Myeloid Neoplasms Flashcards
(41 cards)
Acute leukaemia of ambiguous lineage (ALALs)
> 20% abnormal progenitors that do not show differentiation along a single lineage
<4% of acute leukaemias.
Acute undifferentiated leukemia (AUL) - lack lineage commitment
Mixed phenotype leukaemia (MPAL) - differentiation along more than one lineage. Biphenotypic vs bilineage.
Leukaemias otherwise meeting criteria for t(8;21) AML, inv(16) AML, myeloid/lymphoid neoplasms, CML in blast crisis, AML-MR and tAML should be classified as such, with a notation that they exhibit a mixed lineage immunophenotype
Typically express one myeloid marker (but not two) ie CD13, CD33 and CD117
CD34, HLA-DR and TdT are frequently expressed as well as CD7 and CD56.
lack unusual lineages (NK cells, megakarycoytes etc)
AULs classification
Classification of MPALs
This table is only helpful if you want to give it MORE than one lineage (aka call it a MPAL).
Hence, you can define an AML without MPO if there are two myeloid markers.
What molecular abnormalities are commonly seen in MPAL?
AUL with defining genetic abnormalities:
Poor prognosis
Usually treat with ALL based regimens
Define MRD (in AML)
MRD defined as measurable residual disease and is useful for management of patients in AML.
Defined by the presence of detectable leukaemia below the traditional morphological remission definition of 5% blasts.
Validated and reproducible at a sensitivity of at least 10^-4.
It is measured at specific times during disease course and treatment.
It can be helpful in deciding:
-treatment plan eg whether patients should be considered for allograft
-change in treatment eg. consideration of change in therapy with MRD directed therapy with clinical trials
-Prognostic marker eg. MRD positive post “second course” and comparing baseline molecular risk profile to molecular response risk
-Detecting MRD relapse before morphological relapse, allowing consideration intervention and treatment to take place
What makes a molecular marker a good MRD marker:
-Not a marker of clonal haematopoiesis, as will always be present even with disease free states
-Not germline mutations – again as will always be present
-A defined mutation that can easily be tracked, and does not mutate/evolve during disease treatment
-Is able to be detected with a sensitivity of at least 10^-4
Time points of MRD testing
-Ideally at diagnosis
-At end of post course two
-At end of treatment
-On PB 4-6 weekly intervals following remission after 2 years of completed therapy.
Flow MRD in AML
-Uses a leukaemia-associated immunophenotype (LAIP) or difference from normal (DFN) approach to distinguish leukaemic cells from their normal counterparts (haematogones)
-MRD test positivity is defined as ≥0.1% of CD45-expressing cells
-Detection to 10^-5 to 10^-6
Adv: quick TAT, may still be able to detect disease despite a mutational change (immunophenotypic shift), able to be used in patients without a trackable mutation
Disadva:
-Labor intensive (need high number of events)
-requires a fresh sample (first pull) and
-Degree of uncertainty due to significance of changes in DFNs.
-Need diagnostic sample if following LAIP.
-May not change management eg. Elderly population
Reverse transcriptase quantitive PCR
Target RNA is first converted to complementary DNA
Real time quantification of target gene presence by PCR reaction via denaturing, annealing and elongation
Fluorescent labelled oligonucleotide binds to DNA strand of target sequence
As DNA polymerase polymerises complementary strand of DNA, if oligonucleotide bound to DNA sequence, fluorophore will be released causing a release of light
Quantified by comparing to level of housekeeping gene amplied (e.g ABL) on a standard curve
Used in: NPM1, PML::RARA, CBFB::MYH11, RUNX1::RUNX1T1
Often reported as log reduction using baseline at 100%
Adv: Is sensitive 10^-5 up to 10^-6,
Disadvantages: requires diagnostic sample to identify trackable fusion gene, not applicable to all patients. Sensitivity limited by quality of sample (RNA much more fragile than DNA, needs to reach the lab within 48hours).
