Chapter 9 Answers to discussion questions

Discuss the role of clonality testing in the diagnosis and monitoring of lymphoproliferative disorders.

An understanding of the role of the WHO classification in the diagnosis of lymphoproliferative disorders is required and an appreciation that in approximately 10–15% of cases of lymphoproliferative disorders clonality assessment is needed to confirm precise diagnosis.

Historically, clonality was primarily studied by performing Southern blotting analyses to characterize rearrangements in segments of the IG and TCR genes. Currently, the most commonly used method in the clinical molecular diagnostic laboratory is PCR, which is an extremely sensitive technique for detecting nucleic acids. This technique is rapid, accurate, specific, and sensitive, and it can be used to analyze small biopsies as well as FFPE samples. These advantages make PCR-based approaches the current gold standard for IG/TCR clonality testing.

The presence of a clonal marker can be used to monitor minimal residual disease (MRD) in the context of therapeutic programs that aim to eradicate neoplastic clones. In lymphoid tumours, the primary targets used in PCR-based analyses of MRD are clonal antigen-receptor rearrangements.  Although these MRD targets can be assessed in virtually all patients, the tests can be relatively complex to perform, due to the fact that the junctional rearrangements must be identified prior to performing the patient-specific real time quantitative PCR assays.

It is first necessary to screen the DNA using PCR to determine the precise clonal rearrangement at diagnosis. Then, the PCR product is sequenced to define the junctional regions and to allow for the design of patient-specific and allele specific primers (ASO) primers. These primers are then used to assess MRD in peripheral blood or bone marrow mononuclear cells from sequential specimens collected at different time points post-therapy. They remain widely used because they are reliable, accurate, specific, and extremely sensitive being able to detect 1 cancer cell among 10–4 - 10–5 normal cells.

Discuss the role cytogenetics in the investigation of a lymphoid malignancy providing an example in your answer.

Lymphoid malignancies have a genetic origin involving alterations that drive the transformation of a somatic cell clone. The molecular analysis of chromosomal translocations whose associations with distinct tumour subtypes was firmly established by cytogenetic analysis. Although present in non-lymphoid tumours, chromosomal translocations represent the hallmark of lymphoid malignancies, and these translocations often represent a major pathogenetic determinant.

The answer should provide a description of the various types of chromosomal translocations that either lead to the formation of a chimeric gene or the overexpression of a normal gene. The advantages and disadvantages of each approach should be considered.

Examples (please see table 9.4 for examples)

t(14;18) Example 1

Translocation t(14;18) is the primary tumorigenic event in up to 90% of follicular lymphomas and approximately 25% of diffuse large B-cell lymphomas (DLBCL).  Translocation of the BCL2 gene (18q21.3) to the IGH transcriptional enhancer, as a result of the t(14;18)(q32;q21), causes constitutive overexpression of the anti-apoptotic BCL2 protein. BCL2 normal function is to antagonize apoptosis. In the deregulated state increased BCL2 levels confer extended survival to B-cells and may thus cause accumulation of cells likely to be targets for additional oncogenic events and transformation.

t(11;18) Example 2

Translocation t(11,18)(q21;q21) is a characteristic genetic marker of the MALT lymphomas. The translocation fuses the amino terminal of the API2 gene to the carboxyl terminal of the MALT1 gene and generates a chimeric fusion product. Proteins API2 and MALT1 are normally quickly degraded but their chimeric transcript API1-MALT1 remains stable. The API2-MALT1 fusion product activates nuclear factor κB (NFκB), a transcription factor for several survival-related genes, including those encoding cytokines, growth factors, cell adhesion molecules, and several cell apoptosis inhibitors. NFκB is well described as a candidate proto-oncogene in a number of lymphomas.