Make it count Technical considerations and initial findings for - PowerPoint PPT Presentation

Make it count Technical considerations and initial findings for accurate quantitative comparison of urinary extracellular vesicles to determine minimal residual kidney disease in AL amyloidosis Marina Ramirez Alvarado, PhD Department of Biochemistry and Molecular Biology/Immunology Mayo Clinic, Rochester, MN USA ramirezalvarado.marina@mayo.edu IKMG Montreal May 24 th , 2019

Disclosure of Conflict of Interest ❑ I do not have a relationship with a for-profit and/or a not-for-profit organization to disclose ❑ I have a relationship with a for-profit and/or a not-for-profit organization to disclose Name of for-profit or not-for-profit Description of relationship(s) Nature of relationship(s) organization(s) Any direct financial payments including receipt of honoraria Membership on advisory boards or speakers’ bureaus Funded grants or clinical trials Patents on a drug, product or device All other investments or relationships that could be seen by a reasonable, well- informed participant as having the potential to influence the content of the educational activity

Outline • Extracellular vesicles- exomeres, exosomes, microvesicles, and apoptotic bodies • Urinary Exosomes- why bother? • Urinary extracellular vesicles (uEVs) in light chain (AL) amyloidosis • Minimal residual disease studies using uEVs in AL amyloidosis • Comparing apples with grapes-towards a standardization of the assay • Testing the range of the uEV oligomer detection with different extraction methods • Conclusions • Acknowledgements

200-500 nm 50-120 nm 400-600 nm Journal of Endocrinology 228 R57-R71 Nature Cell Biology volume 20 , pages332 – 343 (2018)

At the time we started our urinary extracellular vesicles (uEV) research, the main goal was to establish if uEV can be used as a non-invasive tool to study response and progression in renal diseases

Urinary extracellular vesicles (uEVs) from active AL amyloidosis patients present stable oligomeric light chain species Ramirez-Alvarado, et al., PLoS ONE 2012 7(6):e38061

Proof of concept to increase sensitivity to evaluate minimal residual disease using mass spectrometry of uEVs • Mass spec of uEVs and serum: monoclonal immunoglobulin Rapid Accurate Mass Measurement (miRAMM) • Laser microdissection followed by mass spectrometry • cDNA characterization of CD138+ cells from bone marrow • uEVs extraction, characterization using WB and miRAMM • Longitudinal studies of serum samples using miRAMM Ramirez-Alvarado et al., Am J Hematol. 2017 Jun;92(6):536-541

2008 2013 Ramirez-Alvarado et al., Am J Hematol. 2017 Jun;92(6):536-541

Minimal residual disease-uEVs analyzed with miRAMM allows us to identify the pathogenic light chain post treatment when FLC is undetectable in serum Ramirez-Alvarado et al., Am J Hematol. 2017 Jun;92(6):536-541

The LC mass found on the uEV oligomers matches with the same protein found on the renal amyloid deposits and the cDNA translated sequence from plasma cells <--------FR1----------><---CDR1----><------FR2-----><---CDR2---><----------------FR3-------------><-CDR3-><---FR-----> sss ssssss ssssssssssss sssss sss sss ssssssss ssssssssss sssssssss ssssss ssssss 2 3 4 5 6 7 8 9 10 123456789-123456789012345678901abc23456789012345678901abcde23456789012345678ab901234567890123456789012345abcde67890123 IGLV 6-57 NFMLTQPHS-VSESPGKTVTISCTRSSGSIASN-YVQWYQQRPGSSPTTVIYED-----NQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNYYVFGTGTKVTVL AL-ex11 kidamyl VL NFMLTQPHS-VSESPGKTVTISC A RSSGSIASN-YVQW F QQRPGS A PTTVVY E D-----HQRPSGVPDRFSGSIDSSSNSASLTISGLTADDEADYYCQSYDDSNYYVFGTGTKVTVL AL-ex11 cDNA VL NFMLTQPHS-VSESPGKTVTISCARSSGSIASN-YVQWFQQRPGSAPTTVVYED-----HQRPSGVPDRFSGSIDSSSNSASLTISGLTADDEADYYCQSYDDSNYYVFGTGTKVTVL ssssssssssss hhhh ssssssss sssss ssssssss ssssssssss hhhhhhh sssss sssssssssssss 2 3 4 5 6 7 8 9 10 1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456 IGLC1 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS AL-ex11 kidamyl CL GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS AL-ex11 cDNA CL GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Ramirez-Alvarado et al., Am J Hematol. 2017 Jun;92(6):536-541

Variables to take into account with uEVs to use as a clinical tool to follow renal disease response and progression ➢ Urine is a highly variable fluid ➢ Urine volume varies among patients ➢ 24 hour urine sample addresses this partly (dialysis patients barely generate any urine) ➢ Protein content varies by fluid intake and GFR ➢ % Albumin vs. non-Albumin protein content ➢ Particle numbers (total uEVs in urine) vary by disease state ➢ Types of protein excreted varies by disease state ➢ Confounding factors: Non pathogenic Immunoglobulins in urine/uEVs

