18.06.17 - Mature B cell neoplasms, CLL, MM

Question 1

Immunoglobulins are composed of heavy chains (IGH) and light chains; either kappa (IGK) or lamba (IGL). What are the genomic loci of IGH, IGK and IGL?

Answer 1

IGH - 14q32
IGK - 2p12
IGL - 22q11

Question 2

Give an overview of CLL, including incidence.

Answer 2

1) Chronic mature B-cell neoplasm
2) Caused by proliferation and accumulation of monomorphic B lymphocytes in PB, BM, LN, spleen.
3) Accumulation of mature, dysfunction B cells prevents the haematopoiesis of other normal blood cells leading to cytopaenias.
4) CLL only differentiated from SLL by leukaemic appearance.
5) Most common leukaemia in Western adults 12.8/100,000 by age 65, accounts forr 6.5 of NHL. Hig(2.1:1.5)
6) Highest genetic predisposition of haematological malignancies 5-10% CLL have a family history

Question 3

What are the presenting features of CLL?

Answer 3

May be asymptomatic with incidental detection, bit can present with:

1) Fatigue
2) Autoimmune haemolytic anameia
3) Infections
4) Splenomegaly
5) Lymphadenopathy or extra-nodal infiltrates

Question 4

CLL may proceed through distinct clinical phases, describe these.

Answer 4

Pre-malignant - monoclonal B-cell lymphocytosis (MBL)
Overt CLL
Transformation to Richter’s syndrome - aggressive lymphoma - median survival <1 year.

Question 5

What percentage of CLL patients develop a lymphoma pre- or post-treatment?

Answer 5

5-15%

e.g. diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma.

Question 6

What is the potential difficulty of karyotyping for CLL?

Answer 6

Cells may not divide in culture way

Addition of TPA (mitogen) is helpful

Question 7

What percentage of CLL patients will have a clonal abnormality detected by karyotyping or FISH?

Answer 7

50% by karyotyping

>80% by FISH

Question 8

What are the most common chromosome abnormalities detected in CLL patients. What is their associated prognosis?

Answer 8

Isolated del 13q14.3 (mono-or biallelic) - 55% - favourable
del(11q23) (ATM) - 18% - poor
trisomy 12 - 16% - intermediate
del(17p13) (TP53) - 7% - poor

Question 9

Which FISH probes are used for new CLL referrals and to track progression of disease?

Answer 9

ATM/TP53 for detection of gain/loss of signal

12/13 - for detection of gain or loss of signal

Question 10

Which CLL rearrangements are considered to be ‘first-stage’ abnomalities, and which rearrangements may indicate progressive disease?

Answer 10

First-step: T12 and del13q14.3 similarly represented through all stages of disease. Clonal driver mutations.

Progression: del 11q23 (ATM deletion)

Question 11

What is the prognostic significance of a TP53 (17p13) deletion?

Answer 11

Frequently acquired following treatment. Poor prognosis as patients do not respond to standard CLL therapies Associated with a higher genetic complexity. Mutations of TP53 are commonly seen where deletions have been detected on the other allele.

Question 12

The detection of chromosome abnormalities can help to distinguish CLL from other lymphoproliferative disease. GIve an example of a differential diagnosis for CLL.

Answer 12

Mantle cell lymphoma. IGH-CCND1
t(11;14)(q13;q32)

• is also seen in atypical CLL

Question 13

~20% of CLL patients have a rearrangement involving IGH. What is the chromosomal position of IGH, what is the prognosis for CLL patients with IGH rearrangements?

Answer 13

IGH (14q32)

Poor prognosis as often indicative of a complex karyotype

Question 14

Which IGH rearrangement is commonly seen with trisomy 12 in CLL patients? What is its prognosis?

Answer 14

IGH-BCL3
t(14;19)(q32;q13)
inferior prognosis

Question 15

Give three examples of IGH rearrangements that may be seen in CLL patients, and associated prognosis if known.

Answer 15

1) IGH-BCL11A t(2;14)(p16.1;q32)
2) IGH-BCL2 t(14;18)(q32;q21) - no association with complex karyotype or aggressive features
3) IGH-MYC t(8;14)(q24;q32) -subgroup of CLL with poor prognosis. May be secondary abnormality with role in transformation.

Question 16

A number of recurrent somatic mutations have been identified in CLL by NGS. Give some examples and their prognostic significance if known.

Answer 16

4-15% have a mutation of NOTCH1. Truncation of NOTCH1 removes the PEST sequence, which leads to the accumulation of an active protein form. Poor prognosis.

SF3B1 (ubiquitously expressed splicing factor) is found to be mutated in 5-17%, associated with a poor prognosis and drug resistance.

BIRC3

Question 17

With with chromosome aberration are NOTCH1 mutations commonly seen with?

Answer 17

Trisomy 12

More commonly seen with unmutated IGVH CLL

Question 18

Four prognostic subgroups have been developed for CLL that are an integration of cyto and molecular finding. What findings are associated with each category?

Answer 18

High risk - TP53del +/or BIRC3 abn. 10 year survival = 29%

Intermediate risk = NOTCH1 +/or SF3B1 +/or ATM del (11q22-23). 10 year survival 37%

Low risk = +12 or normal cyto. 10 year survival = 57%

Very low risk = isolated 13q14.3del. 10 year survival 69.3%.

Question 19

What diagnostic strategies differentiate CLL from other lymphoproliferative diseases? e.g. hairy-cell leukaemia or leukaemia manifestations of MCL

Answer 19

Blood count
Blood smear
Immunophenotype of circulating lymphoid cells
- CD5+, CD10-, CD19+, CD23+, CD43+, CD103-, BCL6-

Marrow examination not required for diagnosis, but required to determine complete response.

Question 20

Why is FISH used for CLL patients?

Answer 20

To determine cytogenetic aberrations that may not be detectable by karyotyping and may inform therapy.

Question 21

What clinical guidelines are in place for CLL testing.

Answer 21

No ACGS CLL specific guideline

ACGS Haemato-oncology BPG

Question 22

Other than karyotyping and FISH, what other genetic testing methods may be useful in CLL patients?

Answer 22

NGS - detection of somatic mutation
SNP arrays - LoH detection
Other microarray for CN detection

Question 23

What are the advantages and disadvantage of using array-based techniques in CLL patients?

Answer 23

Does not require cell culture, therefore issue of low proliferation overcome.
May not detect mosaicism sensitively enough and low level clones are missed.

Question 24

What is the treatment for CLL?

Answer 24

Incurable.
Patients treated with the onset of symptoms, or disease progression.
Patients usually follow a pattern of remitting and relapsing. Treatment is delivered within context of clinical trial
- FLAIR (fit patients)
- RIAltO (unfit patients)

Question 25

What are the five of the key recommendations for CLL patients stated by the British Committee for Standards in Haematology?

Answer 25

1. All patients are screened for TP53 abnormality pre-treatment.
2. Cyclophoshamide is recommended for fit, previously untreated or relapsed patients who require treatment and who have not entered a clinical trial
3. Alemtuzumab should be considered for previously untreated or relapsed patients with a TP53 abnormality and those with fludaribine-refractory disease who require treatment
4. Suitable patients with poor risk factors such as a TP53 abnormality and those who relapse early after intensive therapy should be considered for allogentic transplantation
5. Patients should be offered entry into clinical trials where possible

Question 26

What are the clinical variants of plasma cell myeloma/multiple myeloma?

Answer 26

1. Nonsecretory myeloma
2. Smoldering myeloma
3. Indolent myeloma
4. Plasma cell leukaemia

Question 27

Give a brief overview of multiple myeloma.

Answer 27

Accounts for ~1% of tumours and 10-15% of malignant neoplasms.
Annual incidence ~60-70/million
More common in men (1.4:1)
More common in AA population
Median age ~70 (rare in <40)
Genetic predisposition. 3.7 fold increase is first degree relative affected.

Question 28

What is MGUS?

Answer 28

Monoclonal gammopathy of undertermined significance
Precursor state to myeloma
Non-malignant and usually asymptomatic
Plasma cell content <10%
Patients have 1% risk by year of progression to myeloma and require observation.

Question 29

What is smoldering myeloma?

