Evidence for the link between markers of inflammation, coagulation and immune activation and end organ disease Session: Immune Activation/Inflammation and HIV Disease IAS Conference, Rome, July 2011 Jens Lundgren, MD Chair – INSIGHT’s scientific steering committee Professor – Univ. of Copenhagen Chief physician – Copenhagen University Hospital Director – Copenhagen HIV Programme

Linking markers to organ disease: prognostic and surrogate biomarkers Prognostic marker –Predicts the risk of disease Causal: marker of process involved in pathogenesis Epiphenomenon: not linked causally (e.g. subclinical stages of disease itself raises levels) Surrogate marker –Change in level reflects the extent of benefit of an clinically beneficial intervention Occurs sooner after intervention is initiated than the disease –Surrogates are usually prognostic markers, but the opposite is seldom true

Validation of surrogate biomarkers Associated with Clinical Events [Case-control or Cohort Studies] Modify with Treatment [Biomarker Studies (Phase II/Pilot/ Vanguard Studies)] Markers that can be reliably measured Stored specimens Prospectively identified and adjudicated events Excellent follow-up Study is large enough to do nested case-controlled studies [Clinical Trials with Clinical Outcomes] Rx Effect on Biomarkers Predicts Rx Effect on Clinical Events

Median CD4+ During Follow-up Avg Difference: 160 cells, p<.001 Time spentIL-2Control < 300 cells6%9% > 600 cells57%36% IL-2: Control: No. pts ESPRIT/INSIGHT study group, NEJM 2009

Primary Endpoint Opportunistic Disease or Death IL-2Control No.Rate*No.Rate*HR (95% CI)p-value (0.75, 1.16)0.52 Predicted HR based on CD4+ difference = 0.74 * rate per 100 person years ESPRIT/INSIGHT study group, NEJM 2009

Soluble Biomarkers of Inflammation & Coagulation in HIV Biological process:Name of marker:Marker of: Activation of: immune cells, endothelium, platelets, and coagulation Interleukin-6 hs-CRP sCD14 D-dimer P-selectin sCD40 ligand Lymphocyte Acute phase reactant Macrophage/monocyte fibrinolysis platelets + endothelium platelets + endothelium + lymphocytes Organ dysfunction hyalyronic acid, FIB-4 NT-proBNP ADMA eGFR, protinuria Liver Heart Endothelium kidney

ESPRIT and SMART Control Group: Deaths by D-dimer Quartile at Study Entry ESPRITSMART D-dimer levels: >0.38 g/mL <0.19 D-dimer levels: >0.37 g/mL <0.13

ESPRIT Control Group: Hazard Ratios (D-dimer Q4/Q1) for Death by 4-Year Intervals HR (4th/1st Quartile) p-value = 0.18 for change in HR over follow-up 1 st 4 years follow-up> 4 years follow-up # deaths, quartile 4:1524 # deaths, quartile 1:44 HR (95% CI):3.48 ( )5.59 ( )

OR for Mortality Associated with Baseline Biomarker Levels for Early (0-2 years) and Late (2 > years) Deaths in SMART Odds Ratio (4th/1st Quartile) Paton et al, IAS 2009 (#MOPEA034) hsCRPIL-6D-dimer Median Level (IQR)≤ 2 yr> 2 yr≤ 2 yr> 2 yr≤ 2 yr> 2 yr Deaths3.13 ( ) 3.69 ( ) 3.58 ( ) 3.72 ( ) 0.45 ( ) 0.31 ( ) Controls2.08 ( ) 1.93 ( ) 2.14 ( ) 2.33 ( ) 0.24 ( ) 0.24 ( # Deaths/Controls95/18871/13792/18467/13394/18869/128

Association with risk of death is reduced the longer the time from measurement of biomarker in persons with peripheral arterial disease Vidula et al, Ann Intern Med 2008

Trajectories in d-dimer Levels in ESPRIT Control Group at study entry, 12 and 36 Months N=244 Upper Limit of normal % above ULN (0.25 µg/mL) 39% 42% No significant change over time

