Leukemia's and other acquired cancers Flashcards
What cell lineage does AML originate from?
AML is cause by clonal expansion of myeloid progenitors (blasts) in the peripheral blood, bone marrow and other tissue
How is AML diagnosed?
when at least 20% blasts are present in the PB or BM
or with <20% blasts + AML related chromosome abnormality
What is the incidence of AML
Median age of diagnosis is 67 and accounts for 25% acute leukemia’s (most common acute leukemia in adults)
Paediatric AML (15 yrs) accounts for 15-20% of all acute leukemia’s
What are the symptoms of AML
shortness of breath, fatigue, easy bruising. increased risk of infection.
can also develop splenomegaly, tender boned and gym hypertrophy due to blast infiltration of organs.
what are the main WHO categories of AML?
- AML with a recurrent cytogenetic abn
- AML with myelodysplastic related chages
- Therapy related AML
- AML not otherwise specifice
what are the 3 recurrent AML rearrangements associated with a good prognosis?
APML
PML-RARA t(15;17)(q24.1;q21.2)
CBFB (16q22)-MYH11 (16p13)
inv(16)(p13q22)/t(16;16)(p13;q22)
RUNX1T1-RUNX1
t(8;21)(q22;q22) (not in pediatric AML)
if secondary abnormaliteis are present these have no prognostic impact
What rearangements are associated with an intermediate prognonsis
All other karyoptypes
what rearrangements are associated with a poor prognosis in paediatric AML (<16yrs)
5q abnormalities -7 t(6;9)(p23;q34) t(9;22)(q34;q11) 12p abnormalities
what rearrangements are associated with a poor prognosis in adult AML
abnormal 3q inv(3)(q21q26)/t(3;3)(q21;q26) add(5q), del(5q), -5, -7, add(7q)/del(7q) t(6;11)(q27;q23) t(10;11)(p11~13;q23) t(11q23) t(9;22)(q34;q11) -17/abn(17p) Complex (≥4 unrelated abnormalities)
What are the AML prognostic groups based on
Grimwade 2010. Stratification for AML17 trial is based on Grimwade 1998. Prognosis in paediatric AML is based on Harrison 2010.
Describe the significance of the PML-RARA rearrangement
PML-RARA is associated with a very aggressive form of AML but a good prognosis as it can be treated with ATRA (all trans retinoic acid). Therefore rapid diagnosis is required.
What treatment is indicated for t98;21)(q22;q22); (RUNX1T1-RUNX1) and inv(16)(p13.1q22)?
Good response with Cytarabine
What is the significance of inv(16)(p13.1q22)?
RUNX1-CBFB
very subtle rearrangement and may be overlooked by standard g-banding of poor preps so requires FISH or RT-PCR to confirm/exclude
diagnostis of AML
+22 is very common 2nd abn and associated with improved outcomes.
The fusion proteins produced by the above rearrangements allow the CBF to bind to the target genes, but the transcriptional activation is lost via a dominant negative inhibition leading to arrest of differentiation and TP53 induction being inhibited resulting to increased cell survival.
What is the etiology and treatment of APL?
PML-RARα protein = RARa function is disrupted and can no longer bind to DNA and represses transcription of retinoic acid target genes. PML also dirupted and can no longer block cell growth and proliferation and induce apoptosis
- uncontrolled cell proliferation.
example of a class 2 mutation Class II mutations: affect transcription factors and primarily serve to block haematopoietic differentiation. ~20-25% of adult AML have mutations in this group.
What is therapy related AML?
10-20% of AML. Develops as a side effect in patients receiving alkylating agents, topoisomerases or radiation to treat a primary cancer.
Often have the came cytogenetic abnormalities as their de novo AML counterparts but with worse prognosis indicating biological differences
What is AML-NOS
encompasses AML patients that do not fall into the other categories. Sub classification is based on morphological, immunological and cytochemical differences.
What myeloid disorders are related to T21?
children with DS have a 50% increased risk of developing AML before the age of 5. Blast cells carry GATA1 mutations.
may be proceeded by transient abnormal myelopoiesis
AML developing > the age of 5 and without a GATA1 mutation is considered conventional DS
What is the diagnostic strategy for AML
- karyotype
10 cells for abnormal (analyse 5 and count 5)
20 cells nor normal (analyse 10 and count 10)
- if single cell abn detected score an additional 10 cells for the abn
FISH may be used if there are insufficient metaphases to complete the analysis
-Y and +15 are commonly found in BM od older patients with no haem malignancy and are considered part of ageing. However they may also be markers of a neoplastic myeloid clone.
What follow-up studies may be required (cyto)
G-banding is not required to establish remission
30 metaphases of 100 iFISH may be scored (only useful if the diagnostic karyotype was abnormal)
What testing should be performed if relapse is queried?
- examine 10 metaphases if diagnostic abn present
if not a significant risk of relapse FISH or RT-PCR for the diagnostic abn should be considered - possibility of second malignancy should be considered in late relapse cases and full diagnostic work-up may be appropriate
What are the reporting times?
3 days fro rapid preliminary result
14 days for urgent referrals (new diagnosis and suspected relapse)
21 days for routine referrals
what are class 1 mutations?
activate signal transduction pathways and result in proliferative advantage to mutated cells
What are class 2 mutations?
Affect transcription factors and primarily function to prevent haemtopoeitic differentiation
Name 2 molecular mutations associated with AML and their prognostic significance?
NPM1- good prognosis
FLT3- poor prognosis.
Describe the significance of the FLT3-ITD
FLT3 (13q12) is a receptor tyr kinase.
Majority of mutations are internal tandem duplications (ITDs) in exons 14 and 15 leading to in-frame insertions within juxtamembrane region of the receptor. Causes loss of structure of autoinhibitory domain and ligand-independent activation of FLT3.
Most affected AML patients have one type of FLT3 mutation, but some have both types. Particularly poor prognosis if wildtype FLT3 allele lost by deletion of 13q or monosomy 13.
Describe the significance of the NPM1 mutations
A third of AML cases, including approx 50% with normal karyotype, have heterozygous mutations in the carboxy-terminus of the nucleolar phosphoprotein, nucleophosmin (5q35.1)
NPM1 thought to have relevant roles in cellular functions, including ribosome biogenesis, centrosome duplication, DNA repair and response to stress.
Wild-type NPM1 protects hematopoietic cells against p53-induced apoptosis under conditions of cellular stress; possible that failure of mutated NPM1 to protect cells may make them more sensitive to high-level genotoxic stress induced by chemotherapy – good prognosis.
Describe MRD in AML
Molecular markers e.g. recurrent rearrangements provide the basis for MRD by measuring the level of the marker at regular intervals compared to a a housekeeping gene (usually ABL). For this knowledge of the specif breakpoints must be determined at diagnosis.
Blood sampling every 6 months for detection of CBFB/MYH11 clones is sufficient a shorter test interval should be performed for patients with t(8;21), NPM1 mutations and t(15;17).NB. Flow cytometry can also be used for MRD monitoring (less sensitive but applicable to more AML cases).
What is CML?
myeloproliferative neoplasm originating from pluripotent bone marrow stem cells.
What are the symptoms of CML?
fatigue, joint pain and fever.
May also ddevelop splenomegaly,m weight loss and loss of apetite
easy bruising and swelling in groin and armpit and neck (lymph nodes) can devlop and symptoms progress
What is the incidence?
1-2 per 100,000 and accounts for 15% of adult leukemias
RARE in children
what are the 3 phases of CML?
CP- chronic phase
90% of diganoses made here due to routine blood test (40% asymptomatic)
AP- accelerated phase
can last months to yrs, some patients progres straight to BC
BC- blast crisis
>20% blast in the BM. more intense treatment and lower success rate. Often has i(17q) present (loss of p53 on 17p)
Describe the Philadelphia chromosome and its significance in CML
the presence of the philadelphia chromosome t(9;22)(q34;q11) is diagnostic of CML and is present in 95% or patients- remaining have the equivalent fusion but may be cryptic.
Encodes the BCR-ABL1 fusion protein (on der 22) which is a constitutively activated tyrosine kinase resulting in uncontrolled cell proliferation and inhibition of apoptosis.
BCr is on ch 22 and ABL1 on chr9
what are the BCR-ABL1 breakpoints
3 common breakpoints
M-bcr = major and account for 95% and encodes a 210kDa fusiuon protein called p210 Bcr-Abl
m-bcr = minor and encodes p190 Bcr-Abl
u-bcr is very rare and encodes p230Bcr-Abl
Other abnormalities in CML
Important to determine if there are other abnormalities present at diagnosis as these are considered warning by ELN guidelines.
What are is the diagnostic strategy from CML
Test BM (van use PB if BM not available) - analyse 5 and count 5 for abnormal analyse 10 and count 10 for normal (+ 5 metaphase FISH is a cryptic rearrangement is suspected)- if one abnormal cells detected, analyse 10 more to see if it is clonal or 100 interphase FISH if insufficient cell
Describe MRD in CML
required to monitor response to treatment and defines remission. Can also be used to identify relapse before it becomes overt. Requires chromosome analysis. FISH can be used for poor preps
What defines a heamatologic response in CML treatment?
Blood counts are taken every week until achieved. A hematological response is achieved when there are normal cell counts and reversal of splenomegaly
What defines a cytogenetic response in CML monitoring?
requires cytogenetic chromosome analysis at 3 and 6 months post treatment, then every 6 months until it has been achieved. It is based on the the level of Ph+ve cells
- no response = 96-100%
- minimal response = 66-95%
- minor response = 36-65%
- partial response = 1-35%
- complete response = 0%
monitoring response at 3, 6 and 2 months is important to determine whether the current treatment should be continued (optimum response) or should be changed (failure/resistance).
ELN have published guidelines defining the overall response to imatinib therapy at different time points in the treatment based on the ph+ve level- described as optimal, suboptimal, warning or failure.
What defines a molecular response in CML monitoring/
Molecular response is measured by RT-PCR, therefore need to identify the specific breakpoints at diagnosis.
measured after cytogenetic response achieved. can ID relapse before it becomes overt
less than major MR- < 3 log reduction
Major MR- ? 3 log reduction in 2 consecutive samples
Complete MR- >45 log reduction- BCR-ABL transcript negative in 2 consecutive samples
which chromosome contains the BCR-ABL fusion? what happens if the ABL-BCR fusion is lost
der(22) small chromosome that resembles a clover carries BCR-ABL. No affect of prognostic significance is the reciprocal fusion on chr 9 is lost (not reported)
What are the reporting times for CML cytogenetic analysis
3 days rapid preliminary result
7 days for urgent (diagnosis or relapse)
14 days for follow-up following a rapid result
21 days for monitoring
what other chromosomal abnormalities may be detected in CML
+8 common in myeloid malignancies and without prognostic impact
+9
+21
-Y also associated with advanced age
i(17)(q10) loss of TP53 and common in BC
What treatments are used for CML at diagnosis
Imatinib in a TKI and is used as a primary treatment for CML
what treatments are available if resistance to imatinib develops
2nd and 3rd generation TKIs have been developed for CML cases which develop resistance mutations. It is important to sequence the BCR ABL transcript to identify the specific resistance mutation as this will inform on secondary therapy choice.
How is RT-PCR used for MRD in CML
quantitavie RT-PCR
- RT the RNA to cDNS
- using primers specific to the fusion protein breakpoint the level of transcript is measured compared to a house keeping gene. Ususally ABL1
- results are normalised using the BCR-ABL1:ABL1 ratio
An international scale can be applied to the major breakpoint transcripts to make standardized interpretation possible (not available for minor or u breakpoints)
Describe the action of imatinib
first line TKI, keeps the protein in its active state but prevent STP binding through competetion. This prevents ATP binding and the TKI cannot phosphorylate downstream targets and activate downstream signalling pathways.
if effective patients should achieve CHR at 3 months, pCGR ant 6 months and CCGR at 12 months, MMR at 18 months.
What other treatments are available for CML
allogenic HSCT can be used for patients in AP or BC and is the only known cure
What is the difference between primary and secondary resistance to a drug?
Primary resistance is the failure to achieve a response e.g. CGR in CML
secondary resistance is the loss of an established response to the therapy.
name 2 second line TKIs used for CML
Dasatinib- 2nd gen- less stringent conformation requirment for TK than imatinib
Nilotinib- exclusively binds active protein = improved specificity. 20-50x more potent that imatinib
what is the significance of RUNXi-RUNX1T1 in AML
t(8;22)(q22;q22)
diagnostic in AML
Associated with auer rods and more common in younger patients
what is the definition of a clone
3 cells with loss of the same chromosome (2 cells can be considered a clone if there is also the same structural abn in both
2 cells with the same abnormality for a gain or structural abnormality
What is the significance of the FLT3-ITD
ITD (internal tandem duplication of exons 14 & 15.
Results in loss of structure of the autoinhibitory domain so it is constitutively active.
what is the significance of an NPM1 mutation
good prognosis- proposed because failure of mutated NPM1 to protect the cell from apoