Monoclonal Gammopathies

Monoclonal gammopathies: definition

 Clonal disease forms of terminal B lymphocytes

that synthesize immunoglobulins
 Monoclonal gammopathies: definition

 Clonal proliferation of plasma cell or lymphoplasmacytic

cell

 Presence of monoclonal protein => monoclonal Ig (Ig or part

of Ig)
Multiple Myeloma – bone marrow
 Monoclonal gammopathies: definition

 Clonal proliferation of plasma cell or lymphoplasmacytic cell

 Presence of monoclonal protein => monoclonal Ig (Ig or

part of Ig)
 Ig G
Ig A
Ig M
Ig D
Ig E

Light chain: kappa

lambda
Heavy chain: gamma 1,2 ,3,4
alfa 1, 2
mu
delta
epsilon
 Monoclonal gammopathies: definition

 Identification of monoclonal protein:

- Serum protein electrophoresis
- immunofixation

 Quantification of monoclonal protein :

- Nephelometry - levels of serum immunoglobulin
- levels of serum light chains
Serum protein electrophoresis – normal
Immunofixation of serum protein – normal
Serum protein immunofixation (IF): establishes the type
of monoclonal protein (heavy and light chain)
How to apply the fixator and AMC
(Monoclonal antibody) KIT
A. Serum sample with fixator
B. AMC sample with anti H γ
C. AMC serum sample with anti H-chain α
D. Anti serum samples from the AMC H chain μ
E. Anti serum samples from the AMC chain L k
F. AMC serum sample with anti L-chain λ.

Strips position obtained after immunofixation of serum

proteins.
Serum protein electrophoresis – polyclonal appearance
Serum protein electrophoresis – Ig A Multiple Myeloma
Serum protein electrophoresis
Kit
SEBI
A
 Serum protein electrophoresis

To interpret correctly an electrophoresis is recommended viewing

chart and not reading the results provided by the laboratory that
usually are not sufficiently informative and can lose diagnosis.
Immunofixation of serum protein – Ig A Multiple Myeloma
 Monoclonal gammopathies: classification

 Independent monoclonal gammopathies:

- Monoclonal gammopathy of unknown significance
(MGUS) 64%
- Multiple Myeloma (MM) 18%
- Waldestrom Disease 2%
- Primary (AL) amyloidosis 8%
- heavy chains disease

 Diseases associated with monoclonal component:

 Monoclonal gammopathies: classification

 Independent monoclonal gammopathies

 Diseases associated with monoclonal component:
- Malignant clonal proliferation - CLL 2%
- NHL 6%
- Controlled clonal proliferation
- Cold agglutinins disease
- Infections
- Liver disease
- Autoimmune diseases
 Multiple Myeloma

 Definition:
 Epidemiology: 3- 4 new cases / 100 000 / year
M> F
50-60 years
 Multiple Myeloma: pathogenesis

 Plasma cell proliferation

=> bone marrow failure
=> Hypercalcaemia
 Secretion of monoclonal immunoglobulin:
=> Adherence to plasma proteins
=> Adherence Er, L, Tb
=> Deposition in organs
=> Increase plasma viscosity
 Multiple Myeloma: clinical picture

 Marrow failure syndrome:

- anemia
- infections
- bleeding
 Pain syndrome - bone lysis / bone fractures
- Neurological lesion- radicular pain,
spinal cord compression
 Renal failure:
 Hypercalcemia syndrome
 Hyperviscosity syndrome
 Multiple Myeloma: tablou clinic
 Marrow failure syndrome
 Pain syndrome
 Kidney failure : - kidney infiltration of plasma cells
- Intratubular light chain deposition (cast
nephropathy) = tubular atrophy
- Light-chain deposition in the glomeruli
(amyloidosis / light chains deposition disease (LCDD)
- Dehydration, hyperuricemia, hypercalcemia,
drugs

