April 24th, 2023

AAN Meeting

Autoantibody Testing for

Autoimmune Myopathies
Andrew Mammen, M.D., Ph.D.
Senior Investigator, NIAMS/NIH
Adjunct Professor of Neurology and Medicine, Johns Hopkins
 Disclosures
• I have patented an anti-HMGCR
autoantibody test, but do not receive
compensation for this
• I will discuss off-label treatments for
myositis
 Overview
• Define the 6 major types of myositis
• Show that each myositis-specific
autoantibody (MSA) has a unique phenotype
• Explain how MSAs may be used for
diagnosis
• Describe a new MSA
• Provide data suggesting that MSAs may
have a functional effect on muscle cells
 Myositis is a
heterogeneous family of
diseases
Six major types of myositis
• Dermatomyositis (DM)
• Antisynthetase syndrome (ASyS)
• Immune-mediated necrotizing myopathy
(IMNM)
• Inclusion body myositis (IBM)
• Overlap myositis
• Immune checkpoint inhibitor-triggered
myositis
• Most cases diagnosed as polymyositis
have
–Inclusion body myositis
–Immune-mediated necrotizing myopathy
–Antisynthetase syndrome without a rash
–Overlap myositis
–Inherited muscle disease
 Dermatomyositis
Muscle Skin Autoantibodies
Anti-NXP2
Anti-Mi-2
Heliotrope rash
Anti-MDA5
Anti-TIF1g
Perifascicular Atrophy
Anti-SAE

Gottron’s papules
ENMC guidelines: three ways to
diagnose DM
1. Classic skin rash and skin biopsy showing
interface dermatitis
2. Classic skin rash, proximal weakness,
muscle biopsy with typical DM features
(e.g., perifascicular atrophy)
3. Classic skin rash and one of the DM-
specific autoantibodies
Mammen et al, Neuromuscular Disorders, 2019
 Anti-NXP2+ DM
• Subcutaneous
edema
• Finger extensor
weakness
• Calcinosis
• Dysphagia
• Modestly increased
cancer risk
Albyda and Pinal-Fernandez, AC&R, 2017
 Anti-Mi2+ DM
• Most severe weakness
• Highest CK values
– Anti-Mi2+ DM mean max CK 3908 (2230-7070) IU/L
– Other DM mean max CK 242 (110-1200) IU/L
• Most necrosis and inflammation in muscle
biopsy
• Minimal cancer risk
Pinal-Fernandez, Neurology, 2019
 Anti-MDA5+ DM

Ulcerating Ulcerating
palmar surface heliotrope
lesion

Ulcerating
Ulcerating
palmar surface
Gottron’s
lesion
 Anti-MDA5+ DM
• Mild weakness (may be
hypomyopathic or
amyopathic)
• Often have severe and
rapidly progressive
interstitial lung disease
(especially in Asian Cato et al., A&R, 2009
populations) Cao et al., AC&R, 2012
Fiorentino et al., JAAD, 2011
– High mortality rate (up to 60%) Betteridge et al., A&R, 2012
Moghadam-Kia, AC&R, 2016
Anti-MDA5+ ILD: tofacitinib may
improve survival

Z Chen et al. N Engl J Med 2019;381:291-293.

Anti-TIF1g (p155/140)+ DM:
increased cancer risk

• Six studies including 312 adult DM cases

• Sensitivity = 78%, Specificity = 89%
• PPD = 58%, NPD = 95%
Arthritis and Rheumatism, 2012
Only ~70% of DM patients have an
MSA…

Can we identify novel DM

autoantibodies?
 Phage immunoprecipitation
sequencing (PhIPseq): a tool to
identify antibodies
a) Oligonucleotide
library synthesis to
encode proteomic-
scale peptide
libraries for display
on bacteriophage
b) Immunoprecipitate
using patient
antibodies
c) Analysis by high-
throughput DNA
sequencing

Mohan et al, Nature

Protocols, 2018
 Transcription factor Sp4 is a novel
autoantigen in anti-TIF1g+ DM
• Collaborator: Ben Larman
• Autoantibodies against Sp4
protein identified by PhIPseq

Anti-Sp4 titer
using serum from 43 DM patients
• Developed anti-Sp4 ELISA
• Screened serum from 371
myositis patients, 80 disease
controls, 200 healthy controls
• Anti-Sp4 only present in DM

y
is

th
s
sit

RA
re
pu

al
og
yo

He
Lu
• All anti-Sp4+ patients were also

Sj
anti-TIF1g+
Hosono et al, ARD, 2023
Anti-Sp4 identifies anti-TIF1g+ patients
without cancer
Perhaps anti-
TIF1g+ DM

