MI 095 Bye Bye Biopsy: Diffusion Tensor & Dynamic Contrast Enhanced Magnetic Resonance Imaging correlate with

molecular events of Inflammation in synovium

Vikas Agarwal1, Rishi Awasthi2, Deepak Tripathi1, Vinita Agrawal3, RKS Rathore4, Kusum Sharma5, CM Pandey6 and, Rakesh Kumar Gupta2
1Department

of Clinical Immunology, 2Radiodiagnosis, 3Pathology, 6Biostatistics, SGPGIMS, Lucknow. 4Department of Mathematics, IIT, Kanpur. 5Department of Medical Microbiology, PGIMER, Chandigarh

Financial Disclosures
No conflicts of interest to disclose

Chronic Inflammation
Infiltration with chronic inflammatory cells
Expression of proinflammatory cytokines & chemokines.

Repair involving neoangiogenesis and fibrosis

Diffusion Tensor Imaging

Protons Aligned in MF
H+ H2O H2O

H+
OHH+ OH-

Restriction in Motion due to cell Membrane RF Pulse

H+ OHH+ OHH+ OH-

H+
OH- + H H+ OHH+ H+ OHOH-

H+
OH-

H+
OH H+

Magnetic field

OHH+

OHH+ H+ OHH+ OHH+ H+ OH OHOH-

H2O

H2O OH-

Diffusion Gradient

H+ OH-

OH-

DTI indices

FA =Fractional Anisotropy
MD = Mean diffusivity CL = Linear anisotropy CP = Planar anisotropy CS= Spherical isotropy

Diffusion anisotropy can be exploited to gain information about the tissue organization at a microscopic level.
(Denis Le Bihan at el. J Magn Reson Imagn 2001)

Perfusion MRI: Dynamic Contrast Enhanced Imaging

Provide information to changes in contrast concentration over time

Implementation of Pharmacokinetic models
(i) Pharmacokinetics (for ktransand kep) (ii) Hemodynamics (for BV and BF)

The DTI indices were measured in the inflamed synovial membrane whereas
inflammatory cytokines were measured in synovial fluid, But not in synovial membrane.

Agarwal, V. et al. Rheumatology (Oxf.) 48,(2009)

DTI could replace synovial histology or synovial fluid analysis for serial assessment of the synovial inflammation

Jenny Buckland; Nature Review Rheumatology volume 5; May 2009

Hypothesis
DTI derived metrics may reflect the aggregation of the inflammatory cells in the inflamed synovium. DCE imaging may provide information regarding neo-angiogenesis in the inflamed synovium. These together may provide information about the ongoing inflammation, and its molecular correlates in situation in vivo.

Objectives
1. To correlate DTI & DCE metrics values with the inflammatory parameters;
Types and numbers of various immune inflammatory cells (CD4, CD8, B cell, Macrophage, Plasma cells) infiltrating in the synovium Proinflammatory molecules [TNF- and IL-1], expressed on these cells Adhesion molecules ICAM-1 (cd54) expressed on these cells. Proinflammatory cytokines in the SF

2.

To establish DTI & DCE metrics values as the potential surrogate marker of active inflammation in-vivo.

Patients
Newly diagnosed subjects with a inflammatory arthritis involving the knee joints were enrolled in the study Age : >18 years Inclusion criteria:

Subjects with chronic inflammatory arthritis, mono- or oligoarthritis

Early inflammatory arthritis of more than 6 weeks duration

Study Population =65 [Mean Age= 39.6 Yrs]

Image Acquisition Conventional MRI including DTI and DCE-MRI was performed on a 3-Tesla MR scanner using a standard 8channel knee coil Image Quantification Masks of synovial regions that enhanced on post contrast T1-weighted imaging were created using an automated segmentation algorithm. Created masks were used to segment the inflamed synovium to extract various DCE (BV, BF, ktrans, ve) and DTI (FA, MD) metrics from the segmented regions

Arthroscopic Knee Synovial Biopsy

Synovial Tissue

Synovial Fluid

Serum

Histopathology H & E staining

Immunohistochemistry [CD3,CD4,CD8,CD20,CD34,CD 54,CD68,CD138,IL-1,TNF-]

Cytokine Analysis [IL-6, TNF-, IL-1]

DTI and DCE Analysis

Synovial Infiltrate Analysis

ROI
(Region of Interest)

Results
Demographical Data
No. of Patients 65

Descriptive Data
DTI indices FA MD (10-3
mm2sec-1)

Mean SD 0.220.03 1.620.50 0.050.02 0.140.05 0.750.02 109.9142.83

Age (Mean SD)

M/F Disease duration months ( Mean SD) Diagnosis RA

39.6113.42(18-76)
45/20 29.236.6 (6-192 months) 6

CL CP CS BF(ml/100gm/
min)

DCE indices
BV(ml/100gm) 9.5444.20 2.491.00 k ep(min-1) 1820.44211.64 PCI Immune cells in synovium 154.9348.65 CD3 63.4132.85 CD4 53.5817.62 CD8 39.3313.96 CD20 52.9317.27 CD34 36.3513.14 CD54 163.2034.61 CD68 36.0614.49 CD138 TOTALCEL 489.66106.38 LS 31.0918.15 IL-1 24.7011.51 TNF-

Chronic Monoarthritis
OA TB USPA Undifferentiated Arthritis JIA AS Reactive Arthritis Leprosy

