Lab diagnosis of CML and monitoring of treatment

Question 1

Overview of CML and monitoring of treatment

Answer 1

• Intro to CML
• Clinical features of CML
• Pathophysiology of CML
• Routine lab tests for CML
• Immunophenotyping in CML
• Cytogenetics in CML
• Molecular techniques in diagnosis of CML
• Treatment of CML
• Principles of monitoring of CML
• How treatment response is monitored and why it’s significant (In CML)
• Conclusion on CML

Question 2

Intro to CML

Answer 2

• Chronic myeloid leukaemia is the clonal and unregulated expansion of mature leucocytes of myeloid lineage, with slow progression.
• 15% of all leukaemias and may occur at any age.
• The objective of this report is to outline the laboratory techniques used in diagnosing CML and to explain the monitoring of response to treatment
• This essay will primarily focus on tyrosine kinase inhibitor treatment
• Other leukaemias will not be included in this disscusion

Question 3

-Clinical features of CML

Answer 3

• The clinical features of CML include:
• systemic symptoms, such as fever, night sweats and weight loss
• splenomegaly,
• signs of anaemia, such as pollor and fatigue,
• easy bruising and gout.
• The disease is triphasic in nature: Chronic, accelerated and blast crisis phase.
• A blast crisis is defined as >20% of blasts in the bone marrow.
• Most presentations occur in chronic phase.

Question 4

-Pathophysiology of CML

Answer 4

• The pathophysiology of CML can be summarised as multiple mutations accumulating in myeloid lineage stem cells.
• Causing the upregulation of oncogenes and the downregulation of tumour suppressor genes,
• Leading to the uncontrolled growth of these cells.
• The most prevalent mutation is the BCR-ABL1 fusion gene caused by the translocation t(9;22)
• The BCR-ABL1 protein is an enzyme with excessive tyrosine kinase activity,
• BCR-ABL1 is capable of causing the upregulation of oncogenic pathways, such as c-myc pathway

Question 5

-Routine lab tests for CML

Answer 5

• There are numerous investigations used to diagnose CML.
• On a full blood count, the main findings suggesting CML would be a raised WCC, with a normochromic, normocytic anaemia
• There may also be a neutrophilia, with a reduced basophil count.
• A bone marrow aspirate would also show hyper cellularity and a predominance of myeloid lineage cells.
• An MPO stain would give a positive result

Question 6

Immunophenotyping in CML

Answer 6

• Another laboratory test that can be performed is immunophenotyping via flow cytometry.
• The most significant findings would include: CD13 and CD33 positivity, with CD34, CD7, CD2 and CD19 negativity;
• The negative results ruling out acute, T-cell lineage and B-cell lineage leukaemias.
• This would confirm the specific species of malignant cell and would confirm the diagnosis of CML.

Question 7

-Cytogenetics in CML

Answer 7

• The specific categorisation of CML is required, in terms of its underlying genotype, to make a final diagnosis.
• This is because specific genotypes have varying prognostic significance.
• A cytogenetic analysis is performed to determine the karyotype of the patient and therefore identify some of the mutations causing the CML.
• BCR-ABL1+ CML is diagnosed on the presence of t(9;22), which can be visualised on cytogenetic analysis.
• In some cases, a normal karyotype is demonstrated and further investigations are needed to elucidate causative mutations.
• Fluorescent in situ hybridisation (FISH) can test for known, specific translocations that cannot be seen using standard cytogenetic analysis.
• BCR-ABL1 fusion can be demonstrated using FISH if a normal karyotype is found on standard cytogenetic analysis.

Question 8

-Molecular techniques in diagnosis of CML

Answer 8

• An additional technique that can be employed is next generation sequencing (NGS).
• This can include RT-PCR to test for the presence of specific mutations on a molecular level.
• This technique is only required if a normal karyotype is found on cytogenetic analysis and FISH,
• or in the case of research to observe how specific mutations contribute to disesase.

Question 9

-Treatment of CML

Answer 9

• Due to the pathogenesis previously described, there is one main method of treatment used in CML that is very effective.
• The excessive activity of the BCR-ABL1 tyrosine kinase can be reduced with the use of tyrosine kinase inhibitors.
• This treatment is only effective in BCR-ABL1 positive cases of CML.
• An example of this treatment approach is imatinib,
• which binds to the ATP binding site of the BCR-ABL1 protein to prevent excessive phosphorylation, leading to a decrease in the amount of unregulated proliferation occurring.

Question 10

-Principles of monitoring of CML

Answer 10

• Tyrosine kinase inhibitors lead to a decrease in the growth of BCR-ABL1 positive cells (ie malignant cells containing the mutation).
• Therefore, measuring the number of BCR-ABL1 genes present in a sample of cells will give an indication of remaining disease activity, and therefore an indication of desirable response to treatment.

Question 11

-How treatment response is monitored and why it’s significant (In CML)

Answer 11

• To monitor response to treatment, RT-PCR can be employed to quantify the levels of the BCR-ABL1 fusion gene in comparison with the normal wild type genes (Either BCR or ABL1).
• The RNA transcripts of each gene are quantitatively measured.
• The two levels of genes are represented as a percentage on a logarithmic scale
• Additionally, a complete cytogenetic response (CCyR) is defined as there being a complete absence of t(9;22) mutations in future cytogenetic analyses of bone marrow samples.
• Response to treatment is usually measured at 3, 6 and 12 months after treatment has begun and at regular intervals afterwards if necessary
• The main limitation of this approach of monitoring response is that it is only viable in CML patients with the BCR-ABL1 mutation.
• Measuring this response to treatment is important in determining prognosis and adjusting treatment regimens or doses to minimise side effects while maximising therapeutic benefit.
• Response to treatment is categorised as either being optimal, failure or an intermediate ‘warning’.
• Patients whose response to treatment is termed ‘failure’ have their treatment changed to incorporate new generations of tyrosine kinase inhibitors

Question 12

Conclusion on CML

Answer 12

• In conclusion, many laboratory techniques can be employed to test for the presence of CML, using the clinical features as a context and evidence.
• The main diagnostic criteria of CML is hypercellularity on bone marrow, with myeloid lineage confirmed by flow cytometry and a cytogenetic analysis to detect any of the significant, large scale mutations, mainly t(9;22)/BCR-ABL1.
• In the presence of a specific mutation that has an established targeted treatment to deter it, measuring the residual levels of the targeted mutation compared with its wild-type can be an effective way of measuring patient response to treatment.