 Hypercalcemia syndrome
 Hyperviscosity syndrome
 Multiple Myeloma: clinical picture
 Marrow failure syndrome
 Pain syndrome
 Kidney failure
 Hypercalcemia syndrome: weakness, confusion and
depression, or an abnormal heart rhythm including
cardiac arrest
 Hyperviscosity syndrome: Headache, visual changes
and retinopathy
 Multiple Myeloma: laboratory analysis

 Blood count
 Bone Marrow aspirate
 Serum protein electrophoresis
 Detection of Bence Jones (light chain) urinary (
nephelometry)
 calcemia
 serum viscosity
 C reactive protein
 Skeletal survey
Multiple Myeloma – bone marrow
Bone marrow aspirate: plasma cell infiltration
Serum protein electrophoresis
Kit
SEBI
A
 Serum protein electrophoresis

To interpret correctly an electrophoresis is recommended viewing

chart and not reading the results provided by the laboratory that
usually are not sufficiently informative and can lose diagnosis.
Serum protein immunofixation (IF): establishes the type
of monoclonal protein (heavy and light chain)
Modul de aplicare al fixatorului si AMC
(anticorpi monoclonali) KIT
A.Proba de ser cu fixator
B.Proba cu AMC anti H γ
C.Proba de ser cu AMC anti lant H α
D.Proba de ser cu AMC anti lant H μ
E.Proba de ser cu AMC anti lant L k
F.Proba de ser cu AMC anti lant L λ.

Pozitia benzilor obtinute in urma imunofixarii proteinelor

serice.
Serum protein immunofixation (IF)
 Multiple Myeloma: Classification by type of
monoclonal protein
=> IgG 56%
=> IgA 24%
=> IgD 2%
=> Ig E / M 1%
=> light chain (or Bence- Jones) myeloma - 16%.
EF is normal or hypo gammaglobuliemie, IF is positive
for kappa or lambda light chain
=> non-secretory myeloma - 1%. Not identify any
monoclonal protein: EF and IF are negative
 Multiple Myeloma: Imaging studies

Skeletal Survey (X –ray) Gold standard for baseline evaluation of

bone disease
MRI Mandatory in selected cases: e.g.,
presumed diagnosis of solitary
plasmacytoma; suspicion of cord
compression; pre – Kyphoplasty; vertebral
collapse in the context of osteoporosis to
confirm myelomatous involvement.
CT (whole body low dose) When MRI is unavailable for assessment of
the spine, or to clarify the extent of soft
tissues. Highest sensitivity for detection of
bone defects.
PET/CT Not to be used routinely. May be useful to
evaluate MRD outside the bone marrow.
Elective technique to assess response to
treatment.
 Skeletal survey

 skull
 backbone
 rib crib
 pelvis
 long bones (femur, humerus)

MRI - it is used in patients with

suspected backbone injury
Humerus fracture - MM
Ulnar lytic lesions - MM
Lytic lesions on the skull - MM
Diagnostic criteria for MGUS, SMM and MM

MGUS SMM MM

Bone marrow palsma < 10% >10 – 60% > 10% or biopsy proven
cells plasmacytoma
AND OR
M - spike < 3 g/ dl serum or < 500 > 3 g/ dl serum or >
mg / day urine 500 mg / day urine
AND AND AND
Myeloma- defining None None ≥1
event

Chesi M, International Journal of Laboratory Hematology, Volume 37, 108–114, May 2015
Diagnostic criteria for MGUS, SMM and MM

• Myeloma – defining event:

– Hypercalcemia (> 11 mg / dL)
– Renal Failure (creatinine clearence < 40 mL / min or creatinine > 2 mg / dL)
– Anemia (Hemoglobin < 10 g / dL)
– ≥ 1 lytic bone lesion on XR, CT or PET-CT
– ≥ 60 % bone marrow plasma cells
– ≥ 100 involved / uninvolved serum – free light chain ratio
– > 1 MRI focal lesion

Chesi M, International Journal of Laboratory Hematology, Volume 37, 108–114, May 2015
  International Staging System (ISS)

Stage ISS Criteria

I β2-microglobulin < 3.5 mg/L; albumin ≥ 3.5 g/L
II neither stage I nor stage III
III β2-microglobulin > 5.5 g/L
Multiple myeloma: Median overall survival