ESPRIT and SMART Control Group: Hazard Ratios for Quartiles (4 th vs. 1 st ) of D-dimer Levels at Study Entry ESPRITSMART EventHRP-valueHRP-value Death4.5< AIDS CVD Non-AIDS malignancy Serious Non-AIDS (SNA) SNA or death from non-AIDS

Change in D-Dimer* from Study Entry to 1 Month ≤ ,00010,000-50,000>50,000 Month 1 HIV RNA Level (copies/mL) ∆ D-Dimer (µg/mL) P=.0005 for trend * DC patients on ART at baseline with HIV RNA ≤ 400 copies/mL Kuller et al, PLoSMed 2008

D-dimer Levels After Starting ART in SMART All participants were off ART at baseline (n=254) Randomized comparison of immediate ART (VS; n=128) or deferral (D No difference in IL-6 and hsCRP All participants were off ART at baseline (n=254) Randomized comparison of immediate ART (VS; n=128) or deferral (D No difference in IL-6 and hsCRP Baker et al JAIDS 2011

SMART: Comparison of Associations with Different Outcomes: Univariate ORs (4 th versus 1 st quartile) – Case-Control Analyses All cause mortality CVDAIDS D-dimer IL Small HDL particles sCD Total HDL particles

Cellular markers of end organ disease in HIV Activation of CD4 and/or CD8 cells and markers of organ disease (cross- sectional studies) –Intima-media thickness ( Hsu et al, AIDS 2006, Kaplan et al, JID 2011 ) and arterial stiffness ( Kaplan et al, Atherosclerosis 2011 ) Linked to AIDS/death events –In cross sectional analysis: OR= 1.94 for risk of AIDS/death by 17% more %CD8 + /HLA- DR + /CD38 + ( Kalayjian et al, JID 2010 ) OR=6.5 for AIDS for 4th vs 1st quartile ( Smurzynski et al, JAIDS 2010 ) –In cohort studies HR=1.61, P=0.042 ( Hunt et al, CROI 2011 ) Still unclear –prognostic marker for organ disease –changes reflect surrogacy for benefit from interventions

Use of markers in routine clinical practise Identify persons at increased risk of disease –Intriguing that D-dimer levels predict risk after multiple years –Unclear which interventions specifically benefits such patients over- and-above benefits from common interventions used in internal medicine –Will cause concern Surrogates of interventions –Remains to be established Markers of inflammation and coagulation remains a research tool and are not yet ready for routine use in HIV medicine

Summary Many markers are prognostic but very few are surrogates of benefit for clinically useful interventions –Establishment of surrogacy is critical when assessing for potentially useful anti-inflammatory interventions This research requires large clinical endpoint driven RCT’s Soluble markers of inflammatory and coagulation long-term risk of – particularly fatal - end-organ disease D-dimer: potential for surrogacy of benefits from ART –additional research required (e.g. START) Data emerging on cellular markers of inflammation Markers of inflammation and coagulation: –useful tools to advance co-morbidity research agenda, –role in routine clinical practise remains to be determined.

Acknowledgements INSIGHT network incl. executive, scientific and operational committee members + international coordinating centres and affiliated sites –Jim Neaton, Jason Baker, Jacquie Neuhaus, Debby Wentworth, Andrew Phillips, Calvin Cohen and Steven Deeks Peter Hunt, UCSF Division of AIDS, NIAID

UARTO: High CD8+ T Cell Activation at Month 6 of ART Predicts Subsequent Mortality in Ugandans with VL<400 Hunt et al, CROI 2011 (306) In Cox Proportional Hazards models, each 10% increase in the frequency of activated (%CD38+ HLA-DR+) CD8+ T cells was associated with an increased hazard of death even after adjustment for baseline CD4 count (HR: 1.62, P=0.048) or month 6 CD4 count (HR: 1.61, P=0.042).