Cytogenetic and molecular studies - AML, CLL, lymphoma and myeloma - molecular risk criteria

Genetics defines the disease

Genetics now defines these diseases, not just their prognosis. Four questions it answers: what is it, how bad is it, which drug, and is it still there?

Modality	Detects	Turnaround
Karyotype	Balanced translocations, complex karyotype, monosomies	5-10 days; needs dividing cells
FISH	Named rearrangements/deletions in interphase	1-3 days
PCR/RT-PCR	Fusion transcripts, MRD	Hours-days
NGS panel	Point mutations, indels	1-3 weeks
Flow cytometry	Immunophenotype, MRD	Hours
Optical genome mapping	Structural variants, replacing karyotype in some labs	Days

Epidemiology

AML: ~1000 new cases/yr in Australia; median age ~68
- Normal karyotype in ~45%; complex karyotype ~10-15% (rises with age)
CLL: the commonest leukaemia in the Western world; del(13q) ~55%, trisomy 12 ~15%, del(11q) ~10%, del(17p) ~5-8% at diagnosis (much higher at relapse)
DLBCL: commonest lymphoma; double-hit in ~8-10%
Mantle cell: ~5-7% of NHL; M>F ~3:1, median age ~65
Myeloma: t(11;14) ~15-20%, del(17p) ~8-10% at diagnosis, t(4;14) ~15%, 1q gain ~35-40%

Mechanisms of oncogenic rearrangement

Mechanism	Result	Example
Fusion gene	New chimeric protein	PML-RARA, BCR-ABL1, RUNX1-RUNX1T1
Promoter juxtaposition	Normal protein, deregulated expression	IGH partner translocations in lymphoma and myeloma
Loss of tumour suppressor	Haploinsufficiency/biallelic loss	del(17p)/TP53, del(11q)/ATM
Activating point mutation	Constitutive signalling	FLT3-ITD, JAK2 V617F, NPM1

IGH (14q32) translocations arise from errors in physiological V(D)J recombination and class-switch recombination in the germinal centre
- -> the partner oncogene comes under the immunoglobulin enhancer

The key partners

t(11;14)(q13;q32) - CCND1(cyclin D1)-IGH
- Mantle cell lymphoma - cyclin D1 overexpression -> G1/S transition
- Also ~15-20% of myeloma (where it is the venetoclax-sensitive subgroup)
t(14;18)(q32;q21) - BCL2-IGH -> follicular lymphoma (~85%); anti-apoptotic
t(8;14)(q24;q32) - MYC-IGH -> Burkitt lymphoma; proliferation index ~100%
t(9;14) - PAX5 -> lymphoplasmacytic; MYD88 L265P in >90% of Waldenstrom
t(4;14) FGFR3/NSD2, t(14;16) MAF -> high-risk myeloma

AML - ELN 2022 genetic risk

Risk	Genetics
Favourable	t(8;21) RUNX1-RUNX1T1; inv(16)/t(16;16) CBFB-MYH11; mutated NPM1 without FLT3-ITD; in-frame bZIP CEBPA
Intermediate	Mutated NPM1 WITH FLT3-ITD; wild-type NPM1 with FLT3-ITD; t(9;11) MLLT3-KMT2A; cytogenetic abnormalities not classified elsewhere
Adverse	Complex (>=3 abnormalities), monosomal karyotype, -5/del(5q), -7, -17/abn(17p); TP53; KMT2A rearrangements other than t(9;11); MECOM(EVI1); myelodysplasia-related mutations (ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2); RUNX1; BCR-ABL1

ELN 2022 changes from 2017: FLT3-ITD allelic ratio dropped; MDS-related mutations added; bZIP CEBPA specified
TP53-mutated AML with complex karyotype behaves worse than the rest of the adverse group - effectively "very adverse"
MRD by flow or NPM1/CBF-fusion PCR drives transplant decisions as much as the baseline genetics

CLL - test before every line of therapy

CLL - test before every line of therapy, not just at diagnosis

FISH panel: del(13q), trisomy 12, del(11q)/ATM, del(17p)/TP53
TP53 sequencing (mutation without deletion carries the same weight)
IGHV mutational status: unmutated (>=98% homology) = adverse with chemoimmunotherapy
del(17p)/TP53-mutated: chemoimmunotherapy must NOT be used
Clonal evolution - repeat FISH and TP53 at each relapse

Lymphoma

Double/triple-hit = concurrent MYC + BCL2 (and/or BCL6) rearrangements on FISH
- ~8-10% of DLBCL; very poor prognosis with R-CHOP
- WHO-HAEM5/ICC: "high-grade B-cell lymphoma with MYC and BCL2 rearrangements" is now its own entity; MYC+BCL6 alone has been moved out
- Test MYC FISH in all DLBCL; reflex to BCL2/BCL6 if MYC-rearranged
Double-EXPRESSOR (MYC + BCL2 protein by IHC, ~30%) is a different and weaker adverse marker - do not confuse with double-hit
Cell of origin (Hans algorithm: CD10, BCL6, MUM1) - GCB vs ABC
Mantle cell: cyclin D1 IHC + t(11;14) FISH; SOX11 positive in cyclin D1-negative cases
TP53 mutation in MCL - the dominant adverse marker, predicts failure of intensive chemoimmunotherapy