Durie-Salmon ISS staging

staging system system

Stage I 60 months 62 months

Stage II 40 months 44 months

Stage III 15 months 29 months

 Multiple Myeloma: Differential Diagnosis
is with other monoclonal gammopathies

 Monoclonal gammopathy of unknown

significance (MGUS)
 Asymptomatic multiple myeloma (smoldering -
MM)
 Waldenstrom's macroglobulinemia
 Solitary plasmacytoma
 Primary amyloidosis (AL)
 Multiple Myeloma - Therapy
Before deciding to therapy we need to consider the following:
1. MM - is a non-curable malignancy

2. Proper diagnosis

3. The presence of criteria for starting therapy => active myeloma

with one or more of the following criteria CRAB:
Increased Calcium (serum calcium> 11.5 mg / dL)
Renal disease (serum creatinine> 2 mg / dL)
Anemia (hemoglobin <10 g / dL or 2 g / dL <normal)
Bone disease - lytic lesions
 Multiple Myeloma - Therapy
4. To establish whether the patient is eligible for autologous bone
marrow:
Criteria for non- eligibility:
Age over 70 years
Direct bilirubin> 2.0 mg / dL (34.2 micromol / liter)
Serum creatinine> 2.5 mg / dL (221 micromol / liter), except cases of
chronic dialysis
Performance status Eastern Cooperative Oncology Group (ECOG) 3
or 4 unless simtomatologia is not connected with bone pain
Class III or IV heart failure (NYHA)

5. Trying to enroll the patients in a clinical trial, if possible.

 Active Multiple Myeloma - Therapy

I. Treatment complications

II. Cytoreductive therapy

 Active Multiple Myeloma - Therapy
I. The treatment for complications of the disease:
 H-E rebalance
 hyperviscosity => plasmapheresis
 bone disease - bone remineralization agents =>
bisphosphonates (Bonefos, Aredia, Zometa)
- Radiotherapy, vertebroplasty =>
Kyphoplastie, surgery (orthopedics / neurosurgery)
 anemia - blood product / erythropoietin
 hypercalcemia: massive hydration, dexamethasone,
furosemide, bisphosphonates
 Active Multiple Myeloma - Therapy
I. Treatment for complications of the disease:
 Renal failure: Identifying and solving reversible causes of
renal injury / dialysis.

 Infection: antibiotic with broad spectru / vaccination

 Active Multiple Myeloma - Therapy
II. Cytoreductive therapy:
1. Eligible patients for autotransplant (ASCT):
- Induction 4-6 cycles of chemotherapy (bortezomib
based therapy, lenalidomide, thalidomide). Avoid the use of
alkylating agents because they injure the hematopoietic stem
cell (HSC)
- HSC mobilization and harvest
- Autotransplant- administration of very high doses
(melphalan) and subsequently CSH (restoring hematopoiesis)
- follow up without specific therapy, bisphosphonates
only (24 months)
 Active Multiple Myeloma - Therapy
II. Cytoreductive therapy:
Patients ineligible for ASCT:
- Induction with 9 cycles of chemotherapy ((bortezomib
based therapy, lenalidomide, thalidomide).
- maintenance therapy
 Solitary Plasmacytoma
 Isolated plasma cell tumors
- bone
- extraosseous (soft tissue)

 Treatment:
- Surgical resection
- Radiotherapy
 Monoclonal gammopathy of unknown
significance (MGUS)

 without bone lesions

 monoclonal component <3 g% stable
 bone marrow plasma cells <10%
 without anemia, without renal disease, without
hypercalcemia

Do not require treatment

 Waldenstrom's disease
 Definition: proliferation of lymphocytes and plasma cells
Clinical:
 Hyperviscosity syndrome: dizziness, hearing disfunction,
confusional status, coma
Ophthalmic examination may reveal decreased
visual acuity, dilated retinal veins, "sausage-linked” of the
retinal veins, or retinal hemorrhages

