B-ALL Flashcards
(18 cards)
B-ALL morphology
Small blasts with scant cytoplasm, condensed nuclear chromatin, and indistinct nucleoli
Larger cells with modest cytoplasm, which is light-blue to blue-grey, occasionally vacuolated, dispersed nuclear chromatin, and multiple variable prominent nucleoli
Hand-mirror cells (with cytoplasmic pseudopods)
B lymphoblast immunophenotype
almost always positive for CD19, CD79a and cCD22, as well as CD10 (also present on neutrophils), sCD22 (also present on basophils), CD24, PAX5 and TdT in most cases
CD20 and CD34 expression is variable
CD45 may be absent and if present is always dim in expression compared to B cells (ergo haematogones)
The myeloid markers CD13+ and CD33+ may be expressed
PAX5 is the most sensitive marker by IHC (however is also positive in t(8;21) AML)
B-ALL definition
B-ALL: more than 20% of B lymphoblasts by flow cytometry with B-cell lineage markers;
B-LBL: Histology effaced lymph node architecture or diffuse infiltration of an organ by a monomorphic population of blasts with B immunophenotype (CD19, CD22, cCD79a, and/or PAX5) and markers of immaturity (TdT, CD34, and/or CD10), surface immunoglobulin negative. CD34 and TdT expression may be absent in rare cases and pose a diagnostic challenge;
Desirable:
>Immunophenotypic profiles associated with specific recurrent genetic abnormalities
>Identification of specific recurrent genetic abnormalities
B-ALL investigations
Clinical presentation
- BM failure = pancytopenia
- variable leucocytosis
- LAD and hepatosplenomegaly
- bone pain and arthralgias
Investigations
- FBE/film
- biochem, coags, LDH
- Flow, CG, FISH, molecular
Abnormal lymphoblasts:
- CD19, CD22, cCD79a, PAX5
- TdT, CD34 variable, bright CD10
- low CD45
- low/no CD20 or sIg expression
B-ALL prognostic markers
B-ALL KMT2A rearrangement
Presence of KMT2A rearrangement
Higher WCC tendency; CNS involvement common
Common in <1 y.o
Immunophenotype -
CD19+, CD10-, CD24-
TdT NEGATIVE (uncommon in BALL)
CG/molecular: t(4;11)(q21;q23)(Most common)
Poorer prognosis/unfavourable
B-ALL with hypodiploidy
< 43 chromosomes
- near haploid vs low hypodiploid
~50% of paeds low hypodiploid have germline TP53
Unfavourable prognosis
SNP
B-ALL with BCR:ABL1 fusion
High WCC
Increased risk with age - most common in adult population
80-90% have p190 transcript
Often express CD13+, CD33+ (myeloid marker)
May have CD25+
Since TKI LT survival rates increased; better than Ph- in some studies
MRD strong predictor of relapse and OS. Goal of therapy is achieve undetectable BCR-ABL1 transcript with sens of at least 0.01%; gives favourable prognosis
B-ALL with BCR:ABL like
Presence of DNA alteration that induce a phenotype similar to that of BCR::ABL1+ B-ALL but lack the pathognomic BCR::ABL1 rearrangement
BCR::ABL1 like ALL is more about the RNA expression prolife - the genes expressed form a similar profile to those created by the BCR::ABL1 mutation.
> JAK-STAT pathways
ABL class gene rearrangement
Majority occur in patients with Down Syndrome
Unfavourable risk profile
B-ALL with ETV6:RUNX 1 fusion
presence of rearrangement between the ETV6 gene on chromosome 12p13.2 and the RUNX1 gene on chromosome 21q22.1
Age 2-10, most common in children
Very favorable
CD27+ and CD44- seen in both ETV6;RUNX1 translocation and ETV6;RUNX1 like B-ALL
> Frequently expresses myeloid antigens CD13/CD33
FISH: identifies translocation
RT-PCR and RNA sequencing
B-ALL with hyperdiploidy
51-65 chromosomes
> chromosomes X, 4, 6, 10, 14, 17, 18 and 21 (mostly even chromosomes) in the absence of subtype defining translocations .
Mostly children
Rare in adults/neonates
B-ALL with iAMP21
Presence of gains and gross rearrangements of the long arm of chromosome 21
Demonstration of ≥5 copies of RUNX1 per cell, with ≥3 or more copies on a single abnormal chromosome 21.
lower WCC, older paediatric pop
CG: grossly abnormal chr 21
FISH: >5 RUNX1 signals per cell
Microarray: help distinguish from hyperdiploid B-ALL
Other B-ALL defined by genetic abnormalities
TCF3::PBX1
> rearrangement between TCF3 gene on chromosome 19 and the PBX1 gene on chromosome 1.
>CD9 expression
> picked on CG or molecular if cryptic translocation
> int to favourable prognosis
> higher rates of CNS relapse
TCF3::HLF
> rearrangement between TCF3 at 19p13.3 and HLF at 17q22.
>RARE
> picked on RT-PCR or NGS RNA sequence
> VERY POOR prognosis - considered incurable
> strong CD19 expression
IGH::IL3
> juxtaposition of the IGH enhancer and the IL3 promoter t(5;14)
> BM eosinophilia
> SUPER RARE
> intermediate prognosis
B-ALL MRD modalities
Monitoring of MRD throughout the treatment process is of the utmost importance as positive MRD has profound implications on treatment decisions. There are 4 common methods used for MRD in monitoring in ALL:
> prognostic
- end of induction
- pre transplant
> management and transplant decisions
> monitoring for relapse
- both chemotherapy and post alloSCT
Options:
1. Flow MRD
2. Real time qPCR for fusion transcripts
3. PCR for IGH/TCR rearrangements
4. NGS
B-ALL - Flow MRD
Uses a leukaemia-associated immunophenotype (LAIP) or difference from normal (DFN) approach to distinguish leukaemic cells from their normal counterparts (haematogones)
B ALL panels often include CD10, CD19, CD45, CD34 and CD38; high CD10 and low CD38 expression may distinguish from haematogones
T ALL panels often include CD34, TdT, CD7, cCD3 and CD1a
Sensitivity: 10^-4
Advantages:
> rapid TAT
Disadvantages:
> Sensitivity is limited by quality of diagnostic sample
> Immunophenotypic shifts can lead to false negative results
» e.g post targeted therapy e.g. anti-CD20, CD19 therapy
> diagnostic sample is required to determine the LAIP; reduced sensitivity if this is not available
> Fresh cells required - 24-48 hours > Requires technical expertise and equipment
B-ALL MRD with PCR of fusion transcripts.
Principle
> Real time quantification of target gene (BCR:ABL) presence by PCR reaction via denaturing, annealing and elongation
> Fluorescent labelled oligonucleotide binds to RNA strand of target sequence
> As TAQman polymerase polymerises complementary strand of RNA, if oligonucleotide bound to RNA sequence, fluorophore will be released causing a release of light
This light released is measured and compared to calibration curve with the amount of light released is compared to the housekeeping ABL gene and the amount of BCR-ABL in sample can be quantified
Method
> BM or PB via EDTA collection
> RNA extracted from patient sample within 72 hours of sample arriving in lab as it quickly degenerates
>RNA mixed with reagents and run on respective machines (i.e. GeneXpert at PMCC for BCR::ABL1)
> Values automatically generated by PCR analyser
QC/QA
> Housekeeping ABL gene ensures that sufficient sample is used in the test (>1000 copies)
> PCC (probe check control) ensures that the fluorescence signal is measured on all probes - verifies that all reagents are present
> For new reagents must run a positive and negative sample on both old and new reagent to ensure new reagents are appropriate
> Enrolled in external quality assurance programs
Strengths
> Highly sensitive - 10^-4 - 10^-5
> Can be batch run
> Relatively easy with little labour
> Cheap once processes in place
> Do not necessarily need a diagnostic sample however need to know what sequence to target
B-ALL MRD allelel specific oligonucleotide
Use real time quantitative PCR (RQ-PCR) for Immunoglobulin/T cell receptor.
The target is an allele specific oligonucleotide
The VDJ region sequenced via Sanger to identify a clonal rearrangement that can be tracked by PCR
This specific rearrangement is then chosen to make a patient specific primer.
qPCR is then used to identify the presence of this clone in subsequent MRD samples
Strengths
> Highly sensitive - 10^-4 - 10^-5
> Widely applicable: 80-90% of patients are able to be tracked by this method
Limitations
> Labor intensive - need specific primer for each patient which takes 4 weeks
> diagnostic sample is required
> False negatives can occur with loss of emergence of new V(D)Jsequences
> expensive
> Limited number of labs in Australia do this test
B-ALL MRD with IGH rearrangement/NGS
Deep sequencing of IGH/TR genes using multiplex PCR
> Identification of clonal IGH and TCR gene rearrangements without the need for a patient specific primer
Primer targets is the conserved variable and joining regions
> IGH for B- ALL
> TCRG - T-ALL
Method:
1. Library prep
2. Amplification
3. Sequencing
4. Analysis
> assess for clonal peak
Strengths
- Widely applicable - 80-90% of patients will have a trackable clonal variant
- Highly sensitive - up to10^-6
- Relatively labor non-intensive once set up
- can track evolution
Limitations
- Expensive
- Requires a diagnostic sample
- Very few laboratories perform this (Austin is our reference laboratory) eg clonoseq
