The Molecular Haemato-Oncology Laboratory performs a wide range of assays applied as either panels or as individual assays depending on the disease type and/or referral reason: 

The aims of the Molecular Haemato-Oncology Laboratory are to provide clinicians with information to:- aid the diagnosis of haematological malignancies, assess prognosis, select the most appropriate therapy, and monitor the response to treatment.

Please refer to the test directory for details of individual tests. 

The Molecular Haemato-Oncology Laboratory operates an integrated Immunophenotyping and Molecular Haemato-Oncology service.  Hence,  the majority of samples with a suspected diagnosis of acute leukaemia are received for molecular analysis from the Immunophenotyping laboratory, in addition to those directly, as a clinician’s request.  

DNA and/or RNA is isolated from the specimens (paraffin embedded material, blood or bone marrow) according to downstream testing requirements, and processed in batches on the same day (if received before 15.30), or on the following day for samples arriving after 15:30 hours.

 


Acute Myeloid Leukaemia (AML)

The following panel of genes are assessed for AML/suspected AML patients at time of diagnosis.

Real-time reverse transcription PCR (RT QPCR) is performed at diagnosis of AML to identify the presence of the following recurrent genetic abnormalities. 

  • PML::RARA fusion gene [t(15;17)(q22;q21)]
  • RUNX1::RUNX1T1 fusion gene [t(8;21)(q22;q22)]
  • CBFB::MYH11 fusion gene [inv(16)(p13;q22) or t(16;16)(p13;q22)]
  • BCR::ABL1 fusion gene [t(9;22)(q34;q11)] (E1A2, E13A2 & E14A2 transcripts)

When one of the above recurrent genetic abnormalities is detected, quantitative assays are used to enable monitoring of minimal/ measureable residual disease . 

A multiplex RT-PCR assay is performed for the detection of KMT2A fusion genes.  If a fusions gene is detected, a singleplex RT-PCR is performed to identify the fusion partner.

  • KMT2A (MLL) [11q23] fusion gene panel.  This panel is able to detect: KMT2A::AFF1 [MLL-AF4], KMT2A::AFDN [MLL-AF6], KMT2A::MLLT3 [MLL-AF9], KMT2A::ELL [MLL-ELL] and KMT2A::MLLT1 [MLL-ENL].

Identification of FLT3 and NPM1 mutations provide prognostic information and play an important role in determining appropriate treatment options. Analysis of these genes is performed by PCR and capillary electrophoresis on all AML/suspected AML patients at diagnosis.

  • FLT3 internal tandem duplication and D835  tyrosine kinase domain mutation detection
  • NPM1 exon 11 mutation (previously termed exon 12).

For AML patients with NPM1 Type A, Type B or Type D mutations, reverse transcriptase QPCR assay is used to monitor minimal/measurable residual disease. 

Patients with core finding factor leukaemia (i.e. with t(8;21) or t(16;16)/inv(16)) with otherwise good prognosis who also have a KIT mutation have a poorer prognosis. Thus, patients who are identified as having CBFB::MYH11 or RUNX1::RUNX1T1 fusion gene are tested for the presence of a KIT mutations by allele specific real time PCR and Sanger sequencing.  

NHS Lothian also offers Next Generation Sequencing testing for all treatment eligible AML.

AML - Monitoring Minimal Residual Disease

When one of the following recurrent genetic abnormalities is detected, quantitative assays are used to enable monitoring of minimal/measurable residual disease. 

  • PML::RARA fusion gene [t(15;17)(q22;q21)]
  • RUNX1::RUNX1T1 fusion gene [t(8;21)(q22;q22)]
  • CBFB::MYH11 fusion gene [inv(16)/t(16;16)]
  • BCR::ABL1 fusion gene [E13A2, E14A2 or E1A2 fusion transcripts]
  • NPM1 mutation Type A or Type B or Type D

Acute Lymphoblastic Leukaemia (ALL)

The following panel of genes are assessed for ALL/suspected ALL patients at diagnosis.

