haematological malignancies Flashcards
what is leukaemia?
it is tumours of the blood or haematopoietic lineages
what are the classifications of leukaemia?
they can be acute or chronic or myeloid (platelets, monocytes, granulocytes and erythrocytes) or lymphoid (B cells, T cells or NK cells)
what is lymphoma?
it is tumours of the lymphocytes or the immune/lymphatic system
what are some types of lymphoma?
Burkitt, diffuse large B cell, marginal zone and follicular
what are the patient determinant outcomes and their classifications?
tumour, treatment and host
in host - age and performance status
in treatment - drug sensitivity and resistance
in tumour - paraneoplastic activity, tumour burden, invasive potential, growth rate and tumour type
what is used in diagnosis?
stain, microscopy, morphology, cytology, EM, PCR, sequencing, alternative proteomics and probe based RNA analysis
what determines the tumour classifications?
immune response, metastatic potential and metabolic activity and oncogenic events
what determines the host characteristics?
the drug metabolism and toxicity
what determines the treatment?
the DNA damage response, apoptotic response, resistance pathways and therapeutic target expression
what are identified in diagnosis?
disease defining point mutations and translocations, myeloproliferative disorders and lymphomas and leukaemias
what aids the prognostication and treatment selection?
the DNA translocations and deletions defining prognostic groups and treatment choices - leukaemia and lymphoma, the immunoglobulin gene mutation load defining risk
how would you monitor, assess response and predict relapse in disease?
monitor the translocations
what are two outcomes in chromosomal translocations?
there are fusion genes and gene deregulation
in myeloid leukaemia what is the result?
aberrant fusion protein from the fused coding regions of two promoter genes
what is the result in B cell lymphoma?
the expression of one gene is controlled by the promoter of the other gene - deregulation
how can you identify these mutated genes?
PCR and logarithmic expansion of target DNA or FISH
what are the characteristics of PCR to define translocations?
only over a small size range, highly sensitive and specific, gene level, need consistent junction and precise sequence
what are the advantages and disadvantages of using FISH?
it is at the chromosomal level over a large size. There is no need to know the specific sequence and have a consistent and it is specific however it is less sensitive.
what are the two types of translocation?
promoter/enhancer substitution or fusion gene translocation
what is the difference between the two types of translocation?
in fusion gene there is a predictable break point and mRNA is processed to remove introns whereas in promoter there is no predictable break point and no abnormal mRNA
where is fusion and promoter translocations most commonly found?
fusion - most commonly found in myeloidleukaemias
promoter - lymphomas
what are the characteristics of the classic paradigm?
the classic paradigm is CML - abdominal pain, hyperuricaemia, gout, splenic infarction, splenomegaly, fatigue and anaemia
what will a blood test show in CML?
leukocytosis and anaemia
what is the epidemiology of CML?
male or female (1.5:1), rare - 1 in 100,000 and less than 10% of cases in under 20s, median age is middle age - 55-60
what is the chromosomal basis of CML?
fusion gene between 22q11.2 and 9q34 therefore getting rearranged chromosomes creating BCR-ABL fusion gene coding for an active tyrosine kinase
what is the basis of treatment for CML?
it is a small molecule gleevec that is specifically designed to block the active site of the tyrosine kinase - important to count how many cells with the transformation are still present after treatment
how can you identify the treatment response in CML?
PCR - qualitative as will reach a plateau quickly no matter the amount of target molecules. qPCR or real time PCR is quantitative for the number of target DNA molecules
what are the advantages of qPCR?
it is sensitive more so than cytogenetics and can identify early relapse
what do you use in molecular diagnostics of CML?
tumour load and classification, treatment selection, risk and response assessment
what are MPNs?
myeloproliferative neoplasms
the myeloproliferative neoplasms spectrum is rapidly expanding, why is this?
new diagnostic techniques identifying new mutations
what is used in chronic myeloproliferative disorders?
morphology assessment of bone marrow
what is NGS?
next generation sequencing - sequencing hundreds of millions of pieces of DNA in a single run
what is WGS?