11
3 15 14 10

1
2 2 1

Cytokines in the Synovial fluid

IL6 IL-1 TNF- 9788.304734.44 25.4622.56 88.4957.25

Correlation between FA and Inflammatory Immune cells in the inflamed synovium

0.35 0.3 0.25 FA 0.2 0.15 0.1 0.05 0 0 50 100 150 200 CD 4 Linear (CD 3) Linear (CD 8) 0.35 0.35 0.3 0.25 FA 0.2 0.15 0.1 0.05 0 0 CD 34 20 CD 54 40 60 80 100 Linear (CD 34) Linear (CD 54) IL-1 beta TNF-alfa Linear (IL-1 beta) Linear (TNF-alfa)
FA
CD34 r=0.55 p=<0.001 CD54 r=0.51 p=<0.001 CD3 r= 0.80 p=<0.001 CD4 r=0.77 p= <0.001 CD8 r=0.85 p= <0.001

0.35 0.3 0.25 FA 0.2 0.15 0.1 0.05

CD20 r=0.27 p= 0.026 CD138 r=0.30 p= 0.013

250

300

0 0 CD 20 20 CD 138 40 Linear (CD 20) 60 80 Linear (CD 138)

CD 3 CD 8 Linear (CD 4)

0.3 0.25 0.2 0.15 0.1 0.05 0 0 20 40 60

IL-1 r= 0.60 p=<0.001 TNF- r=0.41 p=0.001

80

100

0.35 0.3 0.25 FA

CD68 r=0.68 p=<0.001

0.35 0.3 0.25 0.2 FA 0.15 0.1 0.05 0

Total cells r=0.91 p=<0.001

0.2 0.15 0.1 0.05

0
0 50 CD68 100 150 Linear (CD68) 200 250

200 Total Cells

400

600

800

Linear (Total Cells)

DTIMRI

Infiltrated Immune cells in synovium

CD3 CD 4
0.77** -0.39** 0.32** 0.05

CD 8
0.85** -0.46** 0.41** 0.12

CD 20
0.27* -0.08 0.25* 0.02

CD68
0.68** -0.28* 0.14 0.10

CD 138
0.30*
-0.08 0.25* 0.02

CD 54
0.51** -0.20 0.17 0.08

TNF- IL-1
0.41* -0.01 0.23 0.08 0.60** 0.30* 0.51** 0.06

FA MD CL CP

0.80** -0.39** 0.45** 0.04

Total Inflammatory cells 0.91** -0.43** 0.46** 0.07

CS
PCI

-0.30*
-0.07

-0.37*
0.11

-0.35**
0.10

0.07
0.06

-0.35**
-0.03

0.07
0.17

-0.16
-0.16

-0.15
0.15

-0.34*
0.107

-0.32**
0.069

** Correlation is significant at the 0.01 level ; * Correlation is significant at the0.05 level

Correlation between DCE indices and Angiogenesis in the inflamed synovium

250 200 150 100 50 0 0 20 40 CD 34 60 80 100 Blood Volume Blood Flow
CD34 r=0.814 p=<0.001

20 15 10 5 0 0 20 40 CD34 60

CD34 r=0.84 p= <0.001

80

100

Series1

Linear (Series1)

Series1

Linear (Series1)

5 4 Kep 3 2 1 0 0 20 40 CD34 Series1 60

CD34 r=0.30 p= 0.01

DCE -MRI Indices

Angiogenesis Marker in the synovium CD 34

BF BV
80 100

0.81** 0.84**

k ep
PCI

0.30*
0.15

Linear (Series1)

Linear Regression Analysis Model

DTI indices predict the no. of T cells in the synovium
Preditor IHC
(Estimates CD3 CD4 CD8

0
-126.66 -120.28 -56.01 11.50 3.79 -7.32 -12.44 -48.16 -10.08

1
1269.34 828.04 494.02 125.45 145.44 768.64 219.97 357.25 156.83 3168.58

R2
0.64 0.59 0.73 0.00 0.09 0.46 0.26 0.36 0.17 0.83

Sig.
<0.001 <0.001 <0.001 0.026 0.013 <0.001 <0.001 <0.001 0.001 <0.001

SE
(estimates)

FA

29.42 21.02 9.08 13.52 13.89 25.55 11.36 14.58 10.54 43.67

CD20
CD 138 CD68 CD54 IL-1 TNF-

Total Cells - 213..27

DCE MRI indices predict the angiogenesis in the synovium

Dependent Variables (IHC) DCE 0 Metrices (Predictors) 1 R2 Sig. SE (estimates)

CD 34

BF BV Kep PCI

16.83 19.68 39.69 0.01

0.329 3.484 5.307 29.71

0.663 0.719 0.095 0.024

<0.001 <0.001 0.013 0.214

10.10 9.22 16.56 17.20

Conclusions
A high FA on DTI correlated with inflammatory cell aggregation in the inflamed synovium. DCE parameters (BV, BF) correlated with the markers of angiogenesis i.e. CD34. Correlation of both DTI and DCE parameters varied with the intensity of inflammation. Thus, DTI and DCE metrics capture cellular and molecular events of inflammation and correlated with the degree of inflammation.

Acknowledgement
This study was financially supported by Indian Council of Medical Research (ICMR, Grant No. 5/4-5/4/Ortho/08/NCD1), New Delhi, India.