Myeloma - FISH on CD138-selected plasma cells

High risk	Standard risk
del(17p)/TP53, t(4;14), t(14;16), t(14;20), 1q gain/amp, del(1p)	t(11;14), trisomies (hyperdiploidy), t(6;14)

R-ISS / R2-ISS combine ISS stage + LDH + high-risk FISH
"Double-hit" myeloma = >=2 high-risk lesions
t(11;14) predicts venetoclax sensitivity (BCL2-dependent)

Genetics -> drug

Lesion	Therapy
FLT3-ITD/TKD (AML)	Midostaurin with induction; gilteritinib in relapse
IDH1/IDH2 (AML)	Ivosidenib / enasidenib
TP53 or secondary AML	Azacitidine + venetoclax; poor outcomes with intensive chemotherapy
CD33+ core-binding factor AML	Gemtuzumab ozogamicin
del(17p)/TP53 CLL	BTK inhibitor (zanubrutinib, acalabrutinib, ibrutinib) or venetoclax + obinutuzumab - never FCR
t(11;14) myeloma	Venetoclax (off-label/trial in Australia)
Double-hit lymphoma	Intensified regimen (DA-EPOCH-R) rather than R-CHOP; early CAR-T at relapse
TP53-mutated mantle cell	Avoid intensive chemoimmunotherapy -> BTK inhibitor-based, trials
BCR-ABL1	Tyrosine kinase inhibitor

Genetics -> transplant

AML: adverse risk, or MRD-positive after consolidation -> allogeneic HSCT in first remission
Favourable risk (CBF, NPM1 without FLT3-ITD): chemotherapy alone, transplant only for MRD failure or relapse
Intermediate: MRD and comorbidity decide

Genetics -> monitoring

AML: NPM1, RUNX1-RUNX1T1, CBFB-MYH11 transcripts as MRD; rising MRD = pre-emptive intervention
APL: PML-RARA RT-PCR
CML: BCR-ABL1 IS on the international scale
Myeloma: MRD by NGS/flow to 10^-5 or 10^-6

Germline matters

Test for a germline predisposition when: AML/MDS in a young patient, family history of cytopenias or haematological malignancy, or DDX41, RUNX1, GATA2, CEBPA, ANKRD26, ETV6, TP53 found at high VAF
Consequence: do not use a related donor without testing them

Related entities

Therapy-related myeloid neoplasm - alkylators (del(5q)/-7, latency 5-7 yr) vs topoisomerase II inhibitors (KMT2A/11q23 rearrangement, latency 1-3 yr)
Clonal haematopoiesis of indeterminate potential (CHIP) - DNMT3A, TET2, ASXL1; precursor to myeloid neoplasia and an independent cardiovascular risk factor
Germline predisposition syndromes: DDX41, RUNX1, GATA2, CEBPA, Li-Fraumeni, Fanconi anaemia, telomere biology disorders
Richter transformation of CLL (often TP53-disrupted, MYC-rearranged)
MGUS -> myeloma: the same primary FISH lesion is present from the MGUS stage
HIV and EBV - MYC-rearranged lymphomas
Down syndrome: GATA1-mutated transient abnormal myelopoiesis and ML-DS

AML by ELN risk - 5-year overall survival

AML by ELN risk - 5-year overall survival (intensively treated)


Favourable	~55-70%
Intermediate	~35-40%
Adverse	~10-20%
TP53-mutated	median <1 year

CLL prognosis

CLL

del(13q) sole abnormality: median survival >15-20 yr - often never needs treatment
del(17p)/TP53: historically <3-5 yr with chemoimmunotherapy; transformed by BTK inhibitors and venetoclax
Unmutated IGHV -> shorter time to first treatment (prognostic impact reduced, not abolished, by targeted therapy)

Lymphoma prognosis

Lymphoma

HGBL with MYC/BCL2 rearrangement: ~30-40% 5-yr survival vs ~60-70% for standard DLBCL
Mantle cell: median survival now 8-10+ yr; TP53-mutated ~1-2 yr with chemoimmunotherapy
Follicular: indolent, but POD24 (progression within 24 months) marks a high-risk group

Myeloma prognosis

Myeloma

del(17p), t(4;14), 1q amplification shorten progression-free and overall survival
t(4;14) partly rescued by proteasome inhibitor-containing therapy
MRD negativity is now the strongest post-treatment prognostic marker, outweighing baseline FISH risk

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