IBM

Inflammatory Myopathies with Vacuoles, Aggregates & Mitochondrial Pathology (IM-VAMP)

Inclusion Body Myositis (Subtype)

Pathology features

Inflammation
Focal invasion of fibers
General
IBM
Immune Cell types
Endomysial
Perimysial
Muscle fibers
Aggregates
Histochemistry
AMPDA
Amyloid-like
Cytoplasmic bodies
Eosinophilic
Components
αB-crystallin
β-Amyloid
Desmin
LC3
p62
SMI-31
TDP-43
Ubiquitin
VCP
Ultrastructure
Pathology
Focal invasion
MHC Class-1
Mitochondrial
Ultrastructure
Myopathy
Myonuclei
Vacuoles
Capillaries

Variant syndromes
IM-VAMP in HIV
PM-Mito

Ultrastructure
Aggregates
Focal invasion by cells
Mitochondria
Vacuoles

Gomori trichrome stain

IBM (IM-VAMP): Myopathy; Inflammation; Aggregates & Vacuoles

IM-VAMP (IBM): Muscle Pathology

General description
- Inflammatory Myopathy with Vacuoles, Aggregates & Mitochondrial Pathology (IM-VAMP)
Inflammation: Mononuclear cells
- Locations
  - Endomysium: Most common
  - Also in some patients: Perimysium & Perivascular
- Focal invasion of muscle fibers
  - Most common mechanism of muscle fiber damage
  - Cell types: CD4 + CD8 Lymphocytes & Macrophages
  - CD8 cell components ⁵
    - KLRG1 ¹
    - CD57 (HNK-1; LEU-7) ²
      - T cell large granular lymphocytic leukemia cells
      - Late differentiated T-cells: Reduced proliferation capacity
- Cell types in foci
  - T-cells
    - CD4: Most common
    - CD8: Present in many endomysial cell foci
  - B-cells: CD20
    - Usual: Few or none present
    - Foci reported: sIBM associated with chronic lymphocytic leukemia ⁷⁸
  - Plasma cells: CD138
    - Scattered in foci
  - Histiocytes
    - Many in foci & regions of focal invasion of muscle fibers
Muscle fibers
- Myopathic changes
  - Varied muscle fiber size
  - Regenerating muscle fibers
  - Groups of small fibers
  - Endomysial connective tissue: Variably increased or normal
  - Muscle fiber hypertrophy: More common than in other immune myopathies
- MHC-I
  - Expression: On surface, ± cytoplasm, of most muscle fibers
  - Occurs on morphologically normal & abnormal muscle fibers
  - No specific association with inflammatory cells or foci
- Rimmed vacuoles with granular material & filaments
  - Histochemistry: Best visualized with Congo Red stain
  - Contain
    - Filaments: 15 to 18 nm
    - Several proteins β-Amyloid, Desmin; Ubiquitin; Transglutaminases 1 & 2
- Aggregates & Related proteins
  - Protein aggregates: May contain
    - SMI-31 antibody
    - LC-3
    - p62 (SQSTM1)
    - TDP-43
    - AMPDA
    - Other: β-amyloid, VCP, ubiquitin, αB-crystallin, FYCO1
    - Amyloid-like properties: Congo-red birefringence
  - Cadherin I ⁴
    - Present in cytoplasm of some muscle fibers (68% of IBM)
    - Associated with increased SQSTM1 in muscle fiber cytoplasm
  - Anatomy: Aggregates often not related to vacuoles
  - Ultrastructure
  - Also see: Cytoplasmic bodies
- Mitochondrial disorders
  - Pathology location: Muscle fiber mitochondrial Δ
    - Segmental: 75 μm to > 1 mm along fiber length
    - Fiber distribution: Scattered in muscle
  - COX- muscle fibers
    - Frequency: Up to 15% of muscle fibers
    - MT-ND4 (major arc locus): Reduced
    - MT-ND2 (minor arc locus): Normal
    - mtDNA Deletions & Duplications: Present in 85% of fibers
    - Oxidative enzyme activities: Combined complex I & IV deficiency
    - Respiratory chain-deficient fibers: May be smaller than other fibers
    - No association: Invasion of inflammatory cells or rimmed vacuoles
    - Syndromes
      - IM-VAMP (IBM)
      - PM + Mito
  - SDH+ muscle fibers (Mitochondrial proliferation)
  - mtDNA disorders: IBM muscle fibers ^3, ⁹²
    - Copy number in IBM: 42% of control
    - Deletions: Multiple in > 50% of IBM
      - Deletions & Duplications in muscle
        
        IBM: Mean heteroplasmy level of 10% (Range 1%-35%)
        
        Controls: Mean heteroplasmy level of 1% (Range 0.2%-3%)
      - Variations in deletions: Common
        
        Single muscle fiber: May have multiple different deletions
        Different muscle fibers: Have different sizes & locations
      - Patterns in IBM
        
        Most deletions: Located in major arc of mtDNA; Involve ND4
        
        Unusually large deletions (10%): Involve ND1 & ND4
        mtDNA regions: m.534-4,429, m.6,330-13,993, m.8,636-16,072
        
        Regions may be flanked by: Repetitive sequences
        
        Similar regions to: Mutations in controls & POLG1 mutations
      - Breakpoint hot spots
        
        5' end: tRNALeu gene (np3230–np3304)
        
        3' end: np16070
        
        Other: ~np13923; ~np12300
    - Duplications: Present in many sIBM patients
    - Single nucleotide variants: More in IBM than age-matched controls
  - Ultrastructure

IBM: Myopathic features

H & E stain

H & E stain	H & E stain
Myopathy: Chronic; Ongoing Muscle fiber size: Varied Small fibers Shape: Rounded or Polygonal May occur in clusters May be basophilic Large fibers: May be hypertrophied Muscle fiber morphology Internal nuclei: In scattered muscle fibers Internal clear regions: Irregular shape; Possibly vacuoles Endomysial connective tissue: Increased in some regions

Fiber size: Varied
Focal invasion by cells
Vacuoles

2C fibers (Intermediate-stained): Common

ATPase pH 4.3 stain

Alkaline phosphatase stain: Often normal

Alkaline phosphatase stain

IBM: Inflammation
Focal invasion of Muscle fibers Endomysial Perimysial

Endomysial inflammation

H&E stain

Endomysial inflammation
Many cells are lymphocytic
Minority of cells are histiocytic (Acid phosphatase +)
Some immune cells are focally invading muscle fibers (Arrow)

Acid phosphatase (Blue) stain

Gomori trichrome

Inflammatory cells in Endomysium

Linear distribution of cells between muscle fibers (Above, Left; Below)
Larger collections of cells around muscle fibers (Above, Right)

Gomori trichrome

Inflammatory cells in endomysium
Cells are Focally invading a muscle fiber

Congo red

Inflammation: Endomysial & Perimysial
Lymphocytes & Histiocytes in Endomysium
Focal invasion of some muscle fibers by cells (Arrow)

H & E stain

Inflammation: Endomysial
Focal invasion of some muscle fibers (Dark arrow)
Partially fused (Split) muscle fiber (Light arrow)

H & E stain

H & E stain

Esterase stain

Acid phosphatase stain

Acid phosphatase stain

Focal invasion of muscle fibers Inflammatory cells invade a focal region of a muscle fiber (arrows). Other regions of the muscle fiber appear intact. Some cells invading the muscle fiber are CD8+ T-cells. Other cells are macrophages & stain with acid phosphatase.
CD4 stain	CD3 stain

Acid phosphatase	Acid phosphatase
Cells around vessel (Left) are mostly T-cells (unstained) Cells near and within muscle fibers (Right) contain more macrophages (Stained red)

