Plasma cell dyscrasia and renal amyloidosis: What we can learn from basic science Per Westermark Rudbeck Laboratory Uppsala University, Sweden

Definition of Amyloid Characteristic ultrastructure Cross  -Sheet Fibrils (X-ray diffraction) Binding of Congo red with resulting green birefringence Usually extracellular deposits In vivo

Folded, functional protein Normal function and metabolism Newly synthesized protein Folding intermediate Toxic oligomer Amyloid fibrils Merlini & Westermark, JIM 2004

TIME SEED Importance of seeding Lag phase Elongation Steady state

The word ’amyloid’ is used widely: Amyloid in deposits (”real” amyloid) Synthetic ”amyloid” Amyloid-like material in nature (silk etc) ”Functional amyloid”

Amyloid fibril proteins and their precursors in human ____________________________________________________________________________________________ ALImmunoglobulin light chainSystemic AHImmunoglobulin heavy chainSystemic Aβ2Mβ2-microglobulinSystemic ATTRTransthyretinSystemic AA(Apo)serum AASystemic AApoAIApolipoprotein AISystemic AApoAIIApolipoprotein AIISystemic AApoAIVApolipoprotein AIVSystemic AGelGelsolinSystemic ALysLysozymeSystemic AFibFibrinogen α-chainSystemic ACysCystatin CSystemic ABri ABriPPSystemic ADan*ADanPPSystemic ALect2Leucocyte chemotactic factor 2Systemic AβAβ protein precursor (AβPP)Brain APrPPrion proteinBrain ACal(Pro)calcitoninC-cell thyroid tumors AIAPPIslet amyloid polypeptide (amylin)Islets of Langerhans, insulinomas AANFAtrial natriuretic factorCardiac atria AProProlactinAging pituitary, prolactinomas AInsInsulinSkin (Iatrogenic) AMedLactadherinAortic media AKerKerato-epithelinCornea, familial ALacLactoferrinCornea AOaapOdontogenic ameloblast-ass. proteinOdontogenic tumors ASemISemenogelin IVesicula seminalis

Validation of the 3D profile method for prediction of fibrillizing segments. Goldschmidt L et al. PNAS 2010;107: ©2010 by National Academy of Sciences

Some protein factors important for amyloid fibril formation  -sheet propensity Hydrophobicity Charge Destabilisation, e.g. by mutation Concentration Degradation

Systemic amyloidosis 15 different proteins known Precursor circulating in plasma The precursor may be modified before deposition, e.g. by truncation Depositions in many organs

Human systemic amyloid fibril proteins and their precursors ALImmunoglobulin light chainPrimary; myeloma associated AHImmunoglobulin heavy chainPrimary; myeloma associated  microglobulinHemodialysis ATTRTransthyretinFamilial; senile systemic AASerum AASecondary, reactive AApoAIApolipoprotein AIFamilial AApoAIIApolipoprotein AIIFamilial AApoAIVApolipoprotein AIVSporadic? AGelGelsolinFamilial ALysLysozymeFamilial AFibFibrinogen  -chainFamilial ACysCystatin CFamilial ABri ABri PPFamilial ADan ADan PPFamilial ALECT2 Lecocyte chemotactic factor 2Sporadic

Other components in amyloid: Amyloid P-component: AP = SAP is a glycoprotein, produced by the liver. Up to 10% of the dry substance can consist of SAP Proteoglycans: Heparan sulfate proteoglycan (HSPG) Apolipoprotein E Other components

Systemic AL amyloidosis Extreme variation in deposition pattern Sometimes enormous amounts of amyloid (kilograms) Life threatening Early diagnosis important

Immunoglobulin molecule Light chain structure FR Variable (VL)Constant (CL) CDR Antibody molecule Fab: antigen binding Fc: mediates the effect

AL amyloidosis = primary and myeloma associated amyloidosis Primarely a plasma cell disorder: monoclonal expansion Monoclonal immunoglobulin light chain Manifests with extreme variations: heart, liver, kidneys, peripheral nerves etc. Treatment with melphalane + prednisolone and/or peripheral stem cell transplantation