Anti-Sp4 titer (AU)

patients with
anti-Sp4 do not
need
aggressive
cancer
screening With Cancer Without Cancer
Hosono et al, ARD, 2023
 What is relationship between cancer,
autoantibodies, and dermatomyositis?
• Hypothesis
• Tumors precipitate anti-tumor response
including humoral autoimmunity
• Anti-TIF1 immune response may elicit DM
but not eradicate tumor
• Anti-TIF1 + anti-SP4 immune response may
elicit DM but eradicate tumor
 The antisynthetase syndrome
• Defined by autoantibodies
recognizing one of the
aminoacyl-tRNA synthetases
– Histidyl-tRNA synthetase (Jo1)
– Alanyl-tRNA synthetase (PL12)
– Threonyl-tRNA synthetase (PL7)
– Glycyl-tRNA synthetase (EJ)
– Isoleucyl-tRNA synthetase (OJ)
 The antisynthetase syndrome

Myositis Arthritis Interstitial Lung Disease

Mechanic’s Hands Rash Raynaud’s Phenomenon

 Diagnosing the antisynthetase
syndrome
1. Presence of an antisynthetase
autoantibody
2. One or more of the following
a) Myositis
b) Interstitial lung disease
c) Arthritis
Casal-Dominguez et al., Arthritis and Rheumatology, 2022
 Immune-mediated necrotizing
myopathy
• Anti-HMGCR myopathy ENMC
– Proximal muscle weakness and high CK guidelines:
– Anti-HMGCR+
muscle biopsy
• Anti-SRP myopathy not required
– Proximal muscle weakness and high CK for diagnosis
– Anti-SRP+
• Antibody negative IMNM
– Proximal muscle weakness and high CK
– No myositis-specific autoantibodies
– Necrotizing muscle biopsy
Allenbach et al., Neuromuscular Disorders, 2018
 Anti-HMGCR myopathy
• Muscle biopsy: necrotizing (20% with
inflammatory cell infiltrate)
• 100% proximally weak
• 75% with myalgias
• Maximum CK ~10,000
• Minimal extramuscular manifestations
• Associated with statin exposure (~70%)
• ~1 per 50,000 statin users
• Progresses despite stopping statins
• Statins may trigger flares Mammen, NEJM, 2016
 Anti-SRP myopathy
• Muscle biopsy: necrotizing
• Rapidly progressive
• Severe weakness
• Very high CK levels
• Mild interstitial lung disease in ~20%
• Occasional cardiac involvement
• Difficult to treat Targoff et al., A&R, 1990
Miller et al., J Neurol. Neurosurg. Psychiatry, 2002
Kao et al., A&R, 2004
Hengstman et al., Ann. Rheum. Dis., 2006
 Antibody-negative IMNM

• Probably a heterogeneous mixture of diseases

• Compared to seropositive IMNM
• Increased female predominance (3:1)
• More likely associated with connective tissue
disorder (22% vs 9%) and extramuscular
disease activity (50% vs. 16%)
 Inclusion Body Myositis
*

Invasion of myofibers by
Autoantibodies
CD8+KLGR1+ T cells
Anti-NT5C1A
**

Rimmed vacuoles *https://neuromuscular.wustl.edu/pathol/ibm.htm

**Courtesy of Dr. Antony Amato
Anti-NT5C1a: common in IBM, but not
in other neuromuscular disorders
Larman, et al, 2013

• Sensitivity = 72%
• Specificity = 92% Larman, et al., Annals of Neurology, 2013
 Anti-NT5C1a: common in IBM, but
not in other neuromuscular disorders
Larman, et al, 2013

6/36
(17%)
of DM

• Sensitivity = 72%
• Specificity = 92%
Larman, et al., Annals of Neurology, 2013
Anti-NT5C1a autoantibodies may
not be IBM-specific

ARD, 2014
PLOS One, 2023

• Commercially available
ELISA
AC&R, 2016
• 23-36% with Sjogren’s • 40% with IBM
• 15% with DM
• 14-20% with lupus • 7% with IMNM
• 15% with DM
 Do anti-NT5C1a positive IBM patients
have more severe disease?
Yes No
ARD, 2017 Neurology, 2023

PLOS One, 2023

JNNP, 2016

Perhaps INSPIRE-IBM observational study will provide a definitive answer

My favorite IBM diagnostic criteria
• All three of the following features
– Finger flexion OR knee extension weakness
– Endomysial inflammation
– Invasion of non-necrotic muscle fibers OR rimmed
vacuoles on biopsy
• Sensitivity 90%
• Specificity 96%
• Requires muscle biopsy but not antibody testing
Lloyd et al, Neurology, 2014
Autoantibody testing: practical matters
• There are lots of methodologies: ELISA, line blot, RIPA…
• Know what methodology you are using
• Recognize that some methodologies have drawbacks
– High false negative rate for anti-TIF1g using line blot
– Treat all DM cases as high cancer risk if no known antibody
• If the autoantibody test is inconsistent with the clinical
phenotype, consider confirmation using another method
• My gold standard: the Oklahoma Medical Research
Foundation (slow but reliable)
• Minimize false positives by only testing patients with
reasonable pre-test probability
 If you are interested in the intricacies of
myositis autoantibody testing…

