Haematological Malignancy Genetic Testing Flashcards
Outline haematological malignancies.
Complex group of neoplastic conditions occurring in bone marrow derived cells:
- Disruption of normal cellular processes in the bone marrow and immune system.
Clonal diseases:
- Changes occur in a cell that confers a selective advantage, the cell divides and becomes a clone.
Diversity due to:
- The cell that is affected
- The genetic changes that have occurred
- The point in the cells maturation process that the malignant changes have occurred
Make up approximately 7% of all malignancies (approximately 61 per 100,000):
- 5th most common cancer in the UK.
What factors contribute to the heterogeneity of haematological malignancies?
Diversity due to:
- The cell that is affected
- The genetic changes that have occurred
- The point in the cells maturation process that the malignant changes have occurred
What % of all malignancies in the UK do haematological malignancies make up?
Make up approximately 7% of all malignancies (approximately 61 per 100,000):
- 5th most common cancer in the UK.
- Median age around 70 years - haematological malignancies on the whole particularly affect the elderly.
How may haematological malignancies present?
Depending on the cell lineage(s) affected may present in different ways:
- Lymphoma = lumps
- Myeloma = bone fractures and kidney problems
- Leukaemia = fatigue, vulnerability to infection.
- Will get fatigue and vulnerability to infection with all malignancies but is particularly severe in leukaemia.
- Lethargy due to reduced RBC count, susceptibility to infection due to reduced white blood cell count, bruising tendencies due to reduced platelet counts.
- Some are aggressive whilst others are benign and usually picked up as incidental findings.
What treatment regimens may be given for haematological malignancies?
Treatment regimens often complex:
- Traditional chemotherapeutic agents, radiotherapy, monoclonal antibodies, small molecule inhibitors.
- UK is one of the leading countries in clinical trials and many patients are entered into these.
- Clinical trials do not only test new drugs/treatments but also assess whether biomarker monitoring may play a role in the decision of when to treat (increase in biomarker can preceded relapse).
What are the different types of haematological malignancies?
Can be subdivided into 3 main diseases:
1). Leukaemia
2) . Lymphoma
- Chronic Lymphoblastic Leukaemia (CLL)
- Acute Lymphoblastic Leukaemia (ALL)
- Hodgkin’s Lymphoma (HL)
- Non-Hodgkin’s Lymphoma (NHL)
- Multiple Myeloma (MM)
3) . Myeloma
- Acute Myeloid Leukaemia (AML)
- Chronic Myeloid Leukaemia (CML)
- Myelo-proliferative disorders
In leukaemias the cancer replaces the cells that would normally develop into red cells, white cells, and platelets.
Leukaemic cells usually proliferate rapidly in this setting and migrate into the periphery.
What % of blood stem cells does normal BM contain? How is this affected in aplastic anaemia?
- 30-70% depending on age.
- In aplastic anaemia these cells are mostly gone and replaced by fat cells.
What happens in myelodysplasia?
- Myelodysplasia is the accumulation of dysplastic cells in the bone marrow.
- In Myelofibrosis fibrous tissue expands within the bone marrow cavity to displace the normal cells.
Outline where changes occur for different types of haematological malignancies.
Leukaemia - changes occur in cells in the bone marrow.
Lymphoma - changes occur in cells in the lymph nodes or other parts of the immune system.
Myeloma - changes occur in plasma cells which have returned to the BM following antigen activation in lymph nodes or spleen.
What causes myeloma?
Myeloma - changes occur in plasma cells which have returned to the BM following antigen activation in lymph nodes or spleen.
- As lymphocytes mature they migrate from the bone marrow and settle and continue to develop in in lymph nodes or other parts of the immune system such as the lining of the intestine. Transformation of the cell or expansion of the malignant clone can occur within these regions.
- Plasma cells are derived from B-cells which have been activated by antigens in the lymph nodes and spleen and eventually migrate back to the bone marrow where they segrete Ig’s.
- Expansion of this clonal plasma cell population can destroy surrounding BM causing bone pain and fractures in myeloma.
What conditions are linked with an increased risk of developing leukaemias (genetic risk factors)?
Genetic risk factors:
- Downs syndrome
- Blooms syndrome
- Fanconi anaemia
- Ataxia telangiectasia
- Neurofibromatosis
- Klinefelters
- Familial tendency for AML, CLL, HL, and NHL.
Outline the known environmental risk factors of developing haematological malignancies.
- Lymphomas are much more common in individuals who have been exposed to radiation.
- Development of AML and MDS have been linked to the use of benzene.
- Secondary leukaemias may be caused by chemotherapy and radiotherapy used to treat a primary cancer (therapy related).
- In some countries viruses are linked to the development of leukaemia (e.g. EBV and Burkitt lymphoma).
- In the majority of cases there is no direct evidence for the involvement of a genetic or environmental factor.
- Very difficult to attribute a direct association as multiple factors are usually involved.
Outline the diagnosis and monitoring of haematological malignancies.
Complex and requires a systematic, multi-disciplinary approach.
Clinical features and assessment.
Full blood count and BM/LN biopsy:
- Morphology
- Cell phenotyping - assessment of the cell surface expression of CD is very important in determining the type of disease (myeloid/lymphoid) and the stage of cell development that is blocked.
- Cytogenetics/FISH
- Genetic testing
Outline how the detection of PCR products may be used in genetic testing for haematological disorders.
What it FLT3?
- Genetic aberrations in cancer cells may consist of insertions or deletions of genetic material. When such indels are small simple PCR of the DNA region followed by fragment separation in sufficient to identify the mutated region.
