Le gammapatie monoclonali: un palcoscenico per il laboratorio · Monoclonal Gammopathies •...

Le gammapatie monoclonali: un palcoscenico per il laboratorio

Giovanni Palladinigiovanni.palladini@unipv.it

Centro per lo Studio e la Cura delle Amiloidosi SistemicheFondazione IRCCS Policlinico San Matteo

Dipartimento di BiochimicaUniversità di Pavia

CZECell Ac EP

HRAgEP

Kyle & Rajkumar N Engl J Med 2004;351:1860-73.

Blade J. N Engl J Med 2006;355:2765-2770

Bone Marrow Specimen from a Individual with MGUS

Bone Marrow Specimen from a Patient with Multiple Myeloma

Copyright © American Society of Clinical OncologyBergsagel, P. L. et al. J Clin Oncol; 23:6333-6338 2005

Disease stages and timing of oncogenic events

The earliest oncogenic changes are present in MGUS, and involve two minimally overlapping pathways (ovals), both of which substantially overlap the del 13 pathway (striped oval). Primary immunoglobulin (Ig) translocations (TLC) are thought to occur in germinal center B cells (bidirectional arrow) but the timing for the other two pathways (dashed arrows) is unclear.

Weiss et al, Blood. 2009;113:5418-5422

Landgren et al, Blood. 2009;113:5412-541

A monoclonal gammopathy precedes multiple myeloma in most patients

Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study

Blade J. N Engl J Med 2006;355:2765-2770

Diagnostic Criteria for MGUS, Multiple Myeloma, and Other Conditions

Age-specific and sex-specific prevalence of MGUS

Dispenzieri et al, Lancet 2010; 375: 1721–28

Prevalence of MGUS according to sex and age

Dispenzieri et al, Lancet 2010; 375: 1721–28

MGUS Prevalence

Cohen et al, Am J Med, 1998Landgren et al, Blood 2006Landgren et al, Mayo Clinic Proc, 2007

Iwanaga et al, Mayo Clin Proc, 2007

MGUS prevalence is higher (about two fold) in Afro-Americans compared to Caucasian but does not increase with age in Afro-Americans

Prevalence is lower in Japan

Vachon CM; Blood 2009Brown LM, Cancer 1999Grosbois B, Br J Haematol, 1999Shoenfeld Y, Postgrad Med 1982

MGUS prevalence is higher in first degree relatives of subjects with MGUS or MM

- non sex-linked

- facilitated by the environment (cases in communities, wife/husband)

- anticipation phenomenon

MGUS Prevalence

There is no plateau and continuous follow‐up is necessary

Relative Risk of Full Progression bySerum M‐Spike Size

Relative Risk of Full Progression bySerum M‐Spike Size

Serum m-spike valueSerum m-spike valueCP999081-2

Rel

ativ

e ris

k of

full

prog

ress

ion

Rel

ativ

e ris

k of

full

prog

ress

ion

Prob

abili

ty o

f ful

l pro

gres

sion

at 2

0 ye

ars

(%)

Prob

abili

ty o

f ful

l pro

gres

sion

at 2

0 ye

ars

(%)

0.50.5 1.01.0 1.51.5 2.02.0 2.52.5 3.03.0

13.613.6

2525

5050

6565

33

99

55

77

11 13.613.6

15.615.6

41.241.248.848.8

24.624.6

6464

Blade J. N Engl J Med 2006;355:2765-2770

Recommended Testing in Patients with Suspected MGUS

Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

Merlini & Stone Blood 2006;108:2520-2530

Dangerous small B cell clones

• The whole strategy of the follow‐up of MGUS is to prevent end‐stage organ damage

• AL amyloidosis is a silent killer: when cardiac involvement become symptomatic is already irreversibly fatal in 75% of patients

Cause of death in 210 patients with AL amyloidosis who died in the first year after diagnosis

CHF/sudden death: 82%

Treatment related mortality: 2.5%

liver failure: 4%

renal failure:4.5%

other: 7%

Survival of patients with AL amyloidosis according to NT-proBNP cutoff value (152 pmol/L).

Palladini et al, Circulation 2003

…..therefore at the Summit meeting in Barcelona the proposal to include in the follow up of individuals with MGUS also periodic measurements of the cardiac biomarker NT-proBNP has been unanimously approved

MGUS

DIFFERENTIAL DIAGNOSIS

Clone mass

AnemiaCytopeniaPlasmacytomaImmunodeficiency

Cytokines

AnemiaBone destructionConst. symptomsAcute phaseImmunodeficiency

Multiple Myeloma

M-component

Myeloma cast nephropathyIg deposition (AL, LCDD)HyperviscosityImmunodeficiency

Myeloma‐related organ or tissue impairment (ROTI)

• Calcium levels increased: serum calcium  > 0.25  mmol/L above the upper limit  of normal or > 2.75 mmol/L (12 mg/dL)

• Renal insufficiency: creatinine  >  173 mmol/l or 2 mg /dL• Anemia: Hb 2 g/dL below the lower limit of normal or < 10 g/dL

• Bone  lesions: lytic lesions or osteoporosis with compression  fractures

International Myeloma Working Group (BJH, 2003, 21:749)

Plus: amyloidosis, symptomatic hyperviscosity, recurrent bacterial infections (> 2 episodes in 12 months).