NGS allows for the sequencing of an entire genome at low depth coverage
what is WES?
NGS allows for the sequencing of the entire exome of transcribed genes at a higher depth
in CML what are the stages of copy number and tumour burden?
- complete molecular response and undetectable transcript
- major molecular response
- complete cytogenetic response
- complete haematological response
- diagnosis, pretreatment or haematological response
what is important in chronic lymphocytic leukaemia?
the need to distinguish between indolent disease with little risk and agressive disease with high risk as this is relevant to molecular diagnostics and treatment and risk assessment - stratify the risk i the 4 prognostic groups
what is the epidemiology of B cell chronic lymphocytic leukaemia/lymphoma?
common around 3400 cases a year - subclinical disease related to CLL is even more common. In elderly males with male : female 2:1 and majority over 50 with mean age of 72.
what are the symptoms of B cell chronic lymphocytic leukaemia?
asymptomatic or fatigue, autoimmune anaemia, splenomegaly, lymphadenopathy, lymphocytosis
what is the management of chronic lymphocytic leukaemia?
FBC
what is monoclonal B cell lymphocytosis?
it is a condition that is phenotypically similar to CLL but the number of cells is less than 5x10^9. Around 1% will progress to CLL each year but around 5% of the population harbour this small monoclonal B cell expansion
what is the B cell lifecycle?
start off as progenitor B cells for generating functional antigen receptor. This makes naive B cells that are T cell dependent which can either make memory cells with a germinal centre or make plasma cells or T cell independent which make plasma cells. Memory cells can also make plasma cells. The naive ones make mature B cells that are for recognition of antigen and somatic hypermutation for class switching. The plasma cells are for proliferation and differentiation.
what is somatic hypermutation?
it occurs in a special microenvironment called the germinal centre and acts on the immunoglobulin genes in the B cells. The SMH loads are high and B cells that improve the recognition of antigens are selected for.
how can you detect somatic hypermutation?
FISH - red 17p13 probe and green control probe
what is the CLL stratification and risk assessment?
identifying the risk through the mutation and selecting the appropriate treatment
what are the three risks of CLL?
11q/17p deletion, no deletion but mutation Ig or unmutated no 11q/17p deletion
what are the treatment plans for the three types of CLL?
mutated Ig - rituximab, chlorambucil or fludarabine - wait and see - minimally toxic therapy
unmutated and no deletion - rituximan and fludarabine or fludarabin and cyclophosphamide
deletion - high risk clinical trial and CAMPATH anti-CD52 antibody
what do you use for cytogenetic identification of CLL?
FISH - denature DNA in tumour cells, stain gene region with fluorescent probe, count spots in nucleus <2 spots per nucleus is deletion, 2 is normal and more than 2 is amplified
how would you identify prognostic factors of CLL?
use immjnoglobulin VH gene sequencing. Extract DNA from CLL cells (bone marrow, blood, lymph node), amplify genes using PCR, DNA/NGS sequencing of Ig genes, compare to reference, less than 2% divergence to germline line means unmutated
what is the cellular transduction pathway?
there is a ligand that attaches to a receptor on the plasma membrane which then processes at the level of the plasma membrane and may include scaffold. The signal is through tyrosine kinases and the effector is transcription factors which are key regulators of other pathways. The target is the cytoplasm, nucleus or organelle
what is the B cell receptor signalling in CLL?
once the ligand has attached there is tonic survival, activation or antigen presentation. If there is antigen presentation this results in a T cell cognate interaction and then differentiation. Activation will lead to anergy or deletion of the B cell
how does chemo work in CLL?
chemo results in DNA damage and ATM activation (where there is an 11q deletion). This results in p53 activation (where there is a 17p deletion) which results in apoptosis or will result in p21 activation resulting in cell cycle progression if the cell can be repaired and survive
how does CLL affect B cells?
when the ligand attaches the B cell is activated and the receptor signals and activates vital downstream pathways. This results in survival, resistance to apoptosis and therefore cell proliferation