Muscle fibers replaced by immune cells

VvG stain

IM-VAMP (IBM): Lymphocyte Types

CD4
CD4 cells: Present in foci & Scattered in endomysium

CD8
CD8 cells: More in foci than in other endomysial regions

IM-VAMP: Cell types
Lymphocytes: CD4 & CD8 cells
Present in endomysial lymphocyte clusters
CD8 cells
Location
Endomysial cell foci
Focal invasion of muscle fibers
Terminally differentiated (T_EMRA) phenotype
Loss: CD28
Upregulation: Killer cell lectin-like receptor G1 (KLRG1) & CD57
Cytotoxic potential: Perforin; Granzyme B; KLRG1
Limited proliferative capacity
May produce IFN-γ
Large granular lymphocytes (LGLs; CD3+, CD8+, CD57+, CD244+, CD28-, Kv1.3+)
Clinical association: Large granular lymphocytic (T-LGL) leukemia
CD4 cells
Scattered: In endomysium between muscle fibers
CD4+CD28(null) T cells: Endomysial cell foci; Proinflammatory & Cytotoxic
T cell receptor (TCR) V_β repertoire
Restricted (Clonal expansions)
Similar to changes seen in blood
Histiocytic cells
Scatttered: In endomysium
Clusters: Locations
Endomysial regions of focal invasion of muscle fibers by cells
Surround muscle fibers
Types ⁶
M1 macrophages: Near venous endotheial cells, Reactive & Damaged fibers, NMJs
M2 macrophages: Near LAMP3 Dendritic cells, Plasma cells, Type 2 fibers, Adipocytes
Plasma cells: Few scattered in endomysial cell foci
B-cells: NOT present in cell foci
Fibro-Adipogenic Progenitor (FAP) cells (PDGFRA) ⁶
Profibrotic
Molecules: CD55, COL15A1, CXCL14, PDGFRA, NEGR1, DPP4
Cell associations: Immune cells, Venous endothelial cells, Satellite cells, NMJs, CD55+ FAPs
Nerve-associated Fibroblasts
Molecules: NLGN1, NEGR1
Cell associations: Normal & Damaged muscle fibers, M2 macrophages
Damaged Muscle Fibers
Molecules: GADD45A, NORAD, AChE
Features: Atrophy, Cell/genomic stress, Associated T-cells

CD8 stain

CD8 cells in IM-VAMP: In endomysial cell foci & around muscle fibers

CD8 stain

IM-VAMP: Histiocytes (CD163) & CD8 lymphocytes around & on muscle fiber surfaces

CD163 stain

IM-VAMP: Plasma cells (CD138) scattered in endomysial cell focus

CD138 stain

IM-VAMP (IBM): Perimysial inflammation

H&E stain

IBM: Inflammation
Cells: Many lymphocytes
Locations
Endomysial (Above): Surrounds a muscle fiber
Perimysial (Below): Contains small vessels

H&E stain

IBM: Atypical Inflammation
Cells: Granuloma-like histiocytes (Arrow, Above; Esterase+, Below) Surrounded by lymphocytes

Esterase stain

IBM: Vacuoles (or Inclusions)

Vacuoles

Muscle fibers with several rimmed vacuoles (arrows)
Vacuoles contain
- Congo red: Blue amorphous, granular material
- Gomori trichrome: Red-stained material in muscle fibers & around vacuoles
- VvG: Gray-stained material
Nuclei: Some are near vacuoles or enlarged
Amyloid in some vacuoles
- Visualized with polarized light
- Small foci of red stained amyloid (arrows) are in, or around, vacuoles
Some muscle fibers also contain
- Cytoplasmic bodies or inclusions
Differential diagnosis: Vacuoles

Congo red stain

Congo red stain

Congo red stain

Vacuoles (Arrows)
Shape irregular
Granular basophilic debris
Often within, and surrounding, vacuoles
May have amyloid-like, Congo red birefringence

Congo red stain

Granular basophilic debris
In regions surrounding large vacuole

Vacuoles

Muscle fiber with vacuoles (Left; Congo red stain)
Toluidine blue stain: Vacuoles in 2 muscle fibers (Right)
- Vacuoles may be in large or small fibers
- Vacuoles may contain autophagic debris, or 15 to 18 nm filaments

