Le gammapatie monoclonali: un palcoscenico per il laboratorio

Le gammapatie monoclonali: un palcoscenico per
il laboratorio
Giovanni Palladini
[email protected]
Centro per lo Studio e la Cura delle Amiloidosi Sistemiche
Fondazione IRCCS Policlinico San Matteo
Dipartimento di Biochimica
Università di Pavia
HRAgEP
Cell Ac EP
CZE
Kyle & Rajkumar N Engl J Med 2004;351:1860-73.
Bone Marrow Specimen from a
Individual with MGUS
Blade J. N Engl J Med 2006;355:2765-2770
Bone Marrow Specimen from a
Patient with Multiple Myeloma
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.
Bergsagel, P. L. et al. J Clin Oncol; 23:6333-6338 2005
Copyright © American Society of Clinical Oncology
A monoclonal gammopathy precedes multiple myeloma in most patients
Weiss et al, Blood. 2009;113:5418-5422
Monoclonal gammopathy of undetermined significance (MGUS)
consistently precedes multiple myeloma: a prospective study
Landgren et al, Blood. 2009;113:5412-541
Diagnostic Criteria for MGUS, Multiple Myeloma, and Other Conditions
Blade J. N Engl J Med 2006;355:2765-2770
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
MGUS prevalence is higher (about two
fold) in Afro-Americans compared to
Caucasian but does not increase with
age in Afro-Americans
Cohen et al, Am J Med, 1998
Landgren et al, Blood 2006
Landgren et al, Mayo Clinic Proc,
2007
Prevalence is lower in Japan
Iwanaga et al, Mayo Clin Proc, 2007
MGUS Prevalence
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
Vachon CM; Blood 2009
Brown LM, Cancer 1999
Grosbois B, Br J Haematol, 1999
Shoenfeld Y, Postgrad Med 1982
There is no plateau and continuous follow‐up is necessary
Relative risk of full
progression
9
7
64
48.8
5
65
50
41.2
3
24.6
25
15.6
1
13.6
13.6
0.5
1.0
1.5
2.0
2.5
3.0
Probability of full progression
at 20 years (%)
Relative Risk of Full Progression by
Serum M‐Spike Size
Serum m-spike value
CP999081-2
Recommended Testing in Patients with Suspected MGUS
Blade J. N Engl J Med 2006;355:2765-2770
Dangerous small B cell clones
Merlini & Stone Blood 2006;108:2520-2530
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.
• 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
Treatment related mortality: 2.5%
other: 7%
liver failure: 4%
renal failure:4.5%
CHF/sudden death: 82%
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 NTproBNP has been unanimously
approved
MGUS
DIFFERENTIAL DIAGNOSIS
Multiple Myeloma
Clone mass
Anemia
Cytopenia
Plasmacytoma
Immunodeficiency
Cytokines
Anemia
Bone destruction
Const. symptoms
Acute phase
Immunodeficiency
M-component
Myeloma cast nephropathy
Ig deposition (AL, LCDD)
Hyperviscosity
Immunodeficiency
Myeloma‐related organ or tissue impairment (ROTI)
International Myeloma Working Group (BJH, 2003, 21:749)
• 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
Plus: amyloidosis, symptomatic hyperviscosity, recurrent
bacterial infections (> 2 episodes in 12 months).
Detection, Characterization and Follow up
of
Monoclonal Immunoglobulins
College of American Pathologists
Guidelines 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)
4853
St.
HRAgEP of serum and urine samples from patients with AL
AL AMYLOIDOSIS
Diagnosis: problems and pitfalls
AgEP
In 56% of AL patients the serum M-protein is not
detectable by densitometry
IF anti 
S
U
S
U
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 samples
identified the monoclonal type in all samples with peak
concentrations >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 of
treatment with thalidomide.
College of American Pathologists
Guidelines 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 DETECTION
AND TYPING ARE:
HIGH RESOLUTION AGAROSE GEL ELECTROPHORESIS
OR CAPILLARY ZONE ELECTROPHORESIS
HIGH RESOLUTION IMMUNOFIXATION
College of American Pathologists
Guidelines 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 diseases
most frequent associations:
1
2
3
4
multiple myeloma
Waldenström’ macroglobulinemia
AL amyloidosis
light chain deposition disease
rare associations:
lymphomas
chronic lymphatic leukemia
idiopathic (or benign or of undetermined significance)
when to perform a Bence Jones protein
1
2
patients with serum monoclonal component
(at the discovery and during follow up)
patients suspected of having a monoclonal
gammapathy, clinically or from laboratory
findings:
- bone pain, fatigue, recurrent infections,
purpura, edema
- unexpected hypogammaglobulinemia in adults
anemia, unexplained increased ESR, proteinuria
Bence Jones protein: detection
1. urine sample
second morning void / random
+ Na azide (1%)
as passed or concentrate
2. method
Bence Jones protein: detection
1. urine sample
2. method
monoclonal
electrophoresis
free light chains
immunofixation
Intact Immunoglobulin
Kappa
Free Light Chain
Binding Site®, free light chain assay
Exposed surface
Hidden surface
Previously
hidden
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 for
the 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
Diagnostic sensitivity of IFE and FLC / ratio in 115 patients
with systemic AL amyloidosis
Palladini et al, Clin Chem. 2009;55:499-504.
Technique
Overall
(n. 115)
 clones
(n. 30)
clones
(n. 85)
% positive (95% CI)
IFE serum
urine
serum+urine
80 (72-87)
67 (58-75)
96 (91-98)
60 (42-76)
70 (52-84)
90 (75-97)
87 (79-93)
65 (55-75)
98 (92-100)
FLC ratio
88 (68-94)
97 (85-100)
82 (69-89)
IFE serum + FLC 
96 (91-98)
100 (90-100)
94 (97-98)
IFE serum+urine+FLC 
100 (97-100) 100 (90-100)
100 (96-100)
SUMMARY: Uses of Serum Free Light Chain Assay
•
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*