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BALANCING THE ACT:MINIMIZINGPOST-TRANSPLANT VIRAL
INFECTIONS AND ACUTEREJECTION
Vikas R. Dharnidharka, MD, MPHProfessor and Director,
Division of Pediatric [email protected]
My Disclosures
• Consultant: – Bristol-Myers-Squibb, – Atara Biotherapeutics
• Grant Support: – NIDDK/NIH– NCATS/ICTS – Bristol-Myers-Squibb, Novartis– Submitted grant application with CareDx for
Allosure
Items we will discuss
• Trends in IS and infections with eras• Biology of anti-viral immunity versus
alloimmunity• Immunoprophylaxis not available• Chemoprophylaxis only for CMV• PCR surveillance strategies• Missed viral surveillance• Infection confounding of Suthan and Allosure
biomarkers
CD28, CD40L
IL-2 mRNAIL-2R mRNA Steroids
IL-2 Receptor
M
G1S
G2
P13K/Akt
mTOR
Cyclin/CDK
SirolimusEverolimus
MMFAZA
TCR Complex
Calcineurin promoter(IL-2)
NFAT
calcineurinTacrolimusCsA
Polyclonal Antibodies
(i.e., anti-thymocyte Globulin)
Monoclonal antibodies(i.e., basiliximab, alemtuzumab)Costimulation
blockade(i.e., belatacept)
CD52
Cell surface
Cytoplasm
Nucleus
Transplant immunosuppression
Multiple medication adverse effects
• New onset diabetes after transplantation• Cardiovascular:
– Dyslipidemias– Hypertension
• Malignancies– Viral-driven– Non viral-driven
• Infections– Viral – Cytomegalovirus, Epstein-Barr virus, BK virus– UTI– Sepsis– Pneumonia
8065
60
45 45
35
25
1510 10
4060
65
8590 90
96 97 98
0
20
40
60
80
100Pe
rcen
t
Rejection <12 mo1 Year Survival
BKVAN
CMV
PTLD
’60 ‘65 ‘70 ‘75 ‘80 ‘85 ’90 ’95 ’00 ’05 ’10 ‘15
• CsA• OKT3
• Cyclosporine Emulsion• Tacrolimus
• MMF• Dacluzimab• Basiliximab
• Thymoglobulin• Sirolimus
Year
Renal Transplant Outcomes
• Radiation• Prednisone• 6-MP
• AZA•ATGAM
• Everolimus• Belatacept
%
Figure 1. Consequences of Immunosuppression
TOO LITTLE
Acute rejection
Acute graft dysfunction
Chronic graft inflammation
Chronic graft fibrosis and atrophy
Graft loss
Acute inflammation
TOO MUCH
CMV BK virus infection or graft PTLD
Acute inflammation
Figure 1. Consequences of Immunosuppression
TOO LITTLE
Acute rejection
Acute graft dysfunction
Chronic graft inflammation
Chronic graft fibrosis and atrophy
Graft loss
Acute inflammation
TOO MUCH
CMV BK virus infection or graft PTLD
Acute inflammation
Recipient APC
CD4+ T-cell
Recipient MHC with donor alloantigen
Recipient CD4+ T-cellswith T-cell receptor
Y
Signal 1 Signal 2
B7.1/CD80
CD28
ActivatedCD4+T-cell
Signal 3(cytokines
)
Th1 cell
Th2 cell
Th3 cell
T-regcell
IL-12IFN-g
IL-2IFN-g
IL-4, IL-5IL-6, IL-10,
IL-13
IL-17, IL-21IL-22
IL-10IL-35
TGF-beta
IL-2IL-4
IL-6TGF-beta
IL-12TGF-beta
Antigen recognition and activation
Th17 cell
IL-10TGF-beta TGF-beta
Adapted from Nankivell and Alexander, NEJM 2010; also Hall CJASN 2015
MHC
EBNAS
EBV
LMP1
LMP2A
Latently-infected B cell
CD8+ T cell
NK cell
NKp46
NKG2D
Fas
FasL
CD4+ T cell
TCR
FasL
TCR
Dharnidharka et al, Nature Reviews Disease Primers, 2016
EBNAS
EBV
LMP1
LMP2A
PD-L1
PD-1
PTLD-associated EBV+
B cell lymphoma
CD8+ T cell
IL-10R
IL-10
NK cell PD-1
NKp46
Immunosuppression
NKG2D
Fas
FasL
CD4+ T cell
TCR
FasL
TCR
Dharnidharka et al, Nature Reviews Disease Primers, 2016
Prevention of PTLD
• Strategy used before any stage of the disease has occurred
• Types– Chemoprophylaxis with oral antivirals– Immunoprophylaxis:
• Active: no EBV vaccine as yet• Passive: IVIG or CMV-Ig
12
Prevention of PTLDChemoprophylaxis
Funch DP, et al. Am J Transplant 2005;5:2894-2900.