Gomori trichrome stain

IBM: Vacuoles
One, or several, in a muscle fiber
Shapes: Irregular
Some contain red-staining material

Gomori trichrome stain

Gomori trichrome stain

IBM aggregates: Cytoplasmic Bodies

Gomori trichrome stain

Gomori trichrome stain

Vacuoles & Cytoplasmic bodies
Cytoplasmic bodies (Arrow) may occur in: Muscle fibers with, or without, vacuoles

Gomori trichrome stain

Atypical vacuoles
Contain irregular green-stained material
Larger than usuallyh found in sIBM

Gomori trichrome stain

Gomori trichrome stain

IM-VAMP: Amyloid

Vacuoles may contain amyloid-like birefringent (red-green) material (Congo red stain)

Muscle fiber with large vacuole (Left)
Birefringence: Red-Green material in, and near, vacuole (Middle & Right)

Congo red stain

Muscle Fiber with several Vacuoles
Granular basophilic debris near vacuoles may have red-green birefringence (Below)

Congo red stain

From: R. Schmidt

Ultrastructure: Muscle fibers with aggregated material from same biopsy as above
Contents include: Filaments; Scattered mitochondria;

From: R. Schmidt

Alcian blue/Nuclear fast red

Red-stained material in cytoplasm & near nuclei

IBM: Aggregates

Aggregates: General features in IBM-like disorders

Location
- Most easily seen in the cytoplasm
- Minority occur neighboring nuclei or vacuoles
Histochemistry: Stain with AMPDA, H&E & Gomori trichrome
Contents
- Common components: SMI-31; TDP-43; LC3; αB-crystallin; p62
- Different components may occur alone or associated with other components
- None are very sensitive for the spectrum of IBM-like disorders
- LC3 aggregates
  - Frequent in IBM/IM-VAMP
  - Multiple small punctate aggregates
  - LC3 aggregates occur in other muscle disorders but have different shapes & sizes
- SMI-31 aggregates
  - May be the most specific for IBM
  - Low sensitivity
- Emerin is not present in aggregates or vacuoles
Cytoplasmic bodies
- Increased frequency in IBM/IM-VAMP
- May occur in fibers that also have vacuoles

Aggregates: Histochemistry

Cytoplasmic Aggregates Dark, eosinophilic hyaline appearance (Arrows)		Cytoplasmic Aggregate Large Near irregular vacuoles
H&E stain	H&E stain	VvG stain

Cytoplasmic bodies
Small, dark-stained structure
May be
Several in individual fibers
Present in fibers with or without vacuoles

Gomori trichrome stain

Gomori trichrome stain

AMPDA stain

AMPDA aggregate
Present in aggregated material in muscle fiber cytoplasm
Some, but not most, aggregated material surrounds vacuoles

AMPDA stain

AMPDA stain

SMI-31 positive aggregates in IBM

SMI-31 + Congo red stains

Congo Red + Fluorescence with Texas red filter

IBM (Top Left; Bottom): SMI-31 staining in several patterns
- Aggregates in the cytoplasm of muscle fibers with no vacuoles
- Occasional small aggregates scattered around the rim of some vacuoles
- Some aggregates near nuclei
- Diffuse increase in cytoplasm of some muscle fibers
IBM (Right): No fluorescence of vacuoles or aggregates in muscle fibers for amyloid
SMI-31 aggregates are not usually present in PM-Mito biopsies

SMI-31 + Congo red stains

SMI-31 + Congo red stains

SMI-31 + Congo red stains

SMI-31 staining in some nuclei

SMI-31 + Congo red stains


αB-crystallin IBM (Left): Clusters of αB-crystallin staining in muscle fibers Normal (Right): No focal staining of muscle fibers


Ubiquitin conjugates: IBM Focal dark staining Staining may be present around rim of vacuoles (arrow) Aggregates are also located in other regions of muscle fibers Increased staining in much of the cytoplasm (right)


Valosin-containing protein (VCP): IBM Focal dark staining Aggregates are mostly small & scattered through muscle fibers Some fibers, especially small, can have increased cytoplasmic staining Occasional vacuoles have some VCP staining

