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8.1 Outline the main subgroups of leukaemia in regards to cell of origin and clinical behaviour.
1. Acute myeloid leukaemia: an aggressive leukaemia derived from primitive cells of the myeloid lineage.
2. Chronic myeloid leukaemia: a relatively specific molecular entity (see section 8.3) typically characterised by elevated mature myeloid cells in the peripheral blood which usually follows an initial “chronic phase”.
3. Acute lymphoblastic leukaemia: an aggressive leukaemia derived from primitive cells of the lymphoid lineage.
4. Chronic lymphoproliferative disorders: indolent leukaemia derived from mature cells of the lymphoid lineage.
Acute leukaemias require urgent treatment whereas chronic leukaemias typically only require treatment in symptomatic patients.
8.2 Describe the role of karyotype in the prognostication of AML.
Karyotype stratifies AML into three groups based on the predicted risk of relapse after first remission: those with a low likelihood of relapse (favourable risk); those with a high likelihood of relapse (adverse risk) and those with an intermediate risk of relapse. Bone marrow transplant is typically only considered for patients with a high risk of relapse as morbidity and mortality resulting from this procedure is high.
8.3 What are the types of AML associated with a favourable risk and what cytogenetic and molecular abnormalities characterise these entities?
Acute promyelocytic leukaemia is characterised by a t(15;17) resulting in a PML-RARA fusion. Other translocations involving RARA have also been identified but these are rare. Core binding factor leukaemia is characterised by either a t(8;21) resulting in a RUNX1-RUNX1T1 fusion or inv(16)/t(16;16) resulting in a CBFB-MYH11 fusion.
8.4 Imagine you are a haematologist and receive molecular testing results for a patient with AML that demonstrate a FLT3-ITD and mutations in NPM1 and DNMT3A. What is the impact of these results on prognosis and treatment?
This combination of mutations is associated with a poor prognosis in AML and an allogeneic stem cell transplant would be considered in first remission in this patient.
8.5 Define a monosomal karyotype. How does this affect prognosis in AML?
A monosomal karyotype is defined as two or more monosomies of autosomal chromosomes or one autosomal monosomy plus at least one additional structural abnormality. Often chromosomes 5, 7 and 17 are involved. A monosomal karyotype identifies one of the worst prognostic groups in patients with AML.
8.6 What is the fundamental molecular abnormality in CML?
The Philadelphia chromosome is the defining molecular abnormality in CML, resulting from a translocation involving chromosomes 9 and 22 (t(9;22)) resulting in fusion of the BCR and ABL1 genes (BCR-ABL1).
8.7 List the common breakpoints that generate a BCR-ABL1 fusion. What are the different methods used to detect the Ph chromosome and what are the relative advantages and disadvantages of each?
The most common BCR-ABL1 transcripts are e13a2 and e14a2. Less common transcripts include e1a2 and e19a2. There are three methods used to detect the Ph chromosome:
- G-banded karyotyping detects a translocation involving chromosomes 9 and 22. It can also detect chromosome abnormalities in addition to the t(9;22). The method has limited sensitivity.
- FISH rapidly detects the BCR-ABL1 fusion gene and is useful when cytogenetics is negative or no metaphase cells can be obtained.
- RT-PCR is the most sensitive technique for detecting the BCR-ABL1 fusion transcript. RT-PCR is able to determine the breakpoints of the fusion, which is required for molecular monitoring in response to treatment.
8.8 Why is it important for patients with CML to achieve a deep molecular response?
Depth of response to Tyrosine Kinase Inhibitors is strongly correlated with risk of relapse. Patients who achieve a deep molecular response within the first year of treatment have excellent long term outcomes. In addition, a deep molecular response decreases the number of leukaemic cells able to give rise to blast phase disease.
8.9 What is the difference between primary and secondary resistance to Tyrosine Kinase Inhibitors? How can these develop?
