Myeloma/Plasma cell dyscrasia

Question 1

MGUS

Answer 1

Precursor lesion with the potential to evolve into a plasma cell neiplasm.

• Presence of a non-IgM
• PP of <30g/L
• clonal bone marrow cells <10%,
• absence of end-organ damage

1% risk of progression to PCM each year

Most will have an IGH rearrangement and acquire a “second hit” genetic or microenvironment event to progress to MM

Question 2

MGUS risk of progression

Answer 2

LOW risk factor
* normal FLC ratio
* serum protein <15g/L
* IgG subtype

Question 3

IMWG Criteria for plasma cell diagnoses

Answer 3

Question 4

Other Myeloma defining events

Answer 4

60% or greater clonal plasma cells in bone marrow

Serum involved to uninvolved light chain ratio > 100

More than 1 focal lesion on MRI that is 5mm or greater in size

Question 5

Risk stratification in smouldering MM

Answer 5

Mayo 20/20/20
BM PCs >20%
PP >20g/L
FLC ratio >20

IMWG score
- increasing FLC ratio
- high PP
- BM PC%
- FISH detected t(4:14), del (13q), t(14:16), +1q, monosomy 13.

Question 6

Smouldering MM

Answer 6

ASYMPTOMATIC clonal plasma cell disorder. Distiguished from MGUS by much higher risk of progression to MM.

Defined by:
* PP >30g/L
* PCs 10-60%
* NO myeloma defining events

Question 7

MM morphology

Answer 7

PB
- rouleaux
- circulating PCs
- LEB

BMA plasma morphology is not a reliable predictor of abnormal plasma cells

Question 8

Normal PC phenotype

Answer 8

CD138+

CD19+
CD56-
CD27+
CD28-
CD45+
CD20-
CD81+
CD117-
CD200-
polyclonal cytLC

True aberrancy is indicated by >2 variants, with uniformity of expression of these variant expression patterns which are traceable within a single subpopulation and demonstration of monoclonal cytoLC

Question 9

Abberrant flow in PCM

Answer 9

(% indicates incidence in true PCM) 

• CD19- (96%)
• CD81- (95%)  
• CD200+ (82%)
• CD38 dim (80%)
• CD45- (80%)
• CD56+ (60%)
• CD28+ (36%)
• CD27- (32%)
• CD117+ (32%)
• CD20+ (~20%)

Question 10

Cytogenetics and molecular

Answer 10

~50% are hyperdiploid and have trisomies (or more) of odd-numbered chromosomes: 3, 5, 7, 9, 11, 15, 19, 21 

~50% have IGH translocations at chr 14q32, usually one of 1 genes being involved:   
* NSD2 on 4p16 
* CCND3 on 6p21
* MAFA on 8q24 
* CCND1 on 11q13
* CCN2on 12p13 
* MAF on 16q23 
* MAFB on 20q11 

Secondary chromosomal abnormalities: 
* Del 13q/monosomy 13 
* Del 17p/monosomy 17 
* Del 1p 
* Amp 1q 

Question 11

Mayo risk stratification of Myeloma

Answer 11

Question 12

Risk stratification for myeloma

Answer 12

Low, Standard and High

ISS, IMWG and R-ISS models
- ISS no molcular/cytogenetics

serum B2M
Albumin
LDH (only in R-ISS)

High risk cytogenetics/FISH:
* del17p
* t(4:14)
* t(14:16)

Question 13

Plasmacytoma

Answer 13

Plasmacytoma is a solitary neoplasm of clonal plasma cells without evidence of plasma cell (multiple) myeloma or end-organ damage due to plasma cell neoplasia.

Solitary plasmacytoma of bone (SPB) vs Extramedullary plasmacytoma (EMP)

No myleoma defining events and <10% clonal plasma cells on BM

Question 14

Plasma cell leukemia

Answer 14

≥5% circulating plasma cells in PB smears in patients otherwise diagnosed with PCM

Question 15

Plasma cell myeloma

Answer 15

Biopsy proven plasmacytoma OR ≥10% BM plasma cells

One or more of the following:
* SFLC ratio ≥100
* >1 focal lesions on MRI
* Hypercalcaemia (>2.75mmol/L)
* Renal insufficiency (Cr >177umol/L)
* Anaemia <100g/L
* One or more osteolytic bone lesions

Question 16

Myeloma MRD

Answer 16

Surrogate for PFS and OS in both transplant-eligible and ineligible patients, regardless of CR or non-CR status by IMWG criteria.