 Real-time reverse transcription PCR (RT QPCR) is performed at diagnosis of

  • ETV6::RUNX1 fusion gene [t(12;21)(p13;q22)]
  • TCF3::PBX1 fusion gene [t(1;19)(q23;p13)]
  • BCR::ABL1 fusion gene [t(9;22)(q34;q11)] (E1A2, E13A2 & E14A2 transcripts)

A multiplex RT-PCR assay is performed for the detection of KMT2A fusion genes.  If a fusions gene is detected, a singleplex RT-PCR is performed to identify the fusion partner. 

  • KMT2A (MLL) [11q23] fusion gene panel.  This panel is able to detect: KMT2A::AFF1 [MLL-AF4], KMT2A::AFDN [MLL-AF6], KMT2A::MLLT3 [MLL-AF9], KMT2A::ELL [MLL-ELL] and KMT2A::MLLT1 [MLL-ENL]. 

ALL - Monitoring Minimal /Measurable  Residual Disease

When one of the following recurrent genetic abnormalities is detected, quantitative assays are used to enable monitoring of minimal/measurable residual disease.

  • ETV6::RUNX1 fusion gene [t(12;21)(p13;q22)]
  • TCF3::PBX1 fusion gene [t(1;19)(q23;p13)]
  • BCR::ABL1 fusion gene [t(9;22)(q34;q11)] (E1A2, E13A2 & E14A2 transcripts)

Chronic Myeloid Leukaemia (CML)

Chronic Myeloid Leukaemia (CML) accounts for 15-20% of adult leukaemias. The Philadelphia (Ph) chromosome results from a reciprocal translocation between the long arms of chromosomes 9 and 22. At the molecular level, the translocation disrupts the normal ABL1 and BCR genes resulting in the chimeric BCR-ABL1 fusion gene.

Over 95% of Ph positive CML patients express BCR::ABL1 E13A2 or E14A2 transcript types, or both types simultaneously, while 1-2% of patients express E1A2 transcripts; translated into 210-kDa (p210) and 190-KDa (p190) fusion protein respectively.

Reverse transcription PCR (RT-PCR) is performed to detect the presence of the BCR::ABL1 fusion gene transcripts, E13A2, E14A2 and E1A2 in patients suspected with CML.

Approximately 2–4% of patients harbor atypical BCR::ABL1 transcripts (E13A3/E14A3 or E1A2, E6A2, E8A2, or E19A2) and may yield false negative results by this assay.

  • BCR::ABL1 fusion gene [t(9;22)(q34;q11)] (E1A2, E13A2 or E14A2 transcripts)

CML - Monitoring Minimal/ Measureable Residual Disease

Following detection of a BCR-ABL1 fusion, quantitative real time PCR assays are used to enable monitoring of minimal/measurable residual disease levels.   

Mutations within the ABL1 kinase domain are associated with resistance to some tyrosine kinase inhibitors in patients diagnosed with CML.  Thus, in patients with limited response to TKIs and patients who show increase BCR-ABL1 transcript levels (indicative of progressive disease), a nested RT-PCR followed by Sanger sequencing is performed to enable the detection of ABL1 domain kinase mutations.

 


 Lymphoproliferative disorders (suspected Lymphoma)

Approximately 5‐15% of cases of lymphoproliferative disorders require additional molecular assays (e.g.B‐ and/or T‐cell clonality assessment) to aid clinicians with the diagnosis or exclusion of lymphoid neoplasia.

B- and T-cell clonality assessment is undertaken using the BIOMED‐2 assays, with a standard PCR protocol and primer sets designed to target the IGH, IGK and IGL genes for B cells, and TCRG, TCRB and TCRD genes for T cells, to establish whether there is a clonal immunoglobulin gene (IG) or T‐cell receptor (TCR) gene rearrangement.