Important differences exist between uEV protein concentration and particle concentration among different plasma cell dyscrasias and healthy controls 2,00E+12 Particle Concentration 2,50 Concentration ( µ g/µL) 1,50E+12 2,00 Total Protein per mL 1,50 1,00E+12 1,00 5,00E+11 0,50 0,00 0,00E+00 AL MGUS HD AL MGUS HD 240 202 101 240 202 101 Cooper et al., under review

… and there is no correlation between We found no correlation between total protein content uEV sample concentration and total and particle concentration… protein content 4,5E+11 0,45 Particle Concentration per mL uEV sample Concentration 4E+11 0,4 3,5E+11 0,35 0,3 3E+11 µ g/ μ L 0,25 2,5E+11 0,2 2E+11 p≤0.55 p≤0.52 0,15 1,5E+11 Pearson Correlation Pearson Correlation 0,1 1E+11 y = -2E+12x + 4E+11 0,05 y = 2,4222x + 0,1675 5E+10 R² = 0,0961 R² = 0,112 0 0 0 0,02 0,04 0,06 0,08 0 0,02 0,04 0,06 0,08 24 hour Urine Protein mg/mL 24 hour Urine Protein mg/mL Cooper et al., under review

uEVs in our samples present exosome particle size HD 101 AL 240 100 Mode Particle Size Particle Size in nm 95 90 85 80 75 70 MGUS 202 AL MGUS HD 240 202 101 Cooper et al., under review

Towards standardization: unifying uEV protein content ID Total Protein % Urine Total Urine Total uEV Resuspension Total Ratio uEV Non-Albumin uEV Sample Urine Urine mg/day Albumin Protein Sample Protein Protein Volume (µL) uEV sample to uEV protein Volume (µL) Volume(mL) Volume Concentration (mL) in Urine Bradford sample Total Urine (µg/µL) Needed for Needed for (mL) (mg/mL) Sample (µg/µL) Protein Protein (%) 15 µ g Assay 15 µ g Assay (µg) (µg) AL D64E 2340 15561 57 6.65 60 399000 4.24 375 1590 0.3985 1.8232 8.23 65.51 AL D64F 2039 15843 54 7.77 60 466200 3.26 375 1222.5 0.2622 1.4996 10.00 52.41 AL D64G 1728 15431 55 8.93 60 535800 4.52 375 1695 0.3163 2.0340 7.37 46.62 AL D64H 4307 14428 53 3.35 60 201000 1.57 375 588.75 0.2929 0.7379 20.33 118.98 µ 𝒉 𝒐𝒑𝒐𝑩𝒎𝒄 𝑭𝑾 𝒒𝒔𝒑𝒖𝒇𝒋𝒐 = 𝟒𝟖𝟔 µ 𝑴 ∗ 𝟐𝟔 µ 𝒉 / 𝟐𝟏 µ 𝑴 = 𝟔𝟕𝟑𝒗𝒉 𝒐𝒑𝒐𝑩𝒎𝒄 𝑭𝑾 𝒒𝒔𝒑𝒖𝒇𝒋𝒐 Fraction of protein in uEVs over total protein in urine is around 0.3% From this information , we can calculate how much urine volume we need to analyze 15 mg of uEV protein Cooper et al., under review

Testing the algorithm using stringent conditions 250 150 100 75 50 37 25 20 15 10 Patient AL AL ALD HD AL AL AL AL AL HD 263 263 64-G 101-B 250 250 D64-F D64-F D64-F 101-B sample TP Total 17.2 28.4 33.3 13.2 21.7 65.1 4.6 7.2 32.6 6.6 m g m g m g m g m g m g m g m g m g m g Protein Non- 15 24.7 15 3.7 11 2.1 3.3 15 m g m g m g m g m g m g m g m g Albumin uEV ANTI-Kappa Free Light Chain ANTI-Lambda Free Light Chain Cooper et al., under review

Testing our protein standardization with historical data (progress so far)

New patients or • uEVs were extracted patients with using filtering stable disease methods instead of oligomers 17 ultracentrifugation in no 11 most cases oligomers Trends are in the right • When both methods Partial response or direction were used, we very good partial in these historical response evaluated the samples that were not oligomers 4 presence/absence of no 5 optimized for our current oligomers in oligomers methods ultracentrifuged samples • Urine volumes used Complete response range from 5-20 mL oligomers 5 no 6 oligomers The evaluation of response was made by Nelson Leung. Additional blind evaluations will be done. The western blot reading of oligomer presence was done blindly Trish Caffes, Shawna Cooper