Cytokine array analysis in

Castleman’s disease and POEMS

syndrome
Professor Roy Sherwood
Consultant Clinical Scientist & Scientific Director, Viapath
Professor of Clinical Biochemistry, KCL
King’s College Hospital
London
 Castleman’s disease
– Group of uncommon
lymphoproliferative disorders
– Lymph node histological features
• 1954: Benjamin Castleman
The follicle is surrounded by a concentric layers of
lymphocytes resulting in an onion-skin appearance

Unicentric: localised to single lymph node

• No symptoms
• Removal is curative in 90%
Multicentric: systemic
• Lymphocyte proliferation due to ↑
proinflammatory cytokines
• Systemic inflammatory symptoms & multiple
organ system dysfunction
 Castleman’s disease

Interleukin-6 (IL-6)

– Diagnosis
• Exclude infectious, autoimmune & neoplastic
disease
• Confirmed by lymph node biopsy
• Human Herpes Virus-8 (HHV-8)
– HHV-8: 50% of MCD. Viral & Human IL-6
 B-cell & plasma cell proliferation,
VEGF & angiogenesis
– Idiopathic MCD (iMCD): 50% HHV-8-
 Castleman’s disease

Skin
lesions Fever

Lymphadenopathy
Weight
loss Symptoms
Loss of
appetite
Hepatosplenomegaly Night
sweats
Fatigue,
weakness
 POEMS syndrome
– Clinical & histopathological overlap with MCD
– Paraneoplastic syndrome – Vascular endothelial
growth factor (VEGF) secretion by malignant
plasma cells
– Plasma-cell proliferative disorder (myeloma >50%)
– Polyneuropathy (symmetrical & distal)
Minor Major
Pulmonary disease, Papilloedema Sclerotic bone lesions
Organomegaly Enlarged liver, spleen, lymph
nodes
Castleman’s Disease

Endocrinopathy Amenorrhoea, adrenal

insufficiency, hypothyroidism
 VEGF

Monoclonal gammopathy
Skin changes Hyperpigmentation, skin
thickening, excess hair

Plasmacytoma right scapula with overlying erythema

as well as gynecomastia, muscle wasting, and ascites.
 Pathophysiology

– Mechanisms unknown
– ?Pathways for  cytokines in Castleman’s disease:
• NF-B: Primary transcription factor for many
cytokines. Gene mutation turning off
transcription  autoinflammatory disorders
• Cases with /norm IL-6, no response to anti-
IL-6  other pathways/cytokines
– POEMS paraprotein: insufficient for multi-organ
features. Cytokines linked to features of POEMS.

– Both: uncommon, poorly understood but can be

fatal
 Treatment

Corticosteroids improve symptoms

Chemotherapies eliminate hypercytokine-secreting
cells but side effects
• Relapse common

Monoclonal Abs (mAbs)

– E.g. Rituximab
• First-line for Castleman’s disease
• Anti-CD20 B cell mAb
• Immune response weakened  ↑ risk of
infection
 Treatment

– Siltuximab
• Anti-IL-6 mAb
• Approved for HIV & HHV-8 –ve Castleman’s
disease by FDA in April 2014
– Tocilizumab
• Anti-IL-6 receptor mAb
Long term administration & not always effective

– Bevacizumab
• Anti-VEGF mAb
 Cytokines

– Signalling peptides communicate via cell surface

receptors
– Chemokines, interferons & ILs
– Immune response & inflammation
IL-6 VEGF

• Lymphokine in 4α-helical
• Signal protein
long-chain family
• Stimulates vasculogenesis
• Regulates B & T cell
& angiogenesis
function, Ig secretion
 Cytokine measurement
– Implicated in pathophysiological conditions. ↑ blood
concentrations  ↑ production
– Concentrations reflect disease severity – biomarker
– Trials involving cytokine inhibitors require cytokine
monitoring during treatment
Hypothesis: measuring cytokines in plasma may aid
in guiding treatment and monitoring disease activity