AL amyloid Each protein is unique; no two sequences are identical: About 50 different genes for light chains Somatic mutations Therefore it is not surprising that each patient has a unique disease Properties of the amyloid vary from patient to patient Affinity for Congo red varies from very weak to strong. The same is true for the green birefringence in polarized light

Enqvist et al., PLoS ONE 2007 Germ line

NC Variable segmentConstant segment Immunoglobulin light chain and AL protein Major part of amyloid fibril AL protein is encircled

Fragmentation pattern of monoclonal light chains in six different AL proteins of kappa type Enqvist et al., J. Path. 219:473; 2009

Distribution of amyloid in patients with AL-amyloidosis coming from the two genes O18-O8 and L2-L16 Enqvist et al., PLoS ONE 2007

Examples of questions to be answered What determines fibril formation from an immunoglobulin light chain? Is cleavage of the light chain of importance? What initiates fibril formation? Nidus? What determines the deposition pattern? Are there toxic oligomers in AL amyloidosis Which is the role of HSPG in AL amyloidosis?

Amyloid Diagnostics Biopsy Rectum Subcutis (abdomen) surgical needle (not too thin) Organ with symptom: kidney, liver, skin

Source of errors: Under or over diagnostics Insufficient material Incorrect staining Errors in microscope (light, polarizers) Unexperienced examiner

Determination of amyloid type Direct method necessary

N Engl J Med 2002, 346: Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis Lachmann HJ, Booth DR, Booth SE, Bybee A, Gilbertson JA, Gillmore JD, Pepys MB, Hawkins Out of 350 patienter with preliminary diagnosis of AL amyloidosis 34 (9.7%) had familial forms! 18 AFib2 AApoAI 13 ATTR1 ALys

Typing of amyloid Clinical findings: Not enough! Immunohistochemistry: Sometimes good but there are pitfalls Immune electron microscopy Immunochemistry: Western blot, ELISA Mass spectrometry and/or N-terminal sequence analysis after protein extraction

Surgical Fat Tissue Biopsy For Typing and for Protein Studies

Immunohistochemistry Limitations with commercial antibodies Light chain antibodies: directed against the C- terminal part Own antibodies: Mab Pwlam AA ATTR

AA-amyloid, MAB Sne5

AL-amyloid lambda, MAB PWlam

ATTR-amyloid, polyklonal 1898

Mass spectrometry Identification of specific peptides, e.g. after tryptic digestion Laser Dissection Microscopy

Enqvist, Hellman et al., unpublished

Amyloid fibril proteins and their precursors in human ____________________________________________________________________________________________ ALImmunoglobulin light chainSystemic AHImmunoglobulin heavy chainSystemic Aβ2Mβ2-microglobulinSystemic ATTRTransthyretinSystemic AA(Apo)serum AASystemic AApoAIApolipoprotein AISystemic AApoAIIApolipoprotein AIISystemic AApoAIVApolipoprotein AIVSystemic AGelGelsolinSystemic ALysLysozymeSystemic AFibFibrinogen α-chainSystemic ACysCystatin CSystemic ABri ABriPPSystemic ADan*ADanPPSystemic ALect2Leucocyte chemotactic factor 2Systemic AβAβ protein precursor (AβPP)Brain APrPPrion proteinBrain ACal(Pro)calcitoninC-cell thyroid tumors AIAPPIslet amyloid polypeptide (amylin)Islets of Langerhans, insulinomas AANFAtrial natriuretic factorCardiac atria AProProlactinAging pituitary, prolactinomas AInsInsulinSkin (Iatrogenic) AMedLactadherinAortic media AKerKerato-epithelinCornea, familial ALacLactoferrinCornea AOaapOdontogenic ameloblast-ass. proteinOdontogenic tumors ASemISemenogelin IVesicula seminalis

Future Directions Other amyloid markers (e.g. polyelectrolytes) Proteomics Molecule-specific diagnosis? Individe-specific treatment?

Thanks to Knut Sletten, Oslo, Norway Ulf Hellman, Uppsala Charles Murphy, Knoxville, TN Gunilla T. Westermark, Uppsala Stina Enqvist, Uppsala All former and present graduate students All other coworkers

Informa Healthcare Informahealthcare.com