Make it countTechnical considerations and initial findings

for accurate quantitative comparison of

urinary extracellular vesicles to determine

minimal residual kidney disease in AL

amyloidosis

Marina Ramirez Alvarado, PhDDepartment 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

Nature of relationship(s)

Name of for-profit or not-for-profit organization(s)

Description of relationship(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

❑ 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

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

Journal of Endocrinology 228 R57-R71

Nature Cell Biology volume 20, pages332–343 (2018)

50-120 nm200-500 nm

400-600 nm

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

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

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

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

To

tal P

rote

in

Co

ncen

trati

on

(µ

g/µ

L)

AL MGUS HD 240 202 101

0,00E+00

5,00E+11

1,00E+12

1,50E+12

2,00E+12

Part

icle

Co

ncen

trati

on

p

er

mL

AL MGUS HD240 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+11R² = 0,0961

0

5E+10

1E+11

1,5E+11

2E+11

2,5E+11

3E+11

3,5E+11

4E+11

4,5E+11

0 0,02 0,04 0,06 0,08

Part

icle

Co

ncen

trati

on

per

mL

24 hour Urine Protein mg/mL

p≤0.55

Pearson Correlation

y = 2,4222x + 0,1675R² = 0,1120

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0 0,02 0,04 0,06 0,08

uE

Vsam

ple

Co

ncen

trati

on

µ

g/μ

L

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

Part

icle

Siz

e in

nm

AL MGUS HD240 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

µ𝒈 𝒏𝒐𝒏𝑨𝒍𝒃 𝑬𝑽 𝒑𝒓𝒐𝒕𝒆𝒊𝒏 = 𝟑𝟕𝟓µ𝑳 ∗ 𝟏𝟓µ𝒈/𝟏𝟎µ𝑳 = 𝟓𝟔𝟐𝒖𝒈 𝒏𝒐𝒏𝑨𝒍𝒃 𝑬𝑽 𝒑𝒓𝒐𝒕𝒆𝒊𝒏

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

Testing our

protein

standardization

with historical

data

(progress so far)

New patients or

patients with

stable disease

oligomers 17

no

oligomers

11

Partial response or

very good partial

response

oligomers 4

no

oligomers

5

Trish Caffes, Shawna Cooper

Complete response

oligomers 5

no

oligomers

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

• uEVs were extracted

using filtering

methods instead of

ultracentrifugation in

most cases

• When both methods

were used, we

evaluated the

presence/absence of

oligomers in

ultracentrifuged

samples

• Urine volumes used

range from 5-20 mL

Trends are in the right

direction

in these historical

samples that were not

optimized for our current

methods

New patients or

patients with

stable disease

oligomers 17

no

oligomers

11

Partial response or

very good partial

response

oligomers 4

no

oligomers

5

Trish Caffes, Shawna Cooper

Complete response

oligomers 5

no

oligomers

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

• uEVs were extracted

using filtering

methods instead of

ultracentrifugation in

most cases

• When both methods

were used, we

evaluated the

presence/absence of

oligomers in

ultracentrifuged

samples

• Urine volumes used

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…

• Patients with active AL amyloidosis present unique features

(oligomeric species) in their uEVs.

• Pathogenic light chains are enriched in uEVs and are

detectable using mass spectrometry

• Non-Albumin protein in urine is an important parameter to

standardize the uEV assay in plasma cell dyscrasias

• We have identified the range of parameters that allows us

to observe oligomers with an assay we hope to move to the

clinic in the near future

Ramírez-Alvarado Team 2019EB AllenLuis Blancas Mejía, PhDShawna Cooper, MSChris J. DickTorri JordanKhansaa MaarPinaki Misra, PhD

Trish Caffes

COLLABORATORSClinical collaboratorsNelson 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 imagingJon Wall (UT-Knoxville)• Angela Williams• Emily Martin

Mesenchymal stromal cellsYi Lin (Hematology)

Solid State NMRChad Rienstra (UIUC)• Dennis Piehl (UIUC)

Mass spectrometryEllen 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 071514NIH R01 GM 128253NSF 1744098

Regenerative medicine-MayoDepartment of lab medicine and pathology- A. DispenzieriMayo Seidler Professorship-GertzMayo Foundation

Generous support from Amyloidosis patients and their families

Ramirez-Alvarado, et al., PLoS ONE 2012 7(6):e38061

miRAMM

Patient : AL-ex11sample date:

2008

2009

2010

2013

Kappa clone +11

Molecular mass

2126.06 Da

2126.10 Da

2126.09 Da

2125.96 Da