M-protein diagnostics of Multiple Myeloma patients treated with biologics

JFM (Hans) Jacobs

Radboud university medical center (The Netherlands)

Laboratory Specialist Medical Immunology (H.Jacobs@Radboudumc.nl)

EFLM webinar March 27th 2018

Moderated by Christopher McCudden (Ottawa, Canada)

B lymphocyte

Bone marrow

Peripheral blood

Plasma Cell

Monoclonal gammopathy/plasma cell dyscrasia (e.g. multiple myeloma)

Serum protein electrophoresis (SPE)

the separation of charged proteins in an electrical field

g:

b:

a2:

Albumin

a1: - Orosomucoid

- Haptoglobin

- Transferrin

- Immunoglobulins (IgG)

• SPE characterized by albumin, α1-, α2-, β- and γ-fraction • Band-intensity corresponds to its concentration

- a1 Antitrypsin

- a2 Macroglobulin - a Lipoprotein - Ceruloplasmin

- C3 Complement - Hemopexin

- b Lipoprotein

Anode + Pole

Cathode - Pole

- Immunoglobulins (IgA)

M-protein diagnostics, gel electrophoresis

Alb

α-1

α-2

β

Normal γ

Serum protein

electrophoresis (SPE)

#1 #2

Detection

#1 normal

#2 M-protein

Densitometry

Quantification

ELP G A M κ λ

Serum #2

Immunofixation

electrophoresis (IFE)

IgG-κ

Typing/Characterisation

M-protein = Myeloma protein = Paraprotein = Monoclonal component = M spike

Diagnosed at Mayo Clinic 2002

Multiple Myeloma 18% (273)

Amyloidosis (AL) 11% (167)

Lymphoma 4% (55)

Smouldering myeloma 6% (87)

Solitary or extramedullary plasmacytoma

1% (23)

Waldenström’s Macroglobulinemia

3% (43)

Other 6% (93)

MGUS 51% (769)

IgA (21%) Biclonal

(1%)

IgE (0.01%)

IgG (59%) IgD (1%)

FLC 15%

Nonsecretory myeloma (3%)

Monoclonal gammopathy / Plasma cell dyscrasia

M-protein quantification is required for

Classification: f.e. MGUS, smouldering myeloma (sMM) or multiple myeloma (MM)

Staging of symptomatic myeloma (stage I, II and III)

To monitor disease / therapy-response / or disease evolution (f.e. from MGUS > sMM > MM)

M-protein quantification: Why?

Durie et al. Leukemia 2006 Rajkumar et al. Blood 2015

M-protein quantification: How?

Alb. α-1 α-2 β γ

M-protein ‘M-spike’ 20% of total serum protein

T G A M κ λ

SPE Immunofixation

IgG-kappa

For example: • Total serum protein = 80 g/L • M-spike = 20% of total serum protein

• IgG-kappa M-protein = 16 g/L

SPE scanning densitometry

ELP G A M κ λ

2011.1A IgG-kappa

100% IgG-kappa Mean: 42.4 g/L VC: 13 %

Dutch External Quality Assessment (EQA) M-protein diagnostics

National program organized by Radboudumc (75 participating labs)

2011.2C IgG-kappa

2011.4B IgG-kappa

>95% IgG-kappa Mean: 2.7 g/L VC: 29 %

92% IgG-kappa Mean: 5.5 g/L VC: 40 %

ELP G A M κ λ

Monitoring patients, requires good test reproducibility…

(n=75 labs) 2009.3A Mean: 6.9 g/L

CV: 23 %

ELP G A M K L

2009.4A Mean: 6.7 g/L CV: 22 %

2010.4A Mean: 6.4 g/L CV: 22 %

2012.1A Mean: 6.4 g/L CV: 22 %

2014.3B Mean: 6.4 g/L CV: 23 %

ELP G A M K L

Over 5 measurements average within-lab VC : 14 %

Bone marrow and lymphoid organs Polyclonal FLCs produced approx. 500 mg/day

Kidney Capacity to absorb and metabolize 10-30 gram/day

T1/2 varies from 2-6 hrs to (2-3 days with renal failure)

Katzmann et al. Clin Chem 2002

Free Light Chain biology

FLC normal ranges (Freelite, TBS)

Kappa: 3.3 – 19.4 mg/L Lambda: 5.7 – 26.3 mg/L Ratio: 0.26 – 1.65

Kappa Lambda

Diagnosed at Mayo Clinic 2002

Multiple Myeloma 18% (273)

Amyloidosis (AL) 11% (167)

Lymphoma 4% (55)

Smouldering myeloma 6% (87)

Solitary or extramedullary plasmacytoma

1% (23)

Waldenström’s Macroglobulinemia

3% (43)

