Pathologic Diagnosis in Amyloidosis Jung-Sun Kim, MD PhD Department of Pathology Samsung Medical Center Sungkyunkwan University School of Medicine

Amyloidosis A heterogeneous group of diseases of diverse origin Caused by extracellular deposition of misfolded, insoluble aggregated proteins with a characteristic -pleated sheet configuration Amyloidosis is a heterogeneous group of diseases of diverse origin which are caused by extracellular deposition of misfolded, insoluble aggregated protein with a characteristic -pleated sheet configuration

Structure of Amyloid Crystallography: Cross--pleated sheet conformation EM: Continuous nonbranching fibrils with a diameter of 7.5 to 10 nm This schematic drawing shows an amyloid fiber composed of several fibrils with beta-pleated sheet conformation wound around one another. The red bars indicate regularly spaced binding of the dye when stained with Congo red. Congo red staining shows apple-green birefringence under polarized light, which is a diagnostic feature of amyloid. Utrastructurally amyloid is seen as continuous nonbranching fibrils with a diameter of 7.5-10 nm.

Amyloid Fibril Formation Soluble precursor proteins are converted to unstable fragments undergoing folding events resulting from mutation, proteolytic event, environmental factors, or excessive concentration. Self-aggregation of folding intermediates yields protofilaments with high-pleated sheet content , which is promoted by glycosaminoglycans (GAGs). Glycosaminoglycans and amyloid P, which are universal components of amyloid deposits, protect amyloid deposits from proteolysis. Dember, Kidney Int 2005;68:1377–90

Amyloid Proteins and their Precursors in Human So far 27 biochemically distinct forms of amyloid proteins have been identified and additional protein types are continually being added to this list. A nomenclature for amyloid has been developed, which is based on the type of protein involved. there is a prefix ‘‘A,’’ for amyloid, followed by an abbreviation derived from the name of the protein; Westermark et al., Amyloid 2007;14:179-83

Amyloid Proteins and their Precursors in Human AL designates amyloid derived from immunoglobulin light chain, Westermark et al., Amyloid 2007;14:179-83

Amyloid Proteins and their Precursors in Human ATTR designates amyloid derived from transthyretin. Westermark et al., Amyloid 2007;14:179-83

Classification of Amyloidosis Clinicopathologic Category Associated Diseases Major Fibril Protein Chemically Related Precursor Protein SYSTEMIC (GENERALIZED) AMYLOIDOSIS Immunocyte dyscrasias with amyloidosis (primary amyloidosis) Multiple myeloma and other monoclonal plasma cell Proliferations AL Immunoglobulin light chains, chiefly λ type Reactive systemic amyloidosis (secondary amyloidosis) Chronic inflammatory Conditions AA SAA Hemodialysis-associated amyloidosis Chronic renal failure Aβ2m β2-microglobulin HEREDITARY AMYLOIDOSIS Familial Mediterranean fever Familial amyloidotic neuropathies (several types) ATTR Transthyretin SENILE SYSTEMIC AMYLOIDOSIS LOCALIZED AMYLOIDOSIS Senile cerebral Alzheimer disease A APP Endocrine   Medullary carcinoma of thyroid A Cal Calcitonin   Islets of Langerhans Type 2 diabetes AIAPP Islet amyloid peptide Isolated atrial amyloidosis AANF Atrial natriuretic factor Although the ‘‘molecular-based’’ classification of amyloidoses is very precise and allows for future expansion to include additional amyloid types, a simpler subdivision into systemic versus localized amyloidosis, and hereditary versus acquired amyloidosis would be more relevant to clinical practice. certain amyloid deposits are exclusively localized (several cerebral amyloidoses and amyloid associated with endocrine tumors), but others (most notably AL) can be either systemic or localized. On clinical grounds, the systemic or generalized pattern is subclassified into primary amyloidosis and secondary amyloidosis. Primary amyloidosis associated with some immunocyte disorders such as multiple myeloma has deposition of AL amyloid protein derived from immunoglobulin light chains, chiefly lambda type. Secondary amyloidosis occurs as a complication of an underlying chronic inflammatory or tissue destructive process, and in this condition amyloid A protein is deposited. A variety of heredofamilial amyloidosis has been described. One example is familial amyloidotic neuropathies of autosomal dominant inheritance in which the fibrils are made up of mutant TTRs. In contrast, senile systemic amyloidosis has the deposition of the same amyloid protein but nonmutant normal TTR.

