Chronic Lymphocytic Leukemia/Lymphoma (CLL)

OVERVIEW

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes and accounts for ~7% of all B-cell lymphomas and 40% of all adult leukemias.  In CLL, mature clonal B lymphocytes accumulate in the blood, spleen and other lymphoid organs and is diagnosed when there are >5×109/L  B cells in the peripheral blood persisting for more than 3 months.  Small lymphocytic lymphoma (SLL) is the nodal variant of CLL with little or no peripheral involvement.

Recurrent copy number changes of 17p, 11q, 13q, chromosome 12 as well as sequence changes within IGHV, TP53, NOTCH1 and a number of other genes provide important prognostic information and guide treatment decisions. 

INDICATION

Testing is offered for patients with a confirmed diagnosis of CLL to aid in treatment decisions and is not indicated for diagnostic purposes. Copy number changes are assessed by a FISH panel and sequence level changes by an NGS panel. This testing should be performed prior to the initiation of treatment, rather than at the time of diagnosis. Repeat testing upon disease progression while on treatment may be indicated.

TEST REQUIREMENTS

  • For peripheral blood (preferred specimen)
    • Peripheral blood (cytogenetics): 1 x 6ml NaHep (FISH)
    • Peripheral blood (DNA): 1 x 6ml EDTA (NGS Panel)
  • For bone marrow
    • Bone marrow (cytogenetics): 2 x 1.0ml in transport media tube (FISH)
    • Bone marrow (DNA): 1 x 0.5ml in EDTA tube (NGS Panel)
  • For FFPE Tissue Block (NGS Panel ONLY)
    • FFPE tissue block: will be sectioned
    • Specimen must not be de-calcified
    • Minimum tumour content is 10-20%
  • Completed CGL Lymphoid Testing requisition form

TURN-AROUND TIME

Results are reported within 14 days for FISH and 21 days for the CLL NGS panel from receipt of the necessary specimen, completed requisition form and all relevant documentation.  

RESULTS REPORTING

FISH Panel

The results of the tested loci are stratified according to increasing risk as follows: 13q deletion (sole abnormality), with relatively good prognosis; normal result or trisomy 12, with an intermediate prognosis; 11q deletion (ATM) and 17p deletion (TP53), with poor prognosis.

Up to 80% of CLL patients will have a cytogenetic abnormality detected by FISH using the standard four-locus panel.  The remainder (~20%) will have normal copy number at these loci, but may have cytogenetic abnormalities in other regions not covered by this panel.

CLL NGS Panel

CGL’s CLL Panel is a custom next-generation sequencing assay designed to identify sequence changes in selected coding regions of the following genes: TP53, BCL2, PLCG2, BTK, SF3B1, NOTCH1, BIRC3 and MYD88.  All clinically relevant variants with a variant allele fraction greater than 5% are reported.  Variant classification is adapted from AMP/ASCO/CAP guidelines (Li (2017) PMID:27993330).  See here for further details. NOTE: IGHV analysis is not assessed by this NGS panel.

METHOD

FISH Testing

FISH analysis is performed on interphase nuclei using a commercial kit manufactured by Vysis Inc. to interrogate abnormalities of 11q22 (ATM), 13q14, 17p13 (TP53), and the centromere of chromosome 12.  This technique confidently detects a clonal population (with a genetic alteration at one or more of the tested loci) only if it exceeds ~10% of the nucleated cells in the specimen.

NGS Testing

Sequencing is performed on genomic DNA using an amplicon-based panel that targets the coding regions of the following genes (TP53, BCL2, PLCG2, BTK, SF3B1, NOTCH1, BIRC3 and MYD88). Sequence reads are aligned to the human reference genome and variants are reported according to adapted AMP/ASCO/CAP guidelines (Li (2017) PMID:27993330). The NGS panel will not detect copy number changes and may not identify some more complex variants.

REFERENCES

  1. Dighiero G (2008) Chronic lymphocytic leukaemia. Lancet PMID: 18358929
  2. Fabbri G  (2016)  The molecular pathogenesis of chronic lymphocytic leukaemia. Nat Rev Cancer PMID: 26911189
  3. Döhner H et al (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. PMID: 11136261
  4. Van Dyke et al (2010) A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukaemia Br J Haematol PMID: 19895615
  5. Malcikova J, et al. (2018) ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia – update on methodological approaches and results interpretation. Leukemia PMID: 29467486.
  6. Campo E, et al. (2018) TP53 aberrations in chronic lymphocytic leukemia: an overview of the clinical implications of improved diagnostics. Haematologica PMID: 30442727
  7. Zenz T, et al. (2010) TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol.PMID: 20697090.