Congo red + β-amyloid stain	Congo red + fluorescence
β-Amyloid: IBM Small focal regions of dark staining (Left) Aggregates are mostly small & scattered through muscle fiber Some fibers, especially small, can have increased cytoplasmic staining Regions of β-Amyloid do not show fluorescence with Congo red stain (Right)

TDP-43 aggregates

TDP-43

Congo red + TDP43
TDP-43: IBM Aggregates Small focal regions of dark staining (Left; Above) Aggregates are mostly small & scattered through muscle fiber Large cytoplasmic TDP-43 aggregates (Right; Above) Inflammatory cells (Below): TDP-43 stains inflammatory cells in Endomysium Regions of focal invasion of muscle fibers TDP-43 aggregates are not present in PM-Mito biopsies
Congo red + TDP43
CD4 (Green) + TDP43 (Red) TDP-43: Aggregates & Inflammatory cells in IBM TDP-43 stains Focal aggregates in muscle fibers (Red) Inflammatory cells: Overlap with CD4 (Yellow) CD4 also stains scattered endomysial cells without TDP-43 (Green)
SMI-31 (Green) + TDP43 (Red) TDP-43: Aggregates and cytoplasmic staining with varied relation to SMI-31 staining TDP-43 stains Focal aggregates in muscle fibers: With (Yellow) and without (Red) overlap of SMI-31 SMI-31 also stains Cytoplasm in some fibers without TDP-43 (Green) Rim of internal nucleus (Green; Arrow) associated with neighboring TDP-43 staining (Red)

p62 aggregates

p62 stain

p62 stain

LC3 Aggregates

Differential Diagnosis
sIBM-like syndromes
Immune myopathies
Dermatomyositis + Vascular pathology
Tif1-γ
IMPP
MDA5
Systemic Sclerosis
Hereditary Myopathy
LGMD1A
MSP
OPMD
Lipid disorders
Myosin loss
Targets

LC3 aggregates: IM-VAMP (IBM-like syndromes)

LC3 stain

LC3 stain

LC3 stain

LC3: IBM

Focal regions of dark staining of varied shapes (Left)
- Aggregates are cytoplasmic, small or large & have varied shapes
LC3 aggregates are also present in PM-Mito biopsies (Right), unlike SMI-31 or TDP-43

Congo red + LC3 stain

Desmin stain

IBM: Desmin stain: Patterns
Diffusely in cytoplasm of iommature muscle fibers
Irregular aggregates in scattered muscle fibers
Localized at site of focal invasion of a muscle fiber (below)

Desmin stain

IM-VAMP: Ultrastructure

Focal invasion of muscle fibers by cells
Aggregates & Inclusions, Cytoplasmic

IM-VAMP: Muscle fiber pathology + Focal invasion of Muscle fiber by cells
Cytoplasmic inclusions: Several types
Myeloid
Membrane-like
Tubulo-Vesicular
Filamentous
Autophagic debris
Lipid-like bodies
Mitochondria
Endomysial capillaries
Normal size & walls

Inclusions: Different Types in muscle fibers in IM-VAMP

Filamentous (Black arrow; Above)
Osmophilic (Red arrow; Above): Possible remanants of nuclear membranes
Autophagic
- Membrane-like (White arrow; Above): Multilayered; Irregular shapes; May be within larger, "vacuolar", structures
- Tubulovesicular material (Below)

From: R Schmidt

From: R Schmidt

Aggregates contain
Filaments
A few myeloid structures & mitochondria

From: R Schmidt

Aggregates contain
Filaments
A few myeloid structures & mitochondria

From: R Schmidt

Aggregate Contents
Filaments
Myeloid structures

Tubulofilaments

Aggregates contain
Filaments
Myeloid structures
Mitochondria

Aggregates contain
Mitochondria

Muscle fibers: Focal Invasion by Cells

Inflammatory cells & Focal invasion of Muscle fibers in IM-VAMP (IBM)
Lymphocyte, Large, Possibly Granular (Black Arrow)
Extends a process (White arrow) into a non-necrotic muscle fiber
Other lymphocytes & histiocytes
Whole immune cells may be invading muscle fiber
Present in extracellular regions around muscle fiber
Muscle fiber pathology: Aggregates, various types

