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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
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 24th, 2019
Disclosure of Conflict of Interest
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Name of for-profit or not-for-profit
Description of relationship(s) Any direct financial payments including receipt
Membership on advisory boards or speakers’ bureaus Funded grants or clinical trials Patents on a drug, product
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
❑ 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
apoptotic bodies
methods
Journal of Endocrinology 228 R57-R71
Nature Cell Biology volume 20, pages332–343 (2018)
50-120 nm 200-500 nm 400-600 nm
Ramirez-Alvarado, et al., PLoS ONE 2012 7(6):e38061
Urinary extracellular vesicles (uEVs) from active AL amyloidosis patients present stable oligomeric light chain species
Proof of concept to increase sensitivity to evaluate minimal residual disease using mass spectrometry
immunoglobulin Rapid Accurate Mass Measurement (miRAMM)
marrow
miRAMM
miRAMM
Ramirez-Alvarado et al., Am J Hematol. 2017 Jun;92(6):536-541
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
<--------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-VSESPGKTVTISCARSSGSIASN-YVQWFQQRPGSAPTTVVYED-----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
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
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
0,00 0,50 1,00 1,50 2,00 2,50 Total Protein Concentration (µg/µL) AL MGUS HD 240 202 101 0,00E+00 5,00E+11 1,00E+12 1,50E+12 2,00E+12 Particle Concentration per mL AL MGUS HD 240 202 101
Important differences exist between uEV protein concentration and particle concentration among different plasma cell dyscrasias and healthy controls
Cooper et al., under review
y = -2E+12x + 4E+11 R² = 0,0961
5E+10 1E+11 1,5E+11 2E+11 2,5E+11 3E+11 3,5E+11 4E+11 4,5E+11
0,02 0,04 0,06 0,08 Particle Concentration per mL 24 hour Urine Protein mg/mL p≤0.55 Pearson Correlation y = 2,4222x + 0,1675 R² = 0,112 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45
0,02 0,04 0,06 0,08
uEV sample Concentration µg/μL 24 hour Urine Protein mg/mL
p≤0.52 Pearson Correlation
We found no correlation between total protein content and particle concentration… … and there is no correlation between uEV sample concentration and total protein content
Cooper et al., under review
70 75 80 85 90 95 100 Particle Size in nm AL MGUS HD 240 202 101 Mode Particle Size Cooper et al., under review AL 240 HD 101 MGUS 202
uEVs in our samples present exosome particle size
ID Total Urine Volume (mL) Protein mg/day % Albumin Urine Total Protein Concentration (mg/mL) Urine Sample (mL) Total Protein in Urine Sample (µg) uEV Protein Bradford (µg/µL) Resuspension Volume (µL) Total uEV sample Protein (µg) Ratio uEV sample to Total Urine Protein (%) Non-Albumin uEV protein (µg/µL) uEV Sample Volume (µL) Needed for 15µg Assay Urine Volume(mL) Needed for 15µg Assay 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
µ𝒉 𝒐𝒑𝒐𝑩𝒎𝒄 𝑭𝑾 𝒒𝒔𝒑𝒖𝒇𝒋𝒐 = 𝟒𝟖𝟔µ𝑴 ∗ 𝟐𝟔µ𝒉/𝟐𝟏µ𝑴 = 𝟔𝟕𝟑𝒗𝒉 𝒐𝒑𝒐𝑩𝒎𝒄 𝑭𝑾 𝒒𝒔𝒑𝒖𝒇𝒋𝒐
Cooper et al., under review
Towards standardization: unifying uEV protein content
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
250 150 100 75 50 37 25 20 15 10
Patient sample
AL 263 AL 263 TP ALD 64-G HD 101-B AL 250 AL 250 AL D64-F AL D64-F AL D64-F HD 101-B
Total Protein
17.2 mg 28.4 mg 33.3 mg 13.2 mg 21.7 mg 65.1 mg 4.6 mg 7.2 mg 32.6 mg 6.6 mg Non- Albumin uEV 15 mg 24.7 mg 15 mg 3.7 mg 11 mg 2.1 mg 3.3 mg 15 mg
ANTI-Kappa Free Light Chain ANTI-Lambda Free Light Chain
Cooper et al., under review
Testing the algorithm using stringent conditions
New patients or patients with stable disease
17 no
11 Partial response or very good partial response
4 no
5
Trish Caffes, Shawna Cooper
Complete response
5 no
6
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
using filtering methods instead of ultracentrifugation in most cases
were used, we evaluated the presence/absence of
ultracentrifuged samples
range from 5-20 mL Trends are in the right direction in these historical samples that were not
methods
New patients or patients with stable disease
17 no
11 Partial response or very good partial response
4 no
5
Trish Caffes, Shawna Cooper
Complete response
5 no
6
The evaluation of response was made by Nelson Leung. Additional blind evaluations will be done by two hematologists. The western blot reading of oligomer presence was done blindly
using filtering methods instead of ultracentrifugation in most cases
were used, we evaluated the presence/absence of
ultracentrifuged samples
range from 5-20 mL
Testing the algorithm with historical samples:
If the uEV protein recovery is 0.3% and the amount of non Albumin loaded in the gel is 0.562 mg, Range of volume needed for assay using algorithm: 20-6808 mL urine (avg: 1147 mL) Average 24 hour urine volume from historical patients: 1491 mL Range of % albumin in urine: 3-81% (with active disease); 3-100% for patients in CR. Proteinuria range: 65-29326 mg/day
What I hope you learned today…
(oligomeric species) in their uEVs.
detectable using mass spectrometry
standardize the uEV assay in plasma cell dyscrasias
to observe oligomers with an assay we hope to move to the clinic in the near future
EB Allen Luis Blancas Mejía, PhD Shawna Cooper, MS Chris J. Dick Torri Jordan Khansaa Maar Pinaki Misra, PhD Trish Caffes
COLLABORATORS Clinical collaborators Nelson Leung (Nephrology) Samih Nasr (pathology) Priya Alexander (pathology) Morie Gertz (Hematology)
Angela Dispenzieri (Hematology)
Martha Grogan (Cardiology)
Geoff Johnson (Nuclear medicine)
Chris Ward (nephrology, KUMC) Fibril toxicity and imaging Jon Wall (UT-Knoxville)
Mesenchymal stromal cells Yi Lin (Hematology) Solid State NMR Chad Rienstra (UIUC)
Mass spectrometry Ellen McPhail (Lab medicine) Surendra Dasari (bioinformatics, lab medicine) David Barnidge (Formerly lab medicine, now TBS) David Murray (Lab Medicine) Bob Bergen (Proteomics)
Funding
NIH R01 GM 071514 NIH R01 GM 128253 NSF 1744098 Regenerative medicine-Mayo Department of lab medicine and pathology- A. Dispenzieri Mayo Seidler Professorship-Gertz Mayo Foundation Generous support from Amyloidosis patients and their families
Ramirez-Alvarado, et al., PLoS ONE 2012 7(6):e38061
miRAMM Patient : AL-ex11 sample date: 2008 2009 2010 2013 Kappa clone +11 Molecular mass 2126.06 Da 2126.10 Da 2126.09 Da 2125.96 Da