Chapter 8 Answers to discussion questions

8.1 How is molecular assessment of the immunoglobulin heavy chain locus and T-cell receptor locus utilised in lymphoid malignancy?

A good answer would discuss the use of IGH/TR in establishing a diagnosis of lymphoid malignancy, the prognostic implications of this test and the utility of IGH/TR in minimal residual disease testing.

To determine clonality:

Assessment of the distribution of the length of the PCR products obtained by standardised primers can indicate whether a population of cells is monoclonal or not. This is due to the fact that in mature T-cell and B-cells the TR and IGH loci are rearranged and due to random recombination and inclusion of nucleotides (through terminal deoxytransferase) the length of these PCR products should be normally distributed. The presence of a significant proportion of PCR products of one size indicates the presence of a monoclonal population. This can be used to support a diagnosis of lymphoid malignancy in the right clinicopathological context.

To determine prognosis:

Sequencing of the rearranged IGHV locus in CLL can be used to determine prognosis. Patients with evidence of somatic hypermutation of the IGHV locus (defined as a homology to germline of <98%) have a superior prognosis to patients with unmutated IGHV.

To monitor therapy:

Due to the unique nature of the TR/IGH rearrangement to each lymphoid malignancy, highly sensitive and patient specific assays can be designed in order to monitor response to treatment. This is commonly performed in ALL.

 

8.2 Choose a subtype of leukaemia and describe the techniques and utility of molecular methods used to monitor response to therapy.

AML (t(15;17), t(8;21) or inv(16) qPCR and NPM1 MRD), ALL (TR or IGH MRD), CML (BCR-ABL1 quantitative RT-PCR) and MM (IGH MRD) would be appropriate examples where molecular methods are used to monitor response to therapy. An example answer for CML is given below.

 

At diagnosis:

Assays performed at CML diagnosis include cytogenetics, FISH and RT-PCR, which detect the Ph chromosome, t(9;22) and BCR-ABL1 fusion transcript respectively. Cytogenetics is the gold standard, but has a low sensitivity (5% of Ph+ cells in the cultured sample). FISH and RT-PCR are more sensitive and can rapidly identify the BCR-ABL1 fusion, especially when no metaphase cells can be obtained. They are also required to identify cryptic BCR-ABL1 rearrangements that are detected in 5-10% of patients. RT-PCR is used to determine the molecular profile of the of BCR-ABL1 rearrangement, the most common of which are the e13a2 and e14a2 transcripts corresponding to a p210 fusion protein. Identifying the transcript is important as this is used to monitor response to therapy.

 

During treatment:

Regular and timely molecular testing plays an important role in the assessment of therapeutic efficacy, with RT-PCR being the preferentially used assay for monitoring CML patients who are undergoing TKI treatment. This assay has been standardised to improve reproducibility and comparability of quantitative results obtained by different laboratories. Responses are defined as “optimal”, “warning” and “failure”. Current recommendations include monitoring every 3 months until a major molecular response (or greater) is achieved, then every 3 to 6 months. BCR-ABL1 transcript levels ≤10% at 3 months, <1% at 6 months and <0.1% at 12 months are defined as optimal, whereas ≥10% at 6 months and >1% after 12 months is defined as failure.

 

Treatment resistance:

Molecular testing is also important for establishing the cause of treatment failure, as point mutations in the kinase domain of BCR-ABL1 are one cause of TKI resistance. More than 100 mutations have been described to date. Mutations either occur at positions directly involved in imatinib binding (for example Thr315Ile, the most common mutation), or at residues which alter the conformation of BCR-ABL1 thus preventing imatinib binding. Mutations can be detected by sequencing the translocated ABL kinase domain (Sanger or NGS). The selection of an alternative TKI will depend on the resistance mutation identified, as some mutations cause resistance to more than one TKI.

 

8.3 Describe the broad categories of genomic abnormalities observed in leukaemia and their significance using specific examples.

A good answer would discuss mutations, translocations and copy number aberrations using examples from multiple subtypes of leukaemia. These could include:

Mutations

• In intermediate-risk AML somatic mutations are used to identify patients with a poor prognosis who may benefit from an allogeneic transplant. C-terminal NPM1 mutations, biallelic mutation of CEBPA and GATA2 mutations are associated with favourable prognosis, whereas mutations in DNMT3A and RUNX1, and internal tandem duplications in FLT3 are poor prognostic indicators. The development of selective small molecule inhibitors of IDH has also lead to testing of codon 132, 140 and 172 mutations in IDH1 and IDH2.
• In CLL NOTCH1 PEST domain mutations and SF3B1 HEAT domain mutations are associated with chemorefractory disease and large cell transformation.

Translocations

• In AML t(8;21), inv(16) and t(16;16) are diagnostic of core binding facor leukaemia and t(15;17) diagnostic of acute promyelocytic leukaemia. These subgroups are associated with a favourable prognosis.
• Translocations involving chromosomes 9 and 22 are detected in two very different leukaemias - CML and ALL. The BCR-ABL1 fusion protein is targetable using tyrosine kinase inhibitors such as imatinib.
• Translocations involving the IGH locus are common in myeloma, placing the oncogene partner under the transcriptional control of the IGH enhancer. Partner genes include FGFR3/MMSET in t(4;14), CCND1 in t(11;14), MAF in t(14;16), MAFB in t(14;20) and CCND3 in t(6;14). Translocations resulting in overexpression of MYC are also common.

Copy number aberrations

• Four chromosomal abnormalities are routinely investigated by FISH in patients with CLL. These include deletion of 13q14 (involving miR15a/16-1 and RB1) and trisomy 12 which are associated with a favourable prognosis, and deletion of 11q22 and 17p13, targeting ATM/BIRC3 and TP53 respectively, which predict for a poor prognosis. Detection of 17q13 deletion (and TP53 mutations) is most important, as these patients are refractory to conventional chemotherapy and may be better treated with novel agents such as BTK and PI3K pathway inhibitors and BH3 mimetics.
• Complex and monosomal karyotype AML is considered adverse-risk and is often associated with deletion of 17p or mutation of TP53.
• Total chromosome number defines two groups of ALL – hyperdiploid (47-50 chromosomes) and high hyperdiploid (>50 chromosomes) which are favourable genetic lesions, and hypodiploid (<44 chromosomes) and low hypodiploid (32-39 chromosomes) which are unfavourable. Intrachromosomal amplification of chromosome 21 (iAMP21) is a rare but high risk subtype of B-ALL.