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

Inclusion Body Myositis (Subtype)

Pathology features Inflammation   Focal invasion   Immune Cell types   Endomysial inflammation 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   Focal invasion   MHC Class-1 expression   Mitochondrial pathology     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

• 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
    • CD4: Most common
    • CD8: Present in many endomysial cell foci
    • CD20
      • Usual: Few or none present
      • Foci reported: sIBM associated with chronic lymphocytic leukemia ⁷⁸
• 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
    • SDH+ muscle fibers
    • 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 inflammation

H&E stain

Acid phosphatase (Blue) stain

Gomori trichrome

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

Gomori trichrome

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 the muscle fiber (arrows). Other regions of the muscle fiber appear intact. Some cells invading the muscle fiber are CD8+ T-cells. Occasional 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)

VvG stain

CD4 CD4 cells: Present in foci & Scattered in endomysium

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

CD8 stain CD8 cells: In endomysial cell foci in IM-VAMP

IBM: Vacuoles (or Inclusions)

• 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
  • Inclusion body myositis
  • Distal myopathy (HIBM)
  • Oculopharyngeal muscular dystrophy

Congo red stain

Congo red stain

Congo red stain

Congo red stain

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

Gomori trichrome stain Vacuoles & Cytoplasmic bodies   Cytoplasmic bodies may occur in     Muscle fibers that also have vacuoles     Fibers near other fibers with vacuoles (Arrow)

Gomori trichrome stain

Gomori trichrome stain

Gomori trichrome stain

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

Congo red stain

Congo red stain

From: R. Schmidt

From: R. Schmidt

Alcian blue/Nuclear fast red

IBM: Aggregates

• 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

Gomori trichrome stain

Gomori trichrome stain

AMPDA stain

AMPDA stain

AMPDA stain

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

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 stain

p62 stain

LC3 stain

LC3 stain

LC3 stain

• 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

Desmin stain

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

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

MHC-1 Expression

Normal

IBM

• 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 stain

• MHC-I expression on muscle fibers
• Inflammatory myopathy with Mitochondrial dysfunction (IM-Mito)

IM-VAMP: Myonuclear Pathology

H&E stain

Gomori trichrome stain

Gomori trichrome stain

Congo red stain

H&E stain

H&E stain

H&E stain

H&E stain

H&E stain

IM-VAMP (IBM): Mitochondrial Pathology

Cytochrome oxidase (COX): Some patients have scattered COX- muscle fibers Cytochrome oxidase stain

COX stain

SDH stain

Ultrastructure

SDH stain

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

IBM: Capillary Proliferation & Enlargement

VvG stain

VvG stain

Ulex stain

Ulex stain

From: R Schmidt

From: R Schmidt

From: R Schmidt