Detection of Amyloid Congo red stain: the gold standard Salmon-pink color in bright field Apple-green birefringent deposits under polarized light Good fixation, strong light source in a darkened room, thicker sections, multiple sections Diagnosis of amyloidosis is based on the detection of amyloid protein deposits in tissues. Congo red stain is the gold standard for detection of amyloid deposits. In bright field, amyloid stained with Congo red typically have a salmon-pink color. The bright field appearance in itself is not diagnostic. Congo red–stained slides should be examined under polarized light and only the presence of apple-green birefringent deposits is considered diagnostic of amyloid. for the examination of Congo red–stained slides, good fixation, a proper staining protocol (alkaline Congo red), and appropriate optics are required. Reading under with a polarized microscopy with strong light source in a darkened room, facilitates the detection of smaller deposits. Thicker sections (5–10 m) may be helpful, and multiple sections may be needed because deposits of amyloid are frequently very focal and irregularly distributed.

Detection of Amyloid Electron microscopic demonstration of the fibrillar nature of deposits In keeping with the definition of amyloidosis, electron microscopic demonstration of the fibrillar nature of deposits is another way to prove the deposition of amyloid. Knowledge of the ultrastructural appearance of amyloid is also important in the differential diagnosis of various organized deposits.

Detection of Amyloid Thioflavin S & T Methyl violet Sulphonated Alcian blue less sensitive less specific There are other stains such as thioflavin S and T, methyl violet, and sulphonated Alcian blue, however they are less specific and at times also less sensitive for the detection of amyloid.

Amyloid Typing Need to have stringent criteria Indistinguishable on LM or EM Most direct (but not applicable in reality) Mass spectrometry Amino acid sequencing In clinical setting immunostaining Individual amyloid proteins are indistinguishable on LM with congo red or EM Most direct method for amyloid typing is Mass spectrometry and amino acid sequencing. However These are not applicable in reality.

Amyloid Typing Immunohistochemistry: Currently the standard Kappa, Lambda, Amyloid A, Transthyretin for primary screening Immunohistochemistry is currently the standard for amyloid typing in routine clinical practice. Kappa, Lambda, amyloid A and Transthyretin should be included for primary screening to subtype amyloidosis. Kappa Lambda Amyloid A

Amyloid Typing Immunohistochemistry: Limitation Nonreactive with a truncated light chain Limited antibody panel missing a number of hereditary amyloidoses Background staining : “locking-in” of serum proteins during fixation However, Immunohistochemistry method has limitations AA can be reliably typed by immunohistochemistry. However, immunohistochemical typing of AL is still challenging and the difficulties are frequently compounded by truncation of the light (or heavy) chain. Commercial antibodies are raised against the constant regions of the respective immunoglobulin light chains. Therefore, a subset of AL, in which amyloid fibrils are derived from a truncated light chain containing only variable regions, will be expected to be nonreactive with commercial antibodies. At the same time, a limited antibody panel will also miss a number of hereditary amyloidoses as well. The third troublesome issue is the presence of background stain in the tissue, which in paraffin sections in particular can be significant due to the ‘‘locking-in’’ of serum proteins during fixation.

Amyloid Typing Immunofluorescence Cleaner background Higher Sensitivity Limitation: requirement of frozen tissue The last issue can be alleviated by the use of frozen sections and immunofluorescence stains, which provide a cleaner background. Moreover, immunofluorescence stains on frozen sections have a higher sensitivity when compared with immunoperoxidase stains on paraffin sections. However, the limitation of IF is requirement of fresh frozen tissue instead of paraffin-embedded tissue. Also, the limited antibody panel and no detection of truncated light chains are also the same issues in IF. Collins et al., Cardiovasc Pathol 2009;18:205-16

