Simon
Robert Dean
Eric Siemers
Piotr Lewczuk
William Potter
ADNI investigators include:
(complete listing available at
www.loni.ucla.edu
aboration\ADNI_Manuscript_
Citations.pdf).
It takes a great team effort!
Christians
Supported by the NIH/NIA and families
of our patients
include:
(complete listing available at
www.loni.ucla.edu\ADNI\Coll
ADNI_Manuscript_