Detection, Characterization and Follow up of

Monoclonal Immunoglobulins

College of American PathologistsGuidelines for Laboratory Diagnosis and Monitoring of

Monoclonal Gammopathies

• Detection of monoclonal proteins requires the use of high-resolution electrophoresis (either gel-based or capillary) and immunofixation (or immunosubtraction)

(High-resolution techniques: provide crisp separation of transferrin and C3 bands in beta regions)

HRAgEP of serum and urine samples from patients with AL

St.

4853

In 56% of AL patients the serum M-protein is not detectable by densitometry

AL AMYLOIDOSIS

Diagnosis: problems and pitfalls

S U

AgEP

S U

IF anti

CZE and cellulose acetate electrophoresis gave similar data on 794 samples

The detection limit for MC was 0.5 g/L

MC assessment by immunosubtraction on 403 samplesidentified the monoclonal type in all samples with peakconcentrations >10 g/L

CZE of a patient with chemotherapy resistant IgA myeloma obtained before(green curve) one (blue curve) and three weeks (red curve) after initiation oftreatment with thalidomide.

College of American PathologistsGuidelines for Laboratory Diagnosis and Monitoring of

Monoclonal Gammopathies

• Characterization of Monoclonal Proteins

- Immunofixation is the method of choice

- Immunosubtraction performed on CE is not more sensitive than CE, while IF is ten times more sensitive than serum protein electrophoresis

THE RECOMMENDED METHODS FOR MC DETECTIONAND TYPING ARE:

HIGH RESOLUTION AGAROSE GEL ELECTROPHORESISOR CAPILLARY ZONE ELECTROPHORESIS

HIGH RESOLUTION IMMUNOFIXATION

College of American PathologistsGuidelines for Laboratory Diagnosis and Monitoring of

Monoclonal Gammopathies

• Quantification of the monoclonal component

- Quantification is best accomplished by a densitometric scan of the M-protein

- To calculate the amount of free light chains excreted each day, an accurate 24-hour urine collection with a densitometric scan of the spike representing the free monoclonal light chains is critical

Schema of plasma cells producing intact immunoglobulins and free light chain molecules

Associated diseasesmost frequent associations:

1 multiple myeloma 2 Waldenström’ macroglobulinemia3 AL amyloidosis 4 light chain deposition disease

rare associations:

lymphomaschronic lymphatic leukemiaidiopathic (or benign or of undetermined significance)

when to perform a Bence Jones protein

1 patients with serum monoclonal component(at the discovery and during follow up)

2 patients suspected of having a monoclonal gammapathy, clinically or from laboratory findings:- bone pain, fatigue, recurrent infections,purpura, edema

- unexpected hypogammaglobulinemia in adultsanemia, unexplained increased ESR, proteinuria

Bence Jones protein: detection

1. urine sample

2. method

second morning void / random + Na azide (1%)

as passed or concentrate

Bence Jones protein: detection

1. urine sample

2. methodmonoclonal

free light chains

electrophoresis

immunofixation

Intact Immunoglobulin

Exposed surface

Hidden surface

KappaFree Light ChainBinding Site®, free light chain assay

Previouslyhidden surface

FLC reference range: 3.3 – 19.4 mg/L 5.7 – 26.3 mg/L ratio 0.26 - 1.65

N Latex FLC – new monoclonal high-performance assays forthe determination of free light chain kappa and lambda

Velthuis et al, Clin Chem Lab Med 2011;49(8):1323–1332

Reference ranges of 369 samples: 116 fresh serum samples and 253 fresh EDTA plasma samples from healthy lab donors and healthy blood bank donors

Technique Overall(n. 115)

clones(n. 30)

clones(n. 85)

% positive (95% CI)

IFE serumurineserum+urine

FLC ratio

IFE serum + FLC

IFE serum+urine+FLC

80 (72-87)67 (58-75)96 (91-98)

88 (68-94)

96 (91-98)

100 (97-100)

60 (42-76)70 (52-84)90 (75-97)

97 (85-100)

100 (90-100)

100 (90-100)

87 (79-93)65 (55-75)98 (92-100)

82 (69-89)

94 (97-98)

100 (96-100)

Diagnostic sensitivity of IFE and FLC / ratio in 115 patients with systemic AL amyloidosis

Palladini et al, Clin Chem. 2009;55:499-504.

• SCREENING FOR PCD

• BASELINE VALUES PROGNOSTIC– Monoclonal gammopathy of undermined significance– Smoldering myeloma– Symptomatic myeloma– Plasmacytoma– AL amyloidosis

• HEMATOLOGIC RESPONSE– AL amyloidosis– “Non‐secretory” myeloma*– Stringent complete response in multiple myeloma*– Light chain deposition disease* 

SUMMARY: Uses of Serum Free Light Chain Assay