- The FLT3 internal tandem duplication which can vary in size can be present in AML.
- The presence of FLT3 predicts and aggressive disease.
- Simple fragment analysis on an agarose gel highlight the DNA fragment with the internal fragment duplication. Such mutations are predominantly heterozygous and therefore the WT gene is also amplified. This acts as a good control for the experiment. Alternatively the DNA fragments can be separated using capillary electrophoresis.
- The ARMS PCR is a method of detecting point mutations using fragment analysis. Outer PCR primers will amplify the DNA region of choice as in a normal PCR. Within the ARMS PCR an inner primer is designed to complement the mutated DNA with a non complimentary base at the end of the primer. With the correct PCR conditions this single overhanging base will not inhibit the DNAP from extending the fragment. In the case of the WT fragment there are 2 non complimentary bases at the end of the primer. The DNAP is not able to extend this fragment with this amount of overhang. Where the mutant is present the PCR generates the larger outer fragment and a small fragment derived from the ARMS primer. In the case of the WT DNA only the larger fragment is generated.
- QFPCR - measures the amount of DNA after every extension phase of the cycle - uses tags that fluoresce when bound to double stranded DNA. By following the progression of a PCR we can look at when a product is first detected and calculate how much template was there initially. Used in minimal residual disease monitoring.
What is ARMS PCR?
- The ARMS PCR is a method of detecting point mutations using fragment analysis. Outer PCR primers will amplify the DNA region of choice as in a normal PCR. Within the ARMS PCR an inner primer is designed to complement the mutated DNA with a non complimentary base at the end of the primer. With the correct PCR conditions this single overhanging base will not inhibit the DNAP from extending the fragment. In the case of the WT fragment there are 2 non complimentary bases at the end of the primer. The DNAP is not able to extend this fragment with this amount of overhang. Where the mutant is present the PCR generates the larger outer fragment and a small fragment derived from the ARMS primer. In the case of the WT DNA only the larger fragment is generated.
What molecular techniques can be used for monitoring minimal residual disease?
QFPCR can be used for quantifying gene expression in the detection of minimal residual disease using reverse transcription PCR.
Outline pyrosequencing.
- Pyrosequencing is a sequencing technique used for detecting known point mutations or small insertion deletion polymorphisms in short fragments of DNA.
- Useful when there is a low tumour load as it is able to detect low level mutated DNA of around 2-3%.
- New NGS tech as sensitive - but take longer, more costly (at time of lecture).
Outline Acute Myeloid Leukaemia (AML)
- AML varies considerably in its presentation and development.
- The malignant cells are characterised by morphological differences usually associated with the cells stage of maturation.
- The different sub types of AML are associated with the presence of major genetic aberrations including translocations and mutations, a plethora of which may be identified in the molecular lab and used for MRD monitoring and prognostic assessment.
- The different types of AML are diagnosed according to WHO criteria and take into account morphology, immunophenotype, cytogenetics and molecular findings.
- The majority of newly diagnosed AML patients in the UK get entered into nationally run NCRI AML trials.
Outline the WHO classifications of AML.
1) . AML with recurrent cytogenetic abnormalities:
- t(15;17) - acute promyelocytic lekaemia (good prog)
- t(8;21) - CBF AML (good prog)
- inv 16 - CBF AML (good prog)
- 11q23 AML (MLL rearrangement) (good prog)
2) . AML with multi lineage dysplasia:
- Following MDS or MPD or with dysplasia in >50% cells in 2 lineages
3) . AML - NOS
- Classified morphologically
The leukaemic cells karyotype allows categorisation according to the risk of relapse.
Outline the genes affected by the chromosome translocations in leukaemia.
- Balanced translocations tend to confer a favourable prognosis.
- t(8;21)(RUNXT1/RUNX1) and inv16 (CBFB/MYH11) generate chimeric proteins of the core binding factor family which orchestrate cellular transcription.
- t(15;17) is the cause of acute promyelocytic leukaemia (APML) and the chimeric fusion PMLRARa protein is responsible for blocking cell differentiation.
- APML is treated very successfully with ATRA (retinoic acid) and arsenic.
- Both cytogenetics and PCR techniques are used to identify translocations in AML.
- Quantitative Reverse Transcription PCR is used to monitor MRD post therapy.
What translocation is related to the RUNXT1/RUNX1 chimeric protein formation in AML?
- t(8;21)(RUNXT1/RUNX1) generates chimeric protein of the core binding factor family which orchestrate cellular transcription.
What cytogenetic abnormality is related to the CBFB/MYH11 chimeric protein formation in AML?
- inv16 (CBFB/MYH11) generates chimeric protein of the core binding factor family which orchestrate cellular transcription.
What translocation is associated with APML?
- t(15;17) is the cause of acute promyelocytic leukaemia (APML) and the chimeric fusion PMLRARa protein is responsible for blocking cell differentiation.
How is APML successfully treated?
- APML is treated very successfully with ATRA (retinoic acid) and arsenic.
Where do translocations tend to occur in AML? How can we use PCR to identify breakpoints?
- Genetic translocations can occur at multiple breakpoint regions along one or both of the chromosomes and may be intronic or exonic.
- In the nested PCR primer sets which span all of the possible breakpoint regions are utilised. The size of the PCR product may be determined from the PCR gel and from this the specific breakpoint region involved in the translocation can be established.
- Identification of exact break points is becoming more important as small molecule inhibitors are being developed to inhibit the chimeric proteins generated from the translocated genes.