MRD monitoring options need to take consideration both
(1) disease localisation and
(2) desired sensitivity of assessment.

Multiple myeloma is a disease largely confined to the BM and EM sites without PB involvement

Methods:
1. multiparametric flow cytometry
2. molecular methods
3. cytogenetics
4. PET/CT scans

No fixed timepoints recommended. Surrogate for PFS and OS in both transplant-eligible and ineligible patients, regardless of CR or non-CR status by IMWG criteria. 

Question 17

Myeloma Flow MRD methods

Answer 17

Laboratory Methods include - with increasing sensitivity: 
- FISH (targeted lesions e.g. IGH-dependent translocation) - 1 x 10^-2 (1 in 200 abnormal metaphases) 
- Flow cytometry - Euroflow protocol or otherwise - down to10^-4 
- Molecular testing - ClonoSeq is FDA approved in myeloma MRD but not in routine use in Australia 

Question 18

Myeloma Flow MRD

Answer 18

Increasing sensitivity - increasing cell output
In order to increase sensitivity up to 10^-5

Pros
- Increased sensitivity to 10^-5
- Most of our standardization goals achieved
- Worlds best practice

Cons
- sample required within 28h
- Time consuming - total over 4 hours BEFORE analysis begins
- Relies on BMA sample, and patient may have MRD detected on PET CT with extramedullary disease
- Bulk lysing - 1.5hr
- SM staining - 1 hr
- Cytoplasm staining - extra 1 hr
- Instrument running time - 40min

Question 19

Myeloma Molecular MRD
- ClonoSeq

Answer 19

NGS of IgH, IgL, IgK loci

Once disease-associated clonotypes have been identified in a diagnostic (or ‘ID’) sample from a patient, the assay can be used to detect the level of residual disease in follow-up samples (‘MRD’ samples) from the same patient by tracking the presence and frequency of these clonotypes

Pros
- Applicability
- Specificity - 100%
- Sensitivity 10^-6

Cons
- baseline sample required
- Time consuming
ie so late that cannot make clinical decisions in a timely manner
- Labour intensive
- Lack of final PCR - somatic mutations or poor DNA quality

Question 20

Protein electrophoresis

Answer 20

Relies on the size and charge of proteins moving through gel to separate them using a semi-automated gel electrophoresis method.

Detection of protein
-Patient whole blood is collected and allowed to separate into serum
-Agarose gel
-Patient samples are added to wells at one end of the gel
-Gel has a current run through it with a negative and positive end
-Proteins in patient serum will separate based on size and charge and separate into 4 main areas
1. Albumin
2. Alpha 1
3. Alpha 2
4. Gamma globulins

-In plasma cell discrasias it is the gamma globulin region that is of interest
-Once migration has completed, the gel is dried and stained so that protein bands become visible
-This is then scanned by an analyser to determine protein percentages
-A positive and negative control is run with all patient samples
-If a monoclonal protein is detected in the gamma globulin region, we proceed to immunofixation

Question 21

SPEP bands

Answer 21

Question 22

IMMUNOFIXATION

Answer 22

Immunofixation is used to determine the type of monoclonal protein that is present again via migration of proteins through a gel with specific antisera
-Patient serum is collected and added to a track with 6 different wells: control, IgG, IgA, IgM, Kappa, Lambda
-Current run through gel to separate proteins
-Each track has a specific anti-sera added to it (each different immunoglobulin and kappa/lambda) that will bind the protein and cause it to precipitate
-Gel is washed to remove unbound protein
-Gel is dried and stained and is now ready for visual inspection and interpretation
-Peaks should then be quantified

Limitations
- variability between different labs
- best performed at the same lab

Question 23

Serum Free light chains

Answer 23

Serum sample

Prinicple of testing - use Polyclonal or monoclonal antibodies attached to latex microparticles incubated with serum
-attach to epitopes exposed only when light chains not bound to heavy chains
-form complexes
-quantified using turbidimetry (quantity of light transmitted through sample)
-compared to calibrator standard

Turbidimetry test
- Freelite (polyclonal) in my lab

Issues
- lot to lot variation in polyclonal antisera so cannot compare between labs
-Can cause false positives in renal failure, infections and inflammatory conditions