Primary resistance: a lack of response to initial Tyrosine Kinase Inhibitor treatment, often due to BCR-ABL1 independent mechanisms which are not well understood but may involve drug efflux or insufficient dosage, duplication of the Ph chromosome, activation of alternative signalling pathways and epigenetic modification.
Secondary resistance: initial response to TKI therapy but eventual relapse, most commonly due to the development of point mutations in the tyrosine kinase domain of ABL1.
8.10 Outline the genetic lesions in B-ALL which determine prognosis.
Favourable genetic lesions include hyperdiploid karyotype and translocations involving RUNX1 and ETV6 (t(12;21)).
Unfavourable genetic lesions include Ph+ ALL, Ph-like ALL, iAMP21, hypodiploid karyotype, deletion or mutation of IKZF1.
8.11 What are the associations between T progenitor maturation stage and genetic lesion?
Early T-precursor ALL is characterised by mutations commonly seen in myeloid malignancies such as FLT3-ITDs and DNMT3A. RAS pathway mutations are also involved.
Cortical-type T-ALL may be separated into early and late groups based on aberrant expression of TLX1 and TAL1 respectively. Aberrant TLX1/TAL1 has been associated with translocations involving TRA and TRG. Mutations in NOTCH1 and CDKN2A are common in both subgroups.
8.12 What is the difference between a mutated and an unmutated IGHV? What are the clinical and biological implications of this distinction?
Mutated malignant cells arise from a B-cell that has passed through the germinal centre and has undergone somatic hypermutation of the IGHV locus.
Unmutated malignant cells arise from a B-cell that has not yet passed through the germinal centre and thus has not undergone somatic hypermutation of the IGHV locus.
CLL with an unmutated IGHV is clinically more aggressive with a higher tumour burden and is often treatment refractory, making these patients an important group to identify. IGHV mutation status is determined by PCR and direct sequencing.
8.13 Choose one of the four common cytogenetic abnormalities in CLL and outline the genes thought to be the target of this change, its association with IGHV and impact on prognosis.
11q22
Target gene: ATM and BIRC3
IGHV status: unmutated
Prognosis: poor
Trisomy 12
Target gene: unknown
IGHV status: unmutated
Prognosis: intermediate unless NOTCH1 is mutated in which case prognosis is poor
13q14
Target gene: RB1 and miR15a/miR16-1
IGHV status: mutated
Prognosis: favourable
17p13
Target gene: TP53
IGHV status: unmutated
Prognosis: poor
8.14 Describe the function of NOTCH1. How does mutation alter this function?
NOTCH1 mutations are typically frameshift mutations occurring within the PEST domain. These mutations prevent degradation of the active form of the protein, resulting in unregulated expression of NOTCH1 target genes.
8.15 What are the cellular effects of a BRAF Val600Glu mutation?
BRAF Val600Glu is an activating mutation which results in upregulated signalling through the MAPK pathway leading to an increase in cell proliferation and survival.
8.16 How is assessment of the T cell receptor (TR) used to determine clonality in T-cell leukaemia?
A homogeneous T cell receptor (TR) repertoire is a hallmark of T-cell malignancy as all malignant cells are clonally derived from a single neoplastic cell. However the presence of a clonal TR rearrangement does not necessarily imply malignancy because clonal or oligoclonal T-cell populations are also detected in non-malignant conditions such as HIV and EBV infection and benign/reactive inflammatory disorders.
8.17 Describe the various pathways to cyclin D dysregulation in myeloma.
IGH translocations that directly dysregulate CCND1 (t(11;14)) and CCND3 (t(14;16)) or indirectly through the dysregulation of MAF (t(14;16)) or MAFB (t(14;20)) transcription factors that target CCND2.
8.18 Which molecular feature is used to monitor response in myeloma? Are there other leukaemias that could be monitored using the same method?
The patient’s unique IGH VDJ recombination is used to monitor for minimal residual disease following treatment in myeloma. Currently two techniques are used – allele specific PCR and next generation sequencing. Other clonal lymphoid malignancies with a unique IGH or TR rearrangement, such as ALL, could also be monitored using similar methods.