Risk of PTLD with days on antiviral therapy during first year post-
transplant
• Prophylactic antiviral use associated with up to 83% reduction in the risk of PTLD
• Risk of PTLD during the first year was lower by 38% for every 30 days of ganciclovir treatment
Pre-emptive Therapy• Strategy to prevent progression from an early
precursor stage to full blown disease
2Significant decrease in EBV DNA load after reduction of immunosuppression
141Lee TC, et al. Am J Transplant 2005;5:2222-2228.
2Bakker NA, et al, Transplantation 2007;83:433-438.
1 EBV viral load during immunosuppressionwithdrawal
Published biomarker and source Marker For what? Limitations
Protocol biopsy Histology, staining Acute rejection, BK infection Very invasive, expensive
Blood donor-specific antibodies Anti-HLA Ab Antibody mediated rejection, chronic allograft dysfunction
Information limited to humoral component of immune system
Blood pharmacogenomics CYP3A5 Tacrolimus metabolism Does not look at total immune system
Blood or urine PCR Granzyme B, Fas L, FoxP3, perforin, IP-10, fractalkine
Acute rejection Confounded by ischemia, BK virus infection or chronic allograft dysfunction
Viral load monitoring EBV/CMV/BKV Infection Only looks at over-immunosuppression
Blood Microarray AlloMap* Acute rejection (heart transplants only)
Only looks at under-immunosuppression
Blood Immuknow assay CD4-ATP level* Immune status Better at predicting infection risk than acute rejection risk
Blood or urine ELISA or ELISPOT Soluble CD30, Interferon-γ Acute rejection Only looks at under-immunosuppression
Donor-derived cell-free DNA (ddcfDNA) DNA Acute rejection Confounded by BK virus infection
How to judge the immunosuppression state?
•Immune system is sophisticatedly complex •Perhaps NO single biomarker can tell the immunosuppression state
Dharnidharka VR, Pediatr Transplant 2012
Molecular markersAlloMap• ITGAM, Flt 3,
IL1R2• G6B, PF4• WDR40A, MIR• ARHU• PDCD1• ITGA4• SEMA7A
Organ-I• DUSP1• PBEF1• PSEN1• NKTR• MAPK9
Quest• Granzyme B• Perforin• IP10
(CXCL10)• FOXP3
Biological role
• Oxidative cellular stress responses
• Vascular smooth muscle injury• Cell adhesion• IL2 dependent activation of
cytolytic genes • Apoptosis• Steroid responsiveness• Platelet activation• Hematopoiesis• Morphology/mobility• T cell activation• T cell trafficking• B cell activation• Cytolytic T cell effector molecules• T reg cell genes/cytokines
Organ-I now replaced by kSORT through Immucor
AR and BKVN confounding
Christakoudi et al, Ebiomedicine, March 2019
Donor-derived cell-free DNA
ddcfDNA, by itself, may not be able to distinguish injury associated with the interstitial inflammation and tubulitis caused by BKV from similar degrees of inflammation and tubulitis caused by TCMR, but support the tenet that an elevation in dd-cfDNA may be used to reveal the degree of active allograft injury. Bloom et al, JASN, 2017
Combination of markers?• Israeli et al, Transplant Int 2007• Combined different markers that
utilized different technologies• Panel A: stable patient
– Stable CD4-ATP levels in moderate zone
– No incline in PRA levels– sCD30 not elevated pre-
transplant, came down to undetectable levels
• Panel B: acute cellular rejection on Sept 26, 2004– Spike in CD4-ATP level– Incline in PRA level– sCD30 high pre-transplant, came
down but remained in elevated range
Prospectively
Dharnidharka et al, Transplantation, 2013
Serum kyn/trp ratios were higher before AR (P=0.031) and blood CD4-ATP levels were lower before MIE (P=0.008)
Viral PCR monitoring• Cytomegalovirus/Epstein Barr virus/BK virus• Considered by some as de facto biological measure of over-
immunosuppression• Serial monitoring
– When PCR turns positive, could lower immunosuppression– What about AR risk? Low in studies so far– Measures only one end of spectrum
• Can you use this to lower immunosuppression where graft survival is also patient survival?
• May instead be a marker of when to use pre-emptive anti-viral agents
Cutoff for PTLD risk?