The point mutation MYD88 p.Leu265Pro is found in ∼90% of Waldenström macroglobulinemia (WM) cases and in significant portions of activated B-cell diffuse large B-cell lymphomas (DLBCL) and IgM monoclonal gammopathy of undetermined significance (MGUS). An allele specific real-time PCR used for the identification of the pathogenic MYD88 p.L265P mutation is performed to aid in the differentiation of low grade B Cell Lymphoplasmacytic Lymphoma (LPL), WM and MGUS from marginal cell lymphomas.

Associated tests:


 Chronic Lymphocytic Leukaemia (CLL)

Chronic lymphocytic leukaemia (CLL) is a highly heterogeneous disease due to diverse genetic aberrations in CLL patients. The mutation status of the tumour suppressor gene TP53  is the most important predictive factor for guiding treatment decisions in CLL.  The presence of  a TP53 mutation and/or deletion of chromosome 17p is associated with a poor prognosis and an impaired response to chemotherapy. However, small molecule inhibitors such as the Bruton tyrosine kinase (BTK) inhibitor, Ibrutinib, and the phosphatidylinositol 3-kinase (PI3K) inhibitor Idelalisib are an effective treatment for CLL patients harboring TP53 mutations .

 IGHV gene mutation status is also an important biomarker in CLL. The clinical course is generally more aggressive in patients with unmutated IGHV gene than in those with mutated IGHV gene (however, there are exceptions). TP53 mutations may be found in both IGHV mutated and unmutated CLL, but are usually associated with unmutated CLL. TP53 and IGHV mutation status are determined by Sanger sequencing.  

Associated tests:


 Eosinophilia

To aid the investigation of eosinophilia, a nested reverse transcription PCR assay designed to detect the FIP1L1-PDGFRA fusion gene  is  performed.

  Associated tests:

Additional tests that may help in the investigation of eosinophilia including the detection of  rearrangements of PDGRB and FGFR1 and CHIC2 deletionS are investigated by the South East Scotland Cytogenetics Service

Eosinophilia may rarely be observed in Systemic Mastocytosis (SM) and therefore assessment of the KIT gene may be considered (see Mastocytosis).  Assessment of JAK2 V617F mutation detection (offered by MPN service at RIE) may also be considered


 Mastocytosis

Mutations of KIT gene occur in malignant hematopoietic disorders such as Systemic Mastocytosis (SM), Clonal eosinophilic disorders and AML.  The most common KIT gene mutation in SM is KIT D816V [p. Asp816Val]. This variant causes ligand–independent constitutive phosphorylation and activation of KIT, leading to uncontrolled cell growth. Detection of KIT D816V mutation testing is performed by ASO qPCR to aid the diagnosis of SM.

In addition, patients with core binding factor leukaemia otherwise good prognosis AML such as t(8;21) and t(16;16) who also have a KIT mutation have a poorer prognosis. Thus, patients who are identified as having RUNX1::RUNXT1 and CBFB::MYH11 fusion transcripts are tested for the presence of a KIT D816V mutation.

Associated tests:


Next Generation Sequencing for Myeloid disorders   

Next-generation sequencing (NGS) for myeloid disorders enables the testing of multiple gene mutations and fusion transcripts in a single test. The panel targets 40 key DNA genes and 29 driver RNA gene fusions relevant to myeloid disorders.

An NGS panel for myeloid disorders is available for the following patient categories:

  • Treatment eligible patient with acute myeloid leukaemia
  • Transplant eligible patient with a diagnosis of myelodysplastic syndrome 
  • Transplant eligible patient with a diagnosis of  primary myelofibrosis
  • For differentiation of hypoplastic MDS and aplastic anaemia
  • Atypical myeloproliferative neoplasm (“triple negative” PMF, MDS/MPN overlap)
  • Relapse AML patient to provide therapeutic information

Please specify to which category the patient belongs when requesting using the dedicated NGS request form.

 For further information, please refer to the page dedicated to Myeloid NGS.