Won’t detect relapse of other disease eg. tMN or clonal evolution in CMML pt
Definition of MRD relapse
MRD relapse is defined as either conversion from MRD negativity to MRD positivity or ≥1log rise between two positive samples.
Further workup is usually done with repeat bone marrow biopsy if testing was done on PB, to demonstrate if there is any morphological relapse.
Repeat molecular studies to see if there have been any new acquisition of molecular markers or clonal evolution of disease
(eg. Be able to demonstrate FLT3 and a therapy target, RAS mutants or prognostic markers)
NPM1
Most common in adult AML (1/3rd of AML)
exon 12, usually consists of 4bp insertion causing frameshift mutation resulting in increased nuclear export of NPM1 and abnormal cytoplasmic localization.
3 most common mutations are:
Type A - duplication of 4bp (75%)
Type B and Type D - insertion of 4 new nucleotides.
Frequently occur with FLT3ITD, DNMT3A + IDH 1/2
Methods of NPM1 detection
DNA based
Capillary electrophoresis (quantitative assay
> sensitivity down to 1%
> amplification of DNA at exon 12 by PCR.
> Mutant type will be 174bp vs WT 170 bp
> PCR products are labellled with fluorescent tag
> CE separates DNA fragments via size with high resolution.
IF NPM1 mutation will have both wild type peak and additional longer peak present.
Unable to detect the TYPE of variant
NPM1 MRD testing
*Real time RT-qPCR
*RT-dPCR
ALFRED
RNA based
PB or BMA
RNA extraction and cDNA prepared via reverse transcriptase
cDNA is then amplified and NPM1 and ABL1 gene CT calculated and %of NPM1 is quantified compared to ABL1.
Log reduction based on diagnostic sample.
*GeneXpert
AUSTIN
RNA based
Mutant NPM1 mRNA transcripts (A,B and D) are quantified as a % ratio of NPM1 to ABL1 enodgenous control mRNA transcripts in sample
AML NPM1 negative relapse
NPM1 negative relapses (NPM1 wt)
- AML with NPM1 mutation can relapse with NPM1wt, which could prevent MRD detection.
-The majority of co-mutations persist in molecular remission and at a NPM1wt relapse, suggestive of a clonal hematopoiesis driving AML relapse.
Methods for detection of MRD in AML
ELN risk classification
FAVORABLE
- t(8;21)/RUNX1/RUNX1T1
- inv(16), t(16;16)/ CBFB::MYH11
- mutated NPM1 without FLT3-ITD
- bzip in-frame mutated CEBPA
ELN risk stratification
INTERMEDIATE
- mutated NPM1 with FLT3-ITD
- wild type NPM1 with FLT3-ITD
- t(9;11), MLLT3::KMT2A
-molecular or CG abnormalities that don’t fit in either favorable or adverse
ELN risk stratification
ADVERSE
- t (6;9)/DEK::NUP214
- MECOM rearrangement
- KMT2A rearrangement
- t (9;22)/ BCR:ABL1
- complex karyotype
- mutated tp53
- del5q, -7 or 17p abnormality
- t(8;16)/ KAT6A::CREBBP
- molecular mutations - (only if not occurring with favorable risk AML subtypes) ASXL1, RUNX1, SF3B1, SRSF2, BCOR, EZH2
Molecular studies
- Indications
- Features of molecular path
Indications for molecular testing:
> Diagnosis
» Clonality
»_space; Diagnostic criteria (e.g. JAK2 V617F for PRV, KIT D816V for SM)
> Prognosis
> Predictive/targetable
Features of molecular pathology different to general haematology
> Tests are often only performed once (harder to detect WBIT)
> Contamination is an issue in assays that amplify material (PCR)
> Some testing requires genetic counselling
> Test techniques may differ between laboratories (e.g. FISH versus PCR techniques for hypereosinophilia)
> Tissue sources vary (blood, buccal, prenatal testing [amniocentesis, CVS], cancer specimen)
DNA vs RNA
DNA is stable (each cell has the same DNA content)
RNA is labile, and requires prompt delivery to the laboratory; expression of RNA varies between cells
Sanger sequencing
Principle:
- DNA is amplified using a combination of dNTPs and fluorescently-labelled ddNTPs.
