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Le gammapatie monoclonali: un palcoscenico per il laboratorio
Giovanni [email protected]
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