Answer 29

Intermediate between MGUS and MM
Stable PC content 10-30%
No osteocytic lesions
No anaemia
No other clinical findings characteristic of MM

Question 30

What is myeloma?

Answer 30

Plasma cell neoplasm. Accumulation of clonal plasma cells in BM.
PC content >10%
Expansion of a clone of IG-secreting, heavy-chain class-switched, terminally differentiated B-cells that typically secrete a monoclonal immunoglobulin called paraprotein or M-protein detectable in serum and/or urine.

Question 31

What is the typical presentation of myeloma?

Answer 31

Four leading symptoms:
C- hypercalcaemia
R - renal failure, due to proteinuria
A - anaemia, due to BM replacement. 
B - bone disease - lytic bone lesions, hypercalcaemia, fractures, osteroporosis, bone pain

Question 32

What is extramedullary MM?

Answer 32

More aggressive form and may be called secondary or primary plasma cell leukaemia.

Question 33

What proportion of new MM patients will have an abnormality detected by conventional karyotyping? by FISH?

Answer 33

30-40% karyotyping

>90% FISH

Question 34

What are the two subgroups of MM patients following cyotgenetic analysis? and what are the associated prognoses?

Answer 34

1) Hyperdiploid (~50%) 48-75 chr (usually trisomy of 8 chromosomes, odd numbers - 3, 5, 7, 9, 11, 15, 19, 21) favourable prognosis
2) Non-hyperdiploid (~50%) <48, >75, usually include translocations (IGH, 70%) and hemizygous 13del, poor prognosis

Question 35

What are the five major IGH rearrangement partners seen in MM?

Answer 35

t(4;14)(p16.3;q32) - FGFR3
t(11;14)(q13;q32) - CCND1
t(14;16)(q32;q23) - MAF
t(6;14)(p21;q32) - CCND3
t(14;20)(q32;q11) - MAFB

Question 36

What is the genomic location of FGFR3? What frequency its rearrangement with IGH seen in MM patients, and what is its prognostic significance?

Answer 36

4p16.3

15%

Poor prognosis

Question 37

What is the genomic location of CCND1? What frequency its rearrangement with IGH seen in MM patients, and what is its prognostic significance?

Answer 37

11q13

15%

Good prognosis

Question 38

What is the genomic location of MAF? What frequency its rearrangement with IGH seen in MM patients, and what is its prognostic significance?

Answer 38

16q23

5%

Poor prognosis

Question 39

What is the genomic location of CCND3? What frequency its rearrangement with IGH seen in MM patients, and what is its prognostic significance?

Answer 39

6p21

3%

Good prognosis

Question 40

What is the genomic location of MAFB? What frequency its rearrangement with IGH seen in MM patients, and what is its prognostic significance?

Answer 40

20q11

2%

Poor (some evidence to suggest very aggressive and therefore very poor prognostic indicator).

Question 41

Which genetic changes indicate disease progression in MM patients?

Answer 41

1) Monosomy/del 13q (50%) - close association with t(4;14)
2) Del 17p/TP53 (10%) - rare, late event, v. poor prognosis
3) Loss 1p/gain 1q - (40% of new, 70% at relapse) - poor prognosis
4) MYC translocation/amplifications (45% in advanced MM) - late event in tumour progression

Question 42

Summarise the diagnostic strategy for MM patients.

Answer 42

Low proliferation makes karyotyping difficult (3-4 unstimulated culture recommended)

Instead, FISH on plasma cell interphase nuclei for specfic poor prognostic rearragements. Cell selection prior to FISH to ensure myeloma cells are tested. PCE recommended

ImmunoFISH - tagged cells

Question 43

Which FISH probes are used for MM patients?

Answer 43

EMN recommendation: 
t(4;14) - IGH/FGFR3
t(14;16) - IGH/MAF
TP53
1p/1q 

Extended panel:
t(11;14) - IGH/CCND1
t(14;20) - IGH/MAFB
Ploidy status
12/13

Question 44

What is the treatment for multiple myeloma?

Answer 44

Incurable
Treatment given with the onset of symptoms
Standard chemo, steroids, thalidomide, SCT.
Some promising response with allogenic stem cell transplants and reduced intensity conditioning (RIC-allo) in selected patients, could be a curative approach in PCM.