MPN Flashcards
(42 cards)
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
May present with isolated thrombocytosis
Pseudo Gaucher cells + dwarf megas
Sea blue histiocytes
Blasts < 2% in PB
On BM would be hypercellular, granulocytic proliferation, absent dysplasia. < 5% blasts
CML - essential and desirable diagnostic criteria
Essential:
* Peripheral blood neutrophilic leucocytosis
* Detection of Philadelphia (Ph) chromosome and/or BCR::ABL1 by
cytogenetic and/or molecular genetic techniques
Desirable:
* Bone marrow evaluation at diagnosis or at suspected progression
for assessment of disease phase
* Evaluation for additional chromosomal aberrations (ACAs).
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
EURO score and EUTOS score (TKI era)
> age, inc peripheral blasts, big spleen, thrombocytopenia.
> if higher score may favour second generation TKI
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
Megakaryocyte morphology in MPNs
Reticulin Fibrosis - how to grade
final score is determined by highest grade in at least 30% of the marrow
Chronic Neutrophilic Leukemia
RARE
Sustained peripheral neutrophilia
Hepatosplenomegaly
Exclusion of other MPNs and MPN/MDS
PB - WBC > 25 with 80% being neutrophils
- nil significant dysplasia
BM - hypercellular, toxic granulation, no other sig abnormalities
CSF3R - found in 90% cases of CNL
Chronic Eosinophilic Leukemia
Autonomous, clonal proliferation with abnormal morphology.
Eosinophilia is the dominant haematological abnormality.
Can cause end organ damage.
> leukemic infilitration
> release of cytokines, enzymes, or other proteins
PB - mature eosinophilia, abnormal with sparse granulation, increased size, immature forms or nuclear hype segmentation.
BM - increased M:E ratio, mega dysplasia
Inc eosinophilia
Eosinophilia workup + ddx
MPN non driver mutations
Polycythaemia Vera
- Increased RBC production independent of normal mechanisms that regulate erythropoiesis
- Major sx related to hypertension or vascular abnormalities causes by inc RBC mass (hyperviscosity)
- Venous or arterial thrombosis in ~ 20% cases
Two recognised PV phases:
* polycythaemic phase, assoc with inc Hb, elevated Hct and inc RBC mass
* spent phase or post PV MF - assoc with ineffective haematopoiesis and cytopenias, BM fibrosis, EM haematopoiesis and hypersplenism
PV - genetic profile
JAK2 through EPO/MPL/G-CSF
> 95% of PV has somatic gain of function JAK2 exon 14 V617F
Higher JAK2 V617F VAF = inc puriritus and fibrotic transformation
JAK2 exon 12 mutation - 3% of PV
- usually younger
- similar prognosis to JAK2 V617F
20% patients have cytogentic abnormalities at diagnosis
PV - diagnostic criteria
All 3 major criteria OR first 2 major and minor criterion.
(hence don’t need Bmbx)
MAJOR
1. Elevated Hb concentration (>165 men, >160 women) or elevated Hct (>49% men, >48% women)
2. BMBx - hypercellular, panmyelosis, pleomorphic megas.
3. Presence of JAK2 V617F or JAK2 exon 12 mutation
Minor
- Suppressed EPO level
ie. for diagnosis you must have Bmbx and elevated Hb and then either JAK2 OR if not a low EPO
PV - BM findings
Hypercellular
Panmyelosis
Megas - pleomorphic, loose clusters
May have MF-1
Absent iron stores > 95% of patients
Post PV MF diagnosis
- > 10% blasts in PB or BM indicates transformation to accelerated phase
- > 20% blasts - blast-phase post PV MF
PV risk stratification
IPSS score of overall survival in PV:
(based on)
- age
- leucocytosis >15
- hx of venous thrombosis
MIPSS-PV (mutation enhanced)
- Age
- leucoytosis >15
- abnormal karyotype
- SRSF2 mutation (RNA splicing mutation)
Essential Thrombocytosis findings
PB - plt anisocytsois
BMA - markedly increased megas.
deeply lobulated and hyperlobulated, nuclei large to giant forms with abundant cytoplasm
- no sig increase in other lineages
- no ring sideroblasts
Trephine - Normocellular, Staghorn like nuclei - deeply lobulated
MF 0-1