 Tumoral syndrome: lymphadenopathy

hepatomegaly Splenomegaly
 Waldenstrom's disease: diagnostic

Serum protein electrophoresis and immunofixation

- IgM monoclonal protein
 Bone Marrow: infiltrated lymphocytes and plasma
cells (below 10%)

 The absence of bone lesions

 Waldenstrom's disease: therapy
 Plasmapheresis

 "watch and wait”

 chemotherapy: cyclophosphamide + Fludarabine

(Fludara), Rituximab (Rituxan), ibrutinib (Imbruvica)
 Amyloidosis: Definition

 Amyloid is a generic term for a group of protein substances

(chemically different) capable to aggregate as a fibrilar
material, with a linear beta -sheet secondary structure.

 Amyloid deposits can be found in various organs and tissues

in different amounts, causing a wide variety of diseases known
as the amyloidoses.
 Amyloidosis: Definition

 Histologically - these specific protein aggregates are

stained with Congo red and shows green birefringence
under polarized light .

 Depositing only in the extracellular space.

The proposed structure of beta sheet for amyloid
fibril
 Classification of Amyloidoses

SYSTEMIC AMYLOIDOSES LOCALIZED AMILOIDOSIS

HEREDITARY
PRIMARY ( AL Amiloidosis) C- cell thyroid tumors
Ig light chain Cardiac atria
REACTIVE (Secondary) Alzheimer’s disease
Amyloid A Familial dementia, British
HEREDITARY (Familial) Familial dementia, Danish
Transthyretin Cerebral hemorrhage with amyloid, Dutch
Muckles-Welles syndrome
Apolipoprotein A1
Gelsolin
NON-HEREDITARY
Fibrinogen A
Type 2 diabetes mellitus
Lyzozyme Alzheimer’s disease
Cystatin C Spongioform encephalopathies
BETA 2 MICROGLOBULIN AL localised amiloidosis
Beta 2 microglobulin
SENILE AMILOIDOSIS
Transthyretin
Westermak P, Amyloid, The Journal of Protein Folding Disorders, 2014;
 Light chain amyloidosis (Primary
amyloidosis)
 The precursor protein = immunoglobulin light chains (AL
amyloidosis)

 Amyloid fibrils = variable region of the light chains

 Ratio kappa / lambda = 1/2 (MM = 2/1)

 Production of light chains in plasma cells = bone

marrow, spleen, ...
 Light chain amyloidosis: clinical data

 Incidence 8 new cases / 1 million inhabitants /

year

 The median age: 62 years

 Light chain amyloidosis: clinical data
 Renal disease: progressive proteinuria => tubular injury =>
kidney disease
 Heart demage:
- arrhythmias
- Restrictive cardiomyopathy
- ECG – low QRS voltages in the limb leads /
pseudoinfarction pattern in the chest leads
- ECO - thickening of interventricular septum ,
myocardial structure modified (granular sparkling), atrial
dilatation, diastolic dysfunction, ventricular systolic
dysfunction / NT-proBNP increased
 Gastrointestinal : macroglossia, malabsorption,
hepatosplenomegaly
Periorbital Bleeding, Macroglossia
Skin bleedings
 Light chain amyloidosis: clinical data

 Neurologic- sensorimotor neuropathy, impaired

autonomic SNV,

 Hemorrhagic manifestations

 Carpal Tunnel Syndrome

 Light chain amyloidosis: diagnostic

 Biopsytissue H & E, Congo Red, Immuno-

histochemistry
 Bone marrow examination
 The study of serum and urinary proteins
 Examination of abdominal fat aspirate
 The
determination of the primary structure of
amyloid fibril
 Amyloidosis - Cardiac involvment

Congo red staining,x100 H&E staining,x100

Immunohistochemical staining against Immunohistochemical staining

KAPPA1,x100 negative control,x100
 Abdominal – fat: examination in polarized
light of preparations stained with Congo red
 Light chain amyloidosis: Therapy

 Chemotherapy as in multiple myeloma

 Therapy for inhibiting amyloidogenesis and stimulation of

resorption of amyloid deposits - monoclonal antibody

 Supportive therapy