• Damoiseaux et al.,
Neuromuscular
Disorders, 2022
• PMID: 35644723
• DOI: 10.1016/j.nmd.20
22.05.011
Are myositis autoantibodies
pathogenic?
 Mouse models of myositis

Immunizing mice with Jo1 protein

causes muscle and lung
inflammation (J Autoimmun., 2007)

Immunizing mice with TIF1 Transfer of human anti-HMGCR or

protein causes muscle anti-SRP into mice causes weakness
inflammation (ARD, 2021 ) (ARD, 2019 )
 Is there evidence that
myositis autoantibodies
may have a functional
effect in humans?
 Anti-Mi2 autoantibodies
• Recognize Mi2a and Mi2b
• Mi2a and Mi2b located in
nucleus
• Mi2a and Mi2b are
components of the
nucleosome remodeling
and deacetylation (NuRD)
complex
• The Mi2/NuRD complex is a
Ramiraz and Hagman, Epigenetics, 2008
transcriptomic repressor
 Can anti-Mi2
autoantibodies get into
muscle cells?
 F
Immunoglobulin is deposited in myonuclei
of anti-Mi2+ DM (and cytoplasm of anti-
HMGCR+ IMNM)
HST IGG/LMN/HOECHST
G
F
MERGE IGG HOECHST
*
*
*
H Anti-HMGCR

* Anti-HMGCR+ IMNM
*
Anti-Mi2+ DM
* Anti-Mi2 Pinal-Fernandez, ARD, accpeted 2023
 Can anti-Mi2
autoantibodies bind to
Mi2/NuRD complex and
disrupt its function?
RNAseq on 206 muscle biopsies to
define transcriptomic profiles
• Normal (n=33) • IMNM (n=54)
• DM (n=50) – Anti-SRP (n=10)
– Anti-Mi2 (n=18) – Anti-HMGCR (n=44)
– Anti-NXP2 (n=14) • IBM (n=16)
– Anti-TIF1g (n=12) • Immune checkpoint
– Anti-MDA5 (n=6) inhibitor-triggered
• ASyS (n=18) myositis (n=35)
• Identified a set of
135 genes only
upregulated in anti-
Mi2+ DM muscle
• The gene set is
enriched for those
known to be
transcriptomically
repressed by
Mi2/NuRD
Pinal-Fernandez, ARD, accepted 2023
 0 0
0 0 0
PRR35
SCRT1
CHRM4 P2RX2 IFITM5
PLPPR3
44
4 4
2.5 2.5

2.0
SCRT1: The
4 3
2.0
333
3most highly
3
log2(TMM+1)

1.5 2
1.5

2
2
2 2
1.0
expressed 1.0
2
1 1
0.5
anti-Mi2- 1
0.5
1
0
0 0
0.0
specific
1 gene 0
0.0
PRR35 IFITM5
An T

i2

AS

An T

i2

AS

M
04 0
N

N
M

M
D

IB

IB
IM

IM
ti−

ti−
CHRM4 4 PLPPR3 4
43 is a transcriptional repressor
SCRT1 2.5
+1)

3 Pinal-Fernandez, ARD, accepted 2023

3
 MADCAM1/LMN/HOECHST SCRT1/LMN/HOECHST IGG/LMN

SCRT1 localization
A C F

• Only in anti-Mi2+ muscle

cells D
G

–In the nuclei B

–In the cytoplasm

E
surrounding the nuclei of H

severely damaged fibers

A 4.3 B 3.8 C 3.5

• SCRT1 expression
levels correlate with
D 2.8 E 1.6 F 1.0 anti-Mi2
autoantibody titers
• SCRT1 expression
levels correlate with
G 0.8 H 0.1 I 0.0 degree of necrosis
and inflammation

Pinal-Fernandez, ARD, accepted 2023

 Cell membrane

Nuclear membrane

Mi2/NuRD -
SCRT1
Muscle Cell
Anti-Mi2 Cell membrane

Nuclear membrane

Mi2/NuRD -
SCRT1
Muscle Cell
 Cell membrane

Nuclear membrane

Anti-Mi2 Mi2/NuRD
SCRT1 protein
+
SCRT1
Muscle Cell
 Cell membrane

Nuclear membrane
Muscle damage?
Anti-Mi2 Mi2/NuRD
SCRT1 protein
+
SCRT1
Muscle Cell
 Review of main points
• MSAs define unique myositis subtypes
• MSAs can be useful for diagnosis
• Anti-Sp4 ”protects” against cancer in DM
• New hypothesis: anti-Mi2 autoantibodies
enter muscle cell nuclei, bind to Mi2, and
derepress gene expression, potentially
causing muscle cell damage
Thanks!