Evidence Investigator

– Biochip Array Technology

– Quantitative detection of
multiple analytes from one
sample
 Evidence Investigator

– Biochip test menu includes:

Adhesion molecules Endocrine
Cardiac Metabolic syndrome
Cerebral Thyroid
Cytokine Tumour marker
– Custom arrays – design your own with up to 22
biomarkers
– Investigator features:
• High throughput – multiple results in short time
• Small sample volume
• Quality: CVs <10%, full traceability, QC (multi-
analyte controls & software), reference ranges
 Evidence Investigator

Ready to use
biochip carrier
Sample / reagent Incubation Wash
addition

Signal reagent Light emitted from Quantification

addition chemiluminescence
 Results
IL-4 VEGF INF-y TNFa IL-1a IL-1b MCP-1
Patient IL-2 ng/L ng/L IL-6 ng/L IL-8 ng/L IL-10 ng/L ng/L ng/L ng/L ng/L ng/L ng/L EGF ng/L
A, T 6.5 <2.1 0.6 1.3 <0.4 39.6 <0.4 0.7 <0.2 0.7 72.6 21.0
E, J <0.9 <2.1 1.9 7.2 0.5 205.0 <0.4 1.3 <0.2 0.5 76.3 60.8
E, J <4.8 <6.6 4.8 <4.9 <1.8 207.3 <3.5 <4.4 <0.8 <1.6 107.7 73.1
G, G <0.9 <2.1 44.9 1.2 <0.4 3.5 <0.4 1.2 0.2 <0.26 4.8 1.3
G-O, N <4.8 <6.6 3.0 36.5 <1.8 305.6 <3.5 <4.4 <0.8 <1.6 313.7 22.8
H, T <0.9 <2.1 1.7 17.3 26.1 266.5 <0.4 1.5 <0.2 0.5 60.3 28.9
R, M 2.3 <2.1 1.6 3.4 0.8 283.1 <0.4 1.2 0.3 1.5 26.6 9.9
R, M 11.8 <6.6 3.6 <4.9 5.5 806.5 5.4 <4.4 1.2 3.7 189.6 <2.9
N, N <0.9 <2.1 0.4 4.5 0.9 17.1 <0.4 1.5 <0.2 0.6 49.7 21.7
P, L <4.8 <6.6 74.2 22.5 7592.5 168.1 5.0 <4.4 <0.8 9.7 530.2 <2.9
P, L <4.8 <6.6 16.9 21.9 1515.4 214.1 <3.5 <4.4 <0.8 <1.6 310.7 <2.9
P, L <4.8 <6.6 <1.2 20.6 9.4 31.0 <3.5 <4.4 <0.8 <1.6 200.6 10.1
B, A 18.0 <6.6 4.1 11.2 8.0 700.4 7.8 <4.4 3.1 12.9 228.4 11.9
C, N <0.9 <2.1 0.6 1.2 <0.4 5.0 <0.4 0.8 <0.2 1.3 62.6 <1.0
F, M <0.9 <2.1 0.5 1.4 <0.4 6.5 <0.4 <0.59 <0.2 <0.26 13.9 <1.0
A, A <0.9 <2.1 0.4 0.9 <0.4 2.8 <0.4 0.7 <0.2 <0.26 12.7 <1.0

80 IL-6 in Castleman's disease 6000

VEGF in POEMS syndrome
70 5000

VEGF (ng/L)
4000
60
IL-6 (ng/L)

3000
50
2000
40
1000
30
0
20

10

0 Date
In remission Symptomatic
Stem cell transplant
 Summary

– mAb drugs are expensive & risk of side effects

– Not effective for all patients
• May not respond to anti-IL-6 if  IL-6
• HHV-8/HIV status
• Coexisting diseases

Future work
– Continue with cytokine analysis to baseline patients
prior to therapy
– Monitor cytokines during treatment
– Used to predict disease state before clinical
symptoms
Acknowledgments:

• Gemma Cross
• Tracy Dew
• Sadie Redding
• Prof Schey
• Reuben Benjamin