Laboratory of PCR Diagnostics of Leukemias

The laboratory performs routine molecular detection of fusion genes in acute myeloid leukemia (AML), detection of clonality in lymphoproliferative diseases of B and T cells – in chronic lymphatic leukemia (CLL; in the latter it moreover determines I_gV_H gene mutational status and the presence of mutations in TP53 gene) or, as the case may be, other chronic and acute lymphoproliferative diseases. Furthermore, it tests clonal mutations in Ph‑ myeloproliferative disorders (MPDs) – JAK2^V617F tyrosine kinase mutation and CALR gene mutation.

Apart from the above-mentioned diseases detection tests, the laboratory also performs follow-up molecular monitoring of minimal residual disease (MRD) for both AML and CLL, and in selected cases of Ph- MPDs.

• AML:
  For every patient with AML, the laboratory performs prospective PML/RARα, AML1/ETO and CBFβ/MYH11 fusion gene tests and tests of the presence of internal tandem duplication of FLT3 gene. On the basis of the cytogenetic funding it is possible to test the most diverse fusions of MLL gene with different breakpoints.
  Furthermore, the laboratory can also test alternative fusion genes that can appear (extremely rarely) in atypical forms of acute promyelocytic leukemia (APL) that do not carry the routinely tested PML/RARα fusion gene, but can have PLZF/RARα, NPM1/RARα, NuMA/RARα, FIP1L1/RARα, STA5b/RARα, PRKAR1A/RARα, BCOR/RARα, or OBFC2A/RARα fusions.
  Molecular characterization of a specific fusion gene is then used for monitoring MRD with the help of quantitative real-time RT-PCR (RQ-PCR).
• CLL:
  The laboratory performs routine tests of clonal rearrangements of I_gV_H genes (for patients with chronic lymphatic leukemia or, as the case may be, with certain lymphomas), and it evaluates their mutational status. In conformity with international recommendations, it tests TP53 gene mutations for patients with CLL using direct sequencing of cDNA. In T- cell lymphoproliferative diseases it identifies clonal rearrangements of δ and γ genes of chains of T – cell receptor (TCR).
  By appointment, the laboratory performs tests of MRD with the help of RQ-PCR using allele – specific primers for patients with earlier proved I_gV_H or TCR clonal rearrangement.
• Ph- MPDs:
  The laboratory regularly determined the presence of JAK2^V617F mutation with the help of RQ-PCR method with LNA-modified probes. For selected patients with polyglobulia it is also possible to test rare alternative mutations of JAK2 gene in exon 12 (here we use two methods: the so-called “wild-type blocking PCR” and high resolution melting [HRM] analysis). For patients with thrombocythemia or myelofibrosis, the laboratory detects CALR gene length mutations using fragmentation analysis method.