Amyloid Typing Combined Congo red and IHC Immuno-electron microscopy Western blotting In order to correlate the Congo red positive area with immunohistochemistry, an ‘‘overlay technique’’ can be tried, in which a Congo red stain and immunohistochemistry are performed on the same slide. Good results for amyloid typing using amyloid-specific antibodies are reported with other such as immunoelectronmicroscopy and Western blotting. To conclude, immunohistochemistry, in particular immunofluorescence on frozen sections, is a fast and valid methodology for amyloid typing but should be done with caution and with a full awareness of its limitations and pitfalls

Biopsy of a clinically affected organ The Choice of Tissue Specimen: Fat as a Source of Tissue for Amyloid Detection Biopsy of a clinically affected organ Subcutaneous fat pad biopsy: less invasive screening test To judge systemic versus local amyloidosis For the detection of amyloid, biopsy of a clinically affected organ is the most sensitive method and may also detect concomitant pathologies. However, such a biopsy is invasive and carries the risk of complications, in particular bleeding. A less invasive procedure, subcutaneous fat pad biopsy, obtained via fine-needle aspiration,, has been introduced as a safe cheap, and rapid screening test. Not infrequently, following the initial detection of amyloid in biopsy material, a second biopsy is necessary for further characterization In such cases, an abdominal fat biopsy should be considered SC fat pad biopsy is useful to judge systemic versus local amyloidosis in the case amyloid deposition is known in a certain organ.

Biopsy-Proven Amyloidosis in SMC (2000 Biopsy-Proven Amyloidosis in SMC (2000.1 – present; by Congo red or EM) Kidney: 37 Heart: 35 Gastrointestinal tract: 34 Skin and soft tissue: 13 Respiratory tract: 12 Bone (marrow): 11 Liver, pancreatobiliary tract: 7 Thyroid: 5 (medullary carcinoma) Thymus, Urinary bladder, Seminal Vesicles, Peripheral Nerve, Eye, Lacrimal gland I’d like to wrap up my talk with the summary of biopsy-proven amyloidosis in SMC for 10 years. Amyloidosis was confirmed in 37 kidney biopsies, 35 heart biopsies, 34 GI tract and so on.

Amyloid Typing in Kidney Biopsy by Immunohistochemistry 23 Kidney biopsies in which IHC/IF stainings were done 6 AA type (Amyloid A (+); kappa/lambda (+/-) 17 AL type (Amyloid A (-): kappa/lambda (+)) 1 kappa 6 lambda 10 not determined Out of 23 kidney biopsies in which IHC or IF stainings were done, 6 cases were of AA type with positive staining for amyloid A but no or weak staining for Ig light chains, and 17 cases were of AL type with negative staining for AA, but positive staining for kappa and/or lambda. EM: non-branching fibrils

Amyloid Typing in Heart Biopsy by Immunohistochemistry Patient Age Sex kappa lambda amyloid A Type 1 51 M P N AL kappa 2 72 3 50 F WP 4 58 AL lambda 5 74 6 64 ND 7 60 8 57 9 71 AL 10 Recently 10 heart biopsies were stained immunohistochemically for kappa, lambda, and amyloid A. None of them were positive for AA, 5 cases were categorized to have the deposition of lambda AL proteins, whereas 3 cases have kappa type AL proteins. N, negative; ND, not done; P, positive; WP, weak positive

Summary Detection of Amyloid Diagnosis of the Amyloid type Congo Red staining EM Diagnosis of the Amyloid type Immunohistochemistry Immunofluorescent staining In summary, For detection of amyloid, Congo red staining is the gold standard, but EM can be used. For typing amyloid proteins, immunohistochemistry including immunofluorescent staining is the standard. However, since this method has limitations, other methods can be additionally used.

HE Congo Red Congo Red Kappa Lambda Amyloid A Here is one example of AL amyloidosis lambda type. HE sections shows amorphous pink deposits in the vascular wall and interstitium. By Congo red staining apple-green birefringence was detected by polarized microscopy. Immunostaining for lambda is strongly positive, while kappa staining is weakly positive. AA was not detected by immunostaining. Kappa Lambda Amyloid A