Study Transplant Value DenominatorCesaro et al, Br J Haematol, 2005 Hematopoetic 300gEq Per 105 PBMCsAalto et al, Clin Infect Dis, 2007 Hematopoetic 50,000 Per mL serumHoshino et al, Br J Haematol, 2001 Hematopoetic 10,000 Per μg PBMC DNARuf et al, J Clin Virol, 2012 Hematopoietic
or heart20,0001,000
Per mL whole bloodPer mL plasma
Choquet et al, Am J Transplant 2014 Heart 100,000 Per mL whole bloodMatsukura et al, Clin Transpl 2002 Liver 100 gEq Per μg PBMC DNA
More cutoffs described in Gartner and Preiksaitis, J Clin Virol, 2010Variation in cutoffs is significant; relate to type of assay, underlying population studied, sample source
Variability of EBV Load Testing Between Centers
Prior to EBV International standard, Preiksaitis et al, AJT, 2009
WHO EBV standard for PCR
Current commercial extractors and targets in USA:
Using the International standard, Rychert et al, Clin Transplant, Oct 2014
Variation can be due to differences in:1. Extraction method2. Amplification reagents3. Genes targeted4. Calibrators used
Kit QiAmpVirus
QiAmpDNA Blood
QiagenVirus
QiagenMiniElute
Roche MagNAPure
Target EBNA1 EBNA1 EBNA1 EBNA-LP EBNA1
Amplicon 97 bp 97 bp 97 bp 155 bp 68 bp
Variability persists
Pattern analysis• Retrospective analysis of EBV DNA
quantitation data from 9779 samples; 740 transplant patients between 2003-2013, all whole blood specimens
• Overall, ROC AUC for peak level was 0.752, sensitivity of 58% and specificity of 88% at cutoff 105,000 copies/ml
• Overall, ROC AUC for rate of increase of 29, 280 copies/ml/week was 0.730, sensitivity 60% and specificity 86%
• In both cases, AUC values are good, not great
• In multivariate analysis, only peak level associated with PTLD, odds ratio 6.6, 95% CI 3.11-14
Cho et al, Am J Clin Pathol, 2014
How to interpret?
• Rising viral load, all done by same assay in same lab (red line): higher risk for PTLD
• An absolute cutoff is hard to define across studies
• Other patterns:– Transient viremia (dark green)– Intermittent viremia (light
green)– Chronic high load carrier
(yellow)
Community recommendations
Smith and Dharnidharka, Pediatr Nephrol, 2014
BK virus infection frequency
Hirsch et al, NEJM, 2002
Community recommendations BK
Dharnidharka and Araya, Pediatr Nephrol, 2009
BK virus screening and management practices among US renal transplant programs: a survey
Hodowanec et al, Transplant InternationalVolume 28, Issue 11, pages 1339-1341, 21 MAY 2015 DOI: 10.1111/tri.12602http://onlinelibrary.wiley.com/doi/10.1111/tri.12602/full#tri12602-fig-0001
BK virus monitoring
• The issues of gene target, international standard, assay standardization and appropriate cutoff ALSO apply to BK virus1
• 104 copies/ml viremia cutoff not recommended in a recent review2
– Only 2/22 developed BKVN (Geddes et al)– BKVN developed at 2845 copies (Almeras et al)
1Babel et al, Nature Rev Nephrol 20112Cannon et al, Curr Opin Organ Transpl 2011
What if viral surveillance missed?21 subjects
16Regular
10 Viral
replication
0FBVD
5Missed
3 Viral
replication
2FBVD
62% l Viral Replication incidence 60%
Yes Intervention opportunity No
0% % of Progression into FBVD 40%
Odds ratio = 23.57, p = 0.047
Al Khasawneh et al, Pediatr Transplant 2012
Transplant International, 2016
Choosing Immune Suppression in Renal Transplantation by Efficacy and Morbidity
The CISTEM study
Funded through R01DK102981
3-year predicted outcomes: CISTEM
R1) Thymoglobulin (TMG)+Tac+Myco+Pred after 30 days (reference group)R2) IL2R+Tac+Myco+Pred after 30 days; R3) TMG+Tac+Myco+no Pred after day 30; R4) CsA+Myco+Any induction; R5) de novo SRL+any IS agent+any induction
What does our center do?• CMV: We use valganciclovir prophylaxis except when both
CMV and EBV D-/R-, – No CMV monitoring (others still perform)
• BKV: We test monthly x 12 by PCR, initially urine only, then blood if urine is positive (others do not test urine)– Reduce immunosuppression if any viremia > accurately
quantifiable level• EBV: We test whole blood monthly x 12 by PCR
– Avoid increase in immunosuppression if viremia above accurately quantifiable level (4,000 copies/mL)
– Reduce immunosuppression if log fold increase in viremia, from same lab, with same assay and source
• Type of reduction is individualized
SUMMARY
1.Viral infections represent at least as big a threat as acute rejection – in current era, maybe a bigger threat
2.Delicate balance of immunosuppression needed, no great biomarkers
3.Current strategies: PCR viral surveillance, potential use of surveillance biopsies and novel biomarkers, adjusting immunosuppression