- ddNTPs cause sequence termination.
- Amplicons are arranged in order of length, and fluorescence emission is used to identify their base sequences.
ADVANTAGES:
- longer reads than NGS
- Useful for genes where there may be multiple variants rather than a single hotspot (eg. UBA1)
- method of choice to confirm a variant
DISADVANTAGES
- inefficient and less sensitive (VAF of 10-20%) as fewer read than NGS
- not suitable for MRD monitoring as not sensitive enough
Examples - UBA1 and alpha and beta thal genetic testing
Next Generation Sequencing
A high throughput massively parallel technology consisting of four steps:
(1) fragmentation and library preparation,
(2) amplification/cluster generation,
(3) sequencing by synthesis
(4) data analysis
Can be panel/whole exome/whole genome
ADVANTAGES:
- High throughput (multiple patients and variants can be sequenced at once)
- more in-depth coverage of genes of interest and no secondary findings
DISADVANTAGES:
- may miss novel mutations in genes outside panel of interest
- expensive
- Large indels, small copy number variants, complex rearragements, pseudogenes are difficult variants to identify
- time consuming (variant curation)
- shorter reads than sanger
- can detect unexpected germiline mutations
Useful in:
> Somatic mutations of clonal/prognostic significance
> Germline mutations
> TCR/BCR rearrangements (e.g. IGVH mutation status, B ALL and T ALL MRD)
Fragment analysis
Separating PCR products based on size (amplification followed by capillary electrophoresis)
ADVANTAGES
- can multiplex using different dyes/probes to detect different mutations in a single assay (e.g. NPM1 and FLT3-ITD/TKD can be performed at the same time)
- quick TAT
DISADVANTAGES
- Does not provide sequence-level information
- May not detect unusual variants (e.g. non TKD D835/I836 mutations)
When is it used?
> FLT3 ITD, FLT3 TKD, NPM1
> TCR gene rearrangement
> Chimerism analysis
Melt curve analysis
Quantitative analysis of melt curves of product DNA after PCR amplification to detect point mutations.
Principle that heteroduplexes (formed when there is a mutant gene present) are more unstable and will denature at lower temps.
Uses a fluorescent dye that only binds to double stranded DNA, which generates a curve of decreasing fluorescence intensity.
As amplification product is slowly heated, denaturation occurs to single strands
The melting temperature and shape shifts of the HRM curves can be used to identify both homozygous and heterozygous variants.
ADVANTAGES
> Discriminates dsDNA based on sequence length, GC base content, or strand complementarity
> Highly sensitive, enough to detect single basepair differences
DISADVANTAGES
>doesn’t tell you excavtly what mutation is present
>Requires sequencing (usually Sanger) to determine the specific mutation
Uses:
> CALR variants (e.g. W515K, W515L versus wild-type)
> Prothrombin G20210, FVL
Allele-specific ddPCR
A highly precise quantitative method where the PCR reaction is divided into approx 20,000 drops, each containing the necessary reagents for PCR amplification
PCR amplification occurs across all wells simultaneously.
After this PCR reaction, each droplet is analyzed by a droplet reader which measures the fluorescence amplitude.
> presence of mutation of interest will be one fluorescent ie green
> presence of wild type will be another fluorescence ie red
The proportion of PCR-positive droplets is then determined, and analyzed to determine the concentration of the original template DNA in the sample.
Enables the detection of single nucleotide mutations
that occur as a rare event in a background of
wildtype sequence
ADVANTAGES:
> More sensitive than qPCR; may be useful for MRD or low-prevalence mutations (e.g. MYD88 in PB)
> Does not require a calibration curve/assay standard
> High precision in estimating mutant fraction
DISADVANTAGES
> Fewer variants can be sequenced at once compared to NGS; sensitivity now replicated by some ultra-deep sequencing NGS techniques
Uses
JAK2 Val617Phe
CKIT D816V
MYD88 Leu265Pro
BRAF Val600Glu
RhD NIPT testing
