Molecular Genetics - Chronic Myeloid Leukemia and Minimal Residual Disease (V) Flashcards
Which formed elements are affected by chronic myeloid leukemia?
Expected blood picture?
CML = hematopoietic stem cell disorder
Affect platelets, Neutrophils, eosinophils, Basophils most
Leukocytosis with predominantly neutrophils and myelocytes
Basophilia and eosinophilia
Genetic cause of CML?
Reciprocal translocation t(9;22)
Formation of fusion gene BCR-ABL1»_space; production of the oncoprotein BCR-ABL
BCR- ABL has uncontrolled tyrosine kinase activity
» making abnormal pluripotent hematopoietic progenitor cell
» initiate excessive production of all myeloid lineage cells
Karyotype findings in CML?
Chromosome 22 with shortened arm = Philadelphia chromosome
3 types of treatment for CML
1) Cytotoxic chemotherapy
2) Allogeneic haemopoietic stem cell transplant
3) Tyrosine kinase inhibitor***
- Imatinib (1st line)
- Dasatinib (2nd line)
3 Methods of monitoring treatment response in CML?
Which is gold standard
Monitor minimal residual disease (MRD)
- Hematological response: blood count
- Cytogenetic response: chromosome
- Molecular response: Quantify fusion gene expression
- Gold Standard, most sensitive**
Explain the limitations of monitoring hematological and cytogenetic response after treatment for CML
Hematological: CBC examined
- Limited because bone marrow can still contain leukemic cells, cause relapse after stop treatment
Cytogenetic: Chromosome overlap by karyotype
- Residual leukemic cells not eradicated
Explain why Monitoring MRD at the RNA level is the best approach to assess treatment response for CML.
DNA - BCR-ABL fusion level is highly variable
***mRNA - only 3 isoform of transcript, much easier to design primer for sequencing
Protein - Western blot is difficult and time consuming
Method for monitoring minimal residual disease.
Sample obtained?
Detect and quantify BCR-ABL1 fusion transcripts (mRNA)
Quantitative Reverse-transcription Polymerase Chain Reaction (qRT-PCR)
Primer designed to amplify signal at fusion junction
Sample = peripheral blood WBC
Describe signal production in qRT-PCR reaction.
One primer adjacent to fusion junction carries fluorescent dye and quencher (cancels dye)
One primer with enzymatic (Taq 5’ to 3’ exonuclease) activity adjacent to fusion junction
If fusion gene is present, it binds to fusion junction and elongates to cleave quencher on one primer»_space; quencher released and fluorescent signal is emitted
Relationship between PCR cycle number and intensity of fluorescent signaling from qRT-PCR?
Amount of fusion gene dictates the intensity of fluorescent signaling
Sigmoidal curve: Threshold > Exponential > plateau
Cycle threshold (CT) = number of PCR cycles at which threshold intensity of fluorescent signal is set
Define CT value.
Effect of high fusion gene number on qRT-PCR cycle threshold (CT)?
Cycle threshold (CT) = number of PCR cycles at which threshold intensity of fluorescent signal is set
If the number of fusion gene is higher to start with, then the CT value would be lower
(lower no. of PCR cycles to reach threshold of intensity)
qRT-PCR of BCR-ABL fusion mRNA transcript is performed.
Cycle threshold value is high.
Comment on the amount of fusion gene present in sample?
High CT = Low number of fusion genes in sample
Higher number of PCR cycles to reach threshold intensity
Expected change in CT value in qRT-PCR after treatment of CML with Imatinib?
Treatment = decrease number of BCR-ABL mRNA transcripts in sample
CT = Increase
(Higher number of PCR cycles to reach threshold intensity)
Method to normalize results of qRT-PCR to monitor minimal residual disease?
International standardized ratio (IS ratio)
Reference to common standard between laboratories
BCR-ABL1/ ABL1 ratio
Rationale behind IS ratio to monitor minimal residual disease?
Each lab has own variation of BCR-ABL1/ ABL1 ratio
ABL1 is expressed normally, used as reference
No BCR-ABL1 gene in patient sample after treatment = recovered
Undetectable fusion transcript level = RNA degraded, cannot tell recovered or not