Muscle fiber: Focal Invasion by Cell
Lymphocyte, single, invades muscle fiber
Lymphocyte location: Inside muscle fiber; Above sarcolemma; Neighbors myonucleus

Muscle fiber: Focal Invasion by Cells
Lymphocytes (Light cytoplasm) & Histiocytes (Dark cytoplasm with phaogcytic debris)
Within the muscle fiber
Closely apposed to each other
Also in extracellular space
Sarcomeres near invading cells: Normal structure

Muscle fiber: Focal Invasion by small cell processes

Lymphocyte: Focal Invasion of a Muscle Fiber (Arrows)
Muscle fiber structure is damaged

MHC-1 Expression

Normal

IBM

IBM: MHC Class-1 up regulation by muscle fibers

Normal (Left)
- Staining of capillaries but not muscle fibers
IBM (Right & Below)
- Up-regulation within, and on the surface of, muscle fibers.
- MHC-1 is also present on inflammatory cells, Some focally invading muscle fibers (Arrow)
- Immature, smaller fibers can also express MHC Class-1 on their surface & in cytoplasm

MHC Class I stain

MHC Class I stain

MHC Class I: Expression by muscle fibers
Diffuse: Present in most, or all, muscle fibers
Distribution
Mostly Sarcolemma, or Sarcolemma + Cytoplasm

MHC Class I stain

Also see

IM-VAMP: Myonuclear Pathology

IM-VAMP: Nuclear features
Location: Internal nuclei
Structure
Clear centers
Outlines: Smudged or Pale

H&E stain

IM-VAMP: Nuclei
Internal: One or Several
Structure: Clear centers
May have peripheral cytoplasmic halo

Gomori trichrome stain

Gomori trichrome stain

IM-VAMP Myonuclei: Smudged outlines or Clear centers

Congo red stain

Myonuclei
Indistinct borders
Irregular shapes

H&E stain

H&E stain

IM-VAMP Myonuclei: Smudged outlines; Pale staining (Arrows)

H&E stain

IM-VAMP Myonuclei: Reduced staining in subsarcolemmal regions of muscle fibers

H&E stain

H&E stain

IM-VAMP (IBM): Mitochondrial Pathology

Molecular Pathology
Mitochondrial DNA (mtDNA)
Deletions & Duplications ³
Histology
COX- Muscle fibers
SDH+ Muscle fibers
Mitochondrial Ultrastructure
Proliferation
Morphologic Δ

Cytochrome oxidase stain

Cytochrome oxidase (COX): Many IBM (IM-VAMP) biopsies have scattered COX- muscle fibers

Cytochrome oxidase stain

COX stain

COX-negative muscle fibers
2 muscle fibers with nearly absent staining
Neighboring fibers have normal staining
Changes are likely due to acquired mutations in mtDNA causing loss of synthesis of COX subunits

SDH stain

SDH-positive muscle fibers
2 muscle fibers with increased staining
Small muscle fiber above shows increased staining in entire cytoplasm
Larger muscle fiber below has more mitochondrial accumulation in subsarcolemmal regions
Neighboring fibers have normal staining
Changes are likely due to acquired mutations in mtDNA causing mitochondrial proliferation

Also see: Myositis (IM-VAMP) with Mitochondrial Pathology

Ultrastructure

Mitochondrial Pathology in Muscle fibers from this biopsy: SDH stain

SDH stain

Ultrastructure: Subsarcolemmal Mitochondrial Accumulation
Also see: Mitochonrial ultrastructure pathology, other

From: R Schmidt

Mitochondria: Subsarcolemmal accumulation

From: R Schmidt

Mitochondrial pathology: Subsarcolemmal regions
Some areas have dense collections of mitochondria (Left)
Other subsarcolemmal areas have relatively few mitochondria (Right)