Other 6% (93)

MGUS 51% (769)

IgA (21%) Biclonal

(1%)

IgE (0.01%)

IgG (59%) IgD (1%)

FLC 15%

Nonsecretory myeloma (3%)

Monoclonal gammopathy

Free Light Chain

λ or κ

Multiple myeloma and renal impairment

Multiple myeloma at initial presentation

• 18-50% renal impairment (serum creat ↑) • 12-15% acute renal failure • 8% become dialysis dependent

Dimopoulos et al. J Clin Oncol 2010 Basnayake et al. Kidney Int 2011

hyperCalcemia, Renal impairment, Anemia, Bone disease

(CRAB diagnostic criteria MM)

Block urine flow Interstit. inflam.

Pathology

• Cast nephropathy (myeloma kidney)

• Light chain (AL) amyloidosis

• Light chain deposition disease

• Hypercalcemia

• Nephrotoxic drugs

• Hyperviscosity syndrome

…

Monoclonal Free Light Chains: not always a monoclonal band

‘hidden epitope’

Free Light Chain λ or κ

ELP G A M κ λ FLC FLC κ λ

FLC’s = short T1/2 = low serum concentration = often no ‘M-spike’…

Henry Bence Jones

Bence Jones proteins The very first cancer biomarker: The Lancet; 1847

‘When urine is heated, a white cloud appears and a precipitate forms. The precipitate disappears on boiling and reappears on cooling...’

REF values (freelite) Free kappa: 3,3 – 19,4 mg/l Free lambda: 5,7 – 26,3 mg/l Ratio: 0,26 – 1,65

sFLC nephelometry

Bead

Bead

Patient 192 mg/l 6.6 mg/l 29

Diagnosis: FLC kappa plasmacytoma-Th12

If clinical suspicion of: FLC multiple myeloma AL amyloidosis

ELP G A M κ λ

Dispenzieri et al. Leukemia 2009

No M-protein… no monoclonal gammopathy?

‘Diagnostic requirement: additional band’

Sensitivity 500-2.000 mg/L 150-500 mg/L

M-protein diagnostics: summary

‘hidden epitope’

Free Light Chain λ or κ

Immunoassay (neph/turb/elisa)

Bead

Bead

‘Diagnostic requirement: abnormal FLC κ/λ ratio’

Bradwell et al. 2001 Clin Chem ‘immunoassay for quantification of FLC’ Drayson et al. 2001 Blood ‘identifying and monitoirng ‘non-secretory MM’ Dispenzieri et al. 2009 Leukemia ‘FLC in international guidelines’

Sensitivity 1-3 mg/L

15% LCMM

• Early detection LCMM • Improved monitoring • Prognostic value

85% intact M-protein

Multiple Myeloma 18%

AL Amyloidosis 11%

Lymphoma 4%

Smouldering myeloma 6%

Other 6% (93)

MGUS 51%

plasmacytoma 1%

Waldenström’s

Macroglobulinemia 3%

M-protein diagnostics: screening, diagnosis and staging

Diagnosed at Mayo Clinic 2002

Diagnostic criteria Disease staging

*

* Or myeloma defining event.

Rajkumar et al. Lancet Oncology 2014.

M-protein diagnostics: follow-up and response evaluation

Durie et al. Leukemia 2006 Rajkumar et al. Blood 2015

MGUS MM

Diagnosis Diagnosis

Progression to MM

1st relapse

2nd relapse

Improved treatment regimes for MM patients

IMWG guideline: Ludwig et al. Leukemia 2013

Daratumumab

Tumor specific antibodies

Carter et al. Nat Rev Cancer 2007

*List is not extensive…

Anti-cancer antibodies used in the clinic*

Scott et al. Nat Rev Cancer 2012

1. Direct tumor cell killing

3. Vascular and stromal cell ablation

2. Immune-mediated tumor cell killing

Daratumumab 2015 CD38 Multiple myeloma

Elotuzumab 2015 SLAMF7 Multiple myeloma

4. Immune modulation tumor micro-environm.

Monoclonal antibody therapy in multiple myeloma

Touzeau et al. Review. Leukemia 2017

Daratumumab

Elotuzumab

Production and humanization of monoclonal antibodies

Techniques:

1) Merge binding portion of monoclonal mouse antibody with human antibody producing DNA.

