MPN Flashcards
CML - definition
Myeloproliferative neoplasm defined by BCR:ABL1 fusion gene and neutrophilic granulocytosis.
> 90% cases diagnosed in chronic phase in which there is an insidious onset, neoplastic cells confined in blood, BM, spleen or liver
Increasing prevalence of the disease due to success of TKI therapy.
Patients usually diagnosed in chronic phase with leucocytosis with bimodal peak (myelocytes and segmented neutrophils), no dysplasia, absolute eosinophilia and basophilia. +/- monocytosis
Blasts < 2% in PB
On BM would be hypercellular, granulocytic proliferation, absent dysplasia. < 5% blasts
Pathogenesis/molecular of CML
95% cases have t(9;22) reciprocal translocation = Philadelphia chromosome
Fusion of BCR gene (chr 22) with regions on ABL1 (chr 9)
Cryptic translocations which cannot be identified on karyotyping may require FISH (dual probe) or moelcular.
BCR:ABL1 –> increased tyrosine kinase activity –> constitutive activation of proteins in several signal transduction pathways.
Transformation of CML to blast phase
Associated with clonal evolution, 70-80% cases have ACAs - additional chromosomal aberrations.
High risk ACAs at diagnosis
3q26.2 (MECOM) rearrangements, monosomy 7, isochromosome 17q, and complex karyotypes found at diagnosis are associated with an increased risk of progression to BP
Definition
> 20% myeloid blasts in PB or BM OR
Presence of EM blast proliferation OR
Skin, LN, bone, CNS
Can be myeloid, lymphoid or mixed
OR
Detection of lymphoblasts in peripheral blood or BM (even if <10%) usually B origin.
Bilineage cases can occur.
Molecular of BCR:ABL1
BCR::ABL1 exists in several different isoforms, depending on precise position of genomic breakpoints.
Two most common isoforms: account for 98% of CML involving the M-BCR (major breakpoint cluster region)
e13a2 and e14a2 (p210)
Majority express e14a2
10% cases express both isoforms.
Remaining 2% carry atypical BCR:ABL1 transcripts that arise from BCR breakpoints outside the major breakpoint cluster region or downstream of ABL1 exon 2 such as E1a2 (p190).
P190 (e1a2) predominant isoform in Ph+ ALL but seen in 1% of CML cases.
BCR:ABL1 transcript type in any given patient is stable overtime.
p210 isoforms have the capability to generate p190 by alternative splicing →, very low levels of transcripts (p190) can be detected in most cases of CML before treatment but are generally believed to be of no clinical significance as the p190 expression is ~ 100-1000 x lower.
And also clonal evolution of p210 to p190 is super rare.
Prognosis of CML
Most important prognostic indicator is response to treatment at the haematological, cytogenetic, and molecular levels.
Therapeutic milestones have been developed by the European LeukemiaNet (ELN) to categorize treatment response during treatment as “optimal”, “warning”, or “failure
BCR:ABL1 test methodology
Principle
- RNA based
- reverse transcriptase PCR is used
–> Taqman Probe qPCR
- used for both diagnosis and monitoring of disease
- qPCR or GeneXpert methods
Two independent PCR runs are set up;
- one of patient sample-extracted RNA for BCR::ABL1, and one of patient sample-extracted RNA for a control gene (typically ABL1)
- Housekeeping gene acts as an internal control gene for RNA quality
- ABL1 copies as a measure of assay sensitivity for undetectable BCR::ABL1 for low level MRD
The level of expression of BCR::ABL1 is expressed as a ratio percentage to the control gene.
This is then multiplied by a specific conversion factor, based on the laboratory’s calibration from a standardized panel, to generate the International Standardized BCR::ABL1 (BCR::ABL1 IS)
IS - International Scale developed to harmonize molecular responses (MRs) across laboratories. IS response is derived by applying a laboratory-specific conversion factor to molecular response data from each individual participating laboratory. This conversion factor is derived from comparison to a reference laboratory and is monitored over time for “drift” in IS measurements.
Taqman probe qPCR assay principle
Quantity of the amplicon is measured during each replication cycle by means of a specific fluorophore which binds to the amplicon product
This is then compared to a standardized curve of normalised fluorescence produced by commercial controls of known amplicon concentration to determine the concentration of target nucleic acid in patient sample
Cycle threshold = in real time PCR is the number of cycles required for the fluorescent signal to cross the threshold (ie exceed background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample
GeneXpert for BCR:ABL1
For diagnosis and monitoring
- perform a reverse transcription polymerase chain reaction (RT-PCR) to detect and quantify the BCR-ABL fusion gene transcript comparing it to a reference ABL gene to calculate a ratio
- RNA based test
- 4mL PB
- Rapid TAT ~ 2.5hrs
- Easy to perform
- Closed cartridge system - minimizes risk of contamination
- Quantitative detection of either p210 or p190 transcript. (note rare isoforms not tested)
On board controls for each sample
- Probe check control
- ABL endogenous control
Current kit sensitivity is 0.003% IS (p210) and 0.0065% (p190)
BCR:ABL response to therapy
They are expressed and reported on a log scale where 1%, 0.1%, 0.01%, 0.0032% and 0.001% correspond to a decrease of 2, 3, 4, 4.5 and 5 logs respectively
Early molecular response (EMR; BCR-ABL1 transcripts ≤10%) at 3 months is associated with good prognosis (1 log)
- If BCR-ABL1 transcripts >10% at 3 months considered a warning and are a trigger to examine patient adherence and assess for resistance.
Aim is to achieve =<0.1% (3 log reduction) aka MMR, major molecular response
- a change of treatment may be considered if MMR is not reached by 36-48 months.
DMR - deep molecular response
- 4 log reduction ( 0.01%)
- 4.5 log reduction (0.001%)
ET vs MF morphology
