AL Amyloidosis:Emerging

Approaches

Raymond L Comenzo, MD

The John Conant Davis Myeloma and Amyloid Program

Open Questions & Emerging Approaches

• How can we achieve truly early diagnosis?

• What is the role of imaging?

• What are the dimensions of FLC toxicity?

• What is organ involvement?

• Are anti-amyloid therapies relevant?

• Is the aCR the last word on response?

• How is MRD testing relevant?

• Are there anti-light chain therapies?

How can we achieve truly early diagnosis?

• Risk assessment• Intercepting AL• Populations at risk

– MGUS– Smoldering multiple myeloma– Multiple myeloma– Waldenstrom’s

• Follow biomarkers• Assess light chains• Clonal genetics

Blood 2014;123:305

How can we achieve truly early diagnosis?IMWG Diagnostic Criteria

1 Presence of an amyloid-related systemic syndrome (e.g., renal, liver, heart, GI tract, or peripheral nerve involvement).

2 Positive amyloid staining by Congo red in any tissue (e.g., fat aspirate, bone marrow, or organ biopsy).

3 Evidence that amyloid is light-chain-related, established by direct examination of the amyloid using mass spectrometry-based proteomic analysis or IEM.

4 Evidence of a monoclonal plasma cell proliferative disorder (serum or urine monoclonal protein, abnormal FLC ratio, or clonal plasma cells in the bone marrow).

Lancet Oncol 2014;15:538Acta Haematol 2019;141:93

Condition % positive

AL 84

ATTRm 45

ATTRwt 15

AL Precursor State

??

Abdominal Fat Pad Aspirate

Stained with Congo red

Eur Heart J 2017;38:1905

Follow Biomarkers

• In patients with pre-existing PCD– Interval from symptoms to AL diagnosis = 10 mos

• Pattern is to refer out– Primary care

– Cardiologists

– Neurologists

– Gastroenterologists

• >30% of AL patients take 3+ years to diagnosis

• And >72% see 3 or more MDs to be diagnosed

Am J Hematol 2014;89:1051Patient 2018;11:207

Assess Light Chains

• Focus on plasma cell disease subpopulations– With serum light chains

– Challenges in purification

• Competition assay– Biotinylated fibrils and LC

• MALDI-TOF-MS– Detect glycosylation

– 11% of AL LC

• Long roads to clinical useAmyloid 2018;25:93Leukemia 2019;33:254

The SAVE Pilot Trial: AL-Basehttp://albase.bumc.bu.edu/aldb

Five λ germline genes account for 73% of λ AL

λ GermlineGene

AL(N / Total)

MM(N / Total)

Odds RatioFavoring AL

95% CI P-value

LV6-57 113/616λ 3/117λ 7.2 2.3 to 22.1 < 0.0001

LV1-44 80/616λ 6/117λ 2.53 1.13 to 5.67 0.01

LV3-21 21/616λ 9/117λ 2.36 1.05 to 5.29 0.04

LV3-1 177/616λ 41/117λ 1.22 0.93 to 1.61 0.19

LV2-14 59/616λ 7/117λ 1.7 0.75 to 3.4 0.29

IGVL Gene Usage

Blood 2001 98: 714-720

MX B 1 2 3 4

Blood 2017;129:299-306

IGVL Gene Usage

The SAVE Pilot Trial: A Diagnostic Screening Trial

Seeking AL Amyloidosis Very EarlyNCT02741999

Department of DefenseSerum Repository

• Pre-diagnosis sera retrieved for AL cases

• 20 cases of AL and 20 controls

– 100% of cases and 0% of controls had monoclonal proteins

• Monoclonal protein present

– In 100% of cases < 4 years before diagnosis

– In 80% between 4 and 11 years before diagnosis

– In 42% at more than 11 years before diagnosis

J Clin Oncol 2014;32:2699-2704.

Department of DefenseSerum Repository

J Clin Oncol 2014;32:2699-2704.

dFLC was > 23 mg/L in 85% of cases and 0% of controls

The SAVE Pilot Trial: A Diagnostic Screening Trial

Seeking AL Amyloidosis Very EarlyNCT02741999

The John C. Davis Myeloma and Amyloid Program

Foremost in Patient Care, At the Front Line for Cures

Clinical Members Andreas Klein, MD

Kenneth B. Miller, MD

Kellie Sprague, MD Monika Pilichowska, MD

Terry Fogaren, NP

Raymond L. Comenzo, MD Founding Director

Research & Support Staff Xun Ma, DMD, PhD

Ping Zhou, MD, PhD

Melissa Warner Denis Toskic

Lori Brown

Dear Mr./Ms.____________,

You are receiving this package because you have enrolled in our study “A Diagnostic Screening Trial Seeking AL Amyloidosis Very Early” and have informed us that you have an upcoming blood draw where you can obtain your research sample for this study. This letter will explain each item we have included in this shipping kit and provide you with instructions on how to package your sample after your blood draw. Included in this shipping kit are the following:

1) A prescription for 3 green top tubes of peripheral blood

2) 3 green top tubes 3) Return shipping envelope 4) Rubber band 5) Paper towels 6) Bubble wrap 7) A specimen bag 8) Pathology box

Instructions for obtaining your research sample:

On the day of your blood draw, remember to bring this shipping kit with you to your clinic. It has important materials to store your blood and ship it back to us at Tufts Medical Center.

After you arrive at your clinic and are ready for your blood draw, you can give your phlebotomist (blood draw specialist) the included prescription for 3 green top tubes of blood as well as the 3 green top tubes to store your sample.

Your phlebotomist will then draw your blood and collect your research sample for this study in those 3 green top tubes. If you have other appointments after this blood draw, you can ask your

phlebotomist for a bag to put the tubes in and bring them with you. Carrying them with you for a while will not harm the sample; they will be perfectly fine for a few extra hours.

After you are done with your appointment(s), it is best to assemble your return shipping kit and send it as soon as possible.

In order to assemble your shipping kit, you should:

1) Take the 3 green

top tubes containing your sample and secure them together using the provided rubber band

2) Wrap the provided paper towel around the rubber-banded

tubes

3) Wrap the bubble wrap around the tubes and paper towels

4) Insert bubble-wrapped tubes into specimen bag and seal

(try to press out as much of the air from the bag as possible before sealing; otherwise, the bag may be too bulky for the next step)

5) Insert specimen bag into the labeled pathology box

The SAVE Kit

The SAVE Pilot Trial Lab Work

Extract RNA to make cDNA

Pilot Trial Results

Pilot Trial Results

• 20 asymptomatic patients (3M, 17W)

• Medians: iFLC 113mg/L, κ:λ ratio 0.06

• 17 patients with IGVL genes identified– LV2-14 = 5, LV1-44 = 3, LV3-19 = 2, LV3-21 = 2, and 1

each of 1-47, 2-11, 3-1, 3-25, 4-69

– 12 AL-related germline donors

• 2 undiagnosed AL patients identified (LV2-14)– One with GI involvement (SMM 2016)

– One with early cardiac involvement (NT-proBNP 171) (SMM 2009)

– Both SCT candidates

What is the role of imaging?

• Cardiac MRI is not validated for diagnosis

• 99mTc-PYP/DPD scan is validated for ATTR cardiac

• SAP scan is validated: Reagent limitations

• 18F-florbetapir (Dr Dorbala)– Reliable cardiac and lung signal

– Hepatically metabolized

– No renal signal

• “An exciting area”

• “Breakthroughs on the

horizon” Acta Haematol 2019;141:93Eur J Nucl Med Mol Imaging 2018;45:1129Current Cardiovascular Imaging Reports 2018;11: 17Circ Cardiovasc Imaging. 2015;8:e002954

Screening Concept

• Smoldering myeloma or MGUS

• AL-related light chain gene– Asymptomatic

– Non-invasive testing negative

– Fat pad Congo-red negative

• 18F-florbetapir imaging– Guiding a biopsy?

• EM needed?

– Diagnosing early disease, ‘seeding’? Affinity of ‘seeds’?

– Diagnosing risk of disease?

What are the dimensions of FLC toxicity?What is organ involvement?

• Not just passive deposition

or fibril formation

– Ronglih Liao, PhD

– Direct LC cardiotoxicity

– Pathways & targets

• Archetypal models

Blood 2014;123:3543 Science 2017;355:198

N Engl J Med 2003; 349:583-596

Amyloid fibrils elicit biological activities

Trends Biochem Sci 2015

PLoS One. 2015

Oligomeric species dissociate from fibril ends to generate a localised pool of toxic species.

MEL 200 SCT on a Clinical Trial:Complete Hematologic Response

51 year old man, 8 years s/p SCT

Blood 1996;88:2301-6

1 year post-SCT

10 years post-SCT

1 year post-SCT

10 years post-SCT

Blood 1996;88:2801

43 year old man, AL with

profound polyneuropathy

51 year old man, 8 years s/p SCT

Blood 1996;88:2301-6

1 year post-SCT

10 years post-SCT

1 year post-SCT

10 years post-SCT

Structural Hierarchyλ LC Proteins

PNAS 2016;113:6200-6205

Structural Hierarchyλ LC Proteins

PNAS 2016;113:6200-6205

Terminology

• Protofilaments

• Toxic aggregates

• Toxic intermediates

• Protofibrils

• Small amyloid oligomers

• Toxic oligomers

What are the dimensions of FLC toxicity?

• We do not know– The basis for LC aggregates

– Where aggregates are

– How aggregates vary

– What aggregates do

– If and how they are chaperoned

– How the transition to fibrils occurs

– The organ-specific contextual responses

– The immune response to the aggregate pool

– How to study the aggregate pool directly

Amyloid 2016;23:168-77

What is organ involvement? Quo Vadis?

• Earlier diagnosis– Intercept AL

– An urgent unmet need

• Multicenter SAVE Trial - ‘seed time’– Biobank, cells and plasma

• Blood

• Bone marrow

– Biomarker discoveries

• Organ responses to light chains, seeding and fibrillar deposits

Contextual Genetics• Organ-specific changes

– Stress signaling– Proteolytic pathways– Reprogramming

• RAIN Trial (NEOD001)– Renal biopsies: AL patients with persistent renal disease– Pathologic scoring (Agnes Fogo, Samih Nasr)– Transcriptional profiles

• U of Michigan (Mathias Kretzler, Neptune Program)• Large database

• Supervised analysis– Cell-extracellular matrix related genes

• IKMG, IMW 2019, ASH : Dr Cindy Varga

Are anti-amyloid therapies relevant?• Alzheimer’s trials• NEOD001 and CAEL-101

– In vitro activity– CAEL-101 active in in vivo imaging– Phase I/II data suggest activity– NEOD001 – Deficiencies in study designs? – Dual antibody or drug (eg, GM-CSF) approach needed?

• GSK – CPHPC + anti-SAP– Active– Study design?

• Doxycycline – In ATTR– Not validated in RCT

Are anti-amyloid therapies relevant?

• Timing– Seeding

• Interception of precursor protein– Prevent aggregation– Maintain folded or dimer status

• High concentrations of light chains– Increased aggregate pool?– Increased need for phagocytic

help?– Dual agents? Dual antibodies?– BITEs?

Amyloid 2016;23:168-77

0 12 24 36 48

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NT-proBNP progression (at least 300 ng/L and 30% increase), 169 patients

NT-proBNP stable, 108 patients

NT-proBNP response (at least 300 ng/L and 30% decrease), 100 patients

p<0.001

p<0.001

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CR (97 patients, 3.6 deaths/100 py)

VGPR (233 patients, 9.6 deaths/100 py)

PR (140 patients, 23.7 deaths/100 py)

NR (179 patients, 47.2 deaths/100 py)

p=0.01

p<0.001

p<0.001

Is the aCR the last word on response?

• aCR– Normal ratio– Negative IFEs

• ‘stringent CR’– D +100 post-SCT– Marrow studies– iFLC < 20mg/L

• MRD – In AL – marrow disease– NGS– Flow cytometry

J Clin Oncol 2012;30:4541

Leukemia 2012;26:2317

Leukemia 2016;30:1979

aCR: still useful?

• eGFR– FLC levels– Κ:λ ratio

• IFE sensitivity– WB– 0.1 g/dL

• Daratumumab– Uninvolved FLC < LLN– Incalculable ratio

• iFLC < 20mg/L?• iFLC < 10mg/L?

Nat Rev Nephrology 2009;5:621

Leukemia 2019;33:527

ANDROMEDA: Janssen

JCO 2018;36(15S):8011EHA 2019

Run-in N = 28> VGPR = 96%Normal iFLC = 54%

How is MRD testing relevant?• clonoSEQ® (https://www.clonoseq.com/sites/default/files/clonoSEQ)

– Multiplex PCR and NGS to identify and quantify rearranged Ig genes

• MRD Protocol (NCT02555969): 10 patients at 1 year

MRD: Adaptive Biotechnologies• Dominant trackable sequences

– A median of 3.5 per patient (2-5)

– Clonal IGVL genes were dominant in 4 of 8 cases

• In cases with intact Ig, IGHV sequences were first trackable.

• In 5/6 λ cases, KDE sequences were trackable

• In κ cases, KV sequences were both dominant and first trackable while in only 1/4 κ cases was a KDE among the dominant sequences.

Are there anti-light chain therapies?

• Directly reduce light chains?

– RNAi

• Clear light chains?*

– Monoclonal antibodies

• Prevent aggregation of amyloidogenic proteins?*

– Small peptides

*Grants currently funded by the Amyloidosis Foundation

RNAi: Consensus Sequence Targets in κ and λ Light-chain Genes

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Blood 2014;123:3440-51Gene Therapy 2016;23:727-733

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Blood 2014;123:3440-51Gene Therapy 2016;23:727-733

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Blood 2014;123:3440Gene Therapy 2016;23:727

A Novel Xenograft Mouse ModelFor Testing Approaches Targeting Human Kappa

Light-chain Diseases

Gene Therapy 2019 (in press)

Open Questions & Emerging Approaches

• How can we achieve truly early diagnosis?

• What is the role of imaging?

• What are the dimensions of FLC toxicity?

• What is organ involvement?

• Are anti-amyloid therapies relevant?

• Is the aCR the last word on response?

• How is MRD testing relevant?

• Are there anti-light chain therapies?

Themes – Look to the Future• Early diagnosis of disease and of risk of disease

• Different tool sets – young scientists needed– Genetic screens

– Imaging, Biomarkers

• Lessons of Alzheimer’s– Aducanumab, β-amyloid and coagulation (Sid Strickland)

• The Past:– Retrospection: Death and numbers

– What fibrils are

• The Future:– Intercept precursor disease

– Correlate imaging and biomarkers and seeding

– Understand seeding and propagation

AcknowledgementsDemarest Lloyd Jr Foundation

The Amyloidosis Foundation

Werner and Elaine Dannheiser Fund for

Research on the Biology of Aging

of the Lymphoma Foundation

The Amyloidosis and Myeloma Research Fund

MMRF

The Cam Neely and John Davis

Myeloma Research Fund

Janssen

Neely Center for Clinical Cancer Research

Cindy Varga, MD

Terry Fogaren, NP

Laboratory

Ping Zhou

Xun Ma

Adin Kugelmass

Denis Toskic

Amandeep Godara