Use cell cultures to express this DNA product

2) Genetically engineered mice that produce ‘human’ antibodies / Human hybridomas

Risk of immunological rejection

Risk of loss of specificity

‘…momab’ ‘…ximab’ ‘…zumab’ ‘…umab’

Biologics for MM patients in clinical practice

Lokhorst et al. NEJM 2015 Mateos et al. NEJM 2018

Lonial et al. NEJM 2015

Daratumumab Mechanisms of effect

Laubach et al. Clin Cancer Research 2015 Van de Donk et al. Blood 2018

Immunomodulatory effects

expansion

Xu et al. Clin Phar Ther 2017 Clemens et al. Clin Pharmacokinet 2017

daratumumab

Human IgG1-kappa mAb biologic

Daratumumab pharmacokinetics

16 mg/kg Weekly

16 mg/kg Every 2 wks

16 mg/kg Every 4 wks

Reaching serum [dara] up to 1 g/L

PE G A M κ λ

Patient 1 Before Daratumumab

Enlarged γ-region

Patient 1 Before daratumumab

M-spike 20.4 g/L

1A

Daratumumab and M-protein interference

G κ λ

Patient 1 After daratumumab

Patient 1 After Daratumumab

Enlarged γ-region

Dara-spike 0.4 g/L

M-spike 10.6 g/L

B

G κ λ

Daratumumab spiked in saline

Van de Donk et al. Clin Chem Lab Med 2016

a.o. IFE negative…

IMWG response criteria (Durie et al. 2006)

SP G κ SP G κ

Before daratumumab

After daratumumab

Abrogate interference using mAb against biological

McCudden et al. Clin Chem Lab Med 2016

DIRA ‘DARA shift-assay’ Daratumumab-specific Immunofixation Reflex Assay

Perform DIRA (or similar shift assay)

Indication to use DIRA or similar shift-assay

Adapted from Van de Donk et al. Blood 2018

SP G κ SP G κ

Before daratumumab

After daratumumab

Daratumumab Hydrashift assay (=pos example)

(+ M-protein comigrates with dara)

Outlook: synergistic effect of combined mAb in MM patients ??

Adpated from Touzeau et al. Review. Leukemia 2017

Alternative techniques to multiplex M-protein / mAb monitoring

Zajec et al. J Proteome Res 2018 Willrich et al. CCLM 2016

Daratumumab does not interfere with serum FLC testing

Rosenberg et al. Clin Biochem 2016

Dira spiked sample in Dutch EQA

100% IgG-kappa M-protein Mean M-spike (n=66): 4.9 g/L Inter-lab CV: 22 %

Dara spiked at 5 g/L

M-spike 5.2 g/L

98% IgG-kappa M-protein Mean M-spike (n=44): 1.7 g/L Inter-lab CV: 46% Many labs don’t spike such small M-proteins and reported <2 g/L

Dara spiked at 1 g/L

All participants report a normal [FLC-kappa]: which is in line with observation of Rosenberg et al.: no monoclonal FLC kappa in Daratumumab

Theoretically yes, but they often go unnoticed….

Can mAb’s used for other indications also interfere with serum protein electrophoresis?

Daratumumab in MM patients

• SPE performed to monitor disease • Dara dosed at high concentrations (16 mg/kg i.v. weekly in first 8 weeks)

• Hypogamma globulinemia (caused by disease process and therapy)

Adalimumab (α-TNF) in Rheumatoid Arthritis

• SPE not commonly performed in RA • Adalimumab dosed at lower concentrations (40 mg s.c. weekly or every 2 weeks)

• Hypergamma globulinemia (caused by disease process)

Low background: easy to detect small bands

High background: difficult to detect small bands

McCudden et al. Clin Chem 2010

Daratumumab interferes with blood group compatibility testing

Van de Donk et al. Blood 2018

Minimal Residual Disease in multiple myeloma

THERAPY

Daratumumab

MRD: few remaining cancer cells after therapy, often below detection limit, eventually cause cancer relapse.

Kumar et al. Lancet Oncology 2016

“…>50% of patients achieve sCR…”

Towards curative therapy for MM…

Barlogie et al. Blood 2014 Paiva et al. Blood 2015

…increases the need for detecting MRD

MRD R&D focus on molecular assays on bone marrow

Landren et al. Am J Hematol 2014; Paiva et al. Blood 2016 (flow cytometry) Puig et al. Leukemia 2014 (ASO q-PCR) Martinez-Lopez et al. Blood 2014 (next generation sequencing) Mailankody et al. Nat Reviews 2015

Paiva et al. Blood 2015

Multicolor flow cytometry ASO qPCR Next Generation Sequencing

Focus: Rearranged B-cell receptor on MM cells

Bone marrow not preferred for monitoring MM

Sampling error caused by tumor heterogeneity

Cumbersome and time-consuming procedure for

repetitive monitoring

Mass spectrometry as alternative sensitive assay to detect M-proteins in serum*

Based on unique M-protein mass

*List of publications is not extensive…

Based on unique M-protein peptides

M-protein diagnostics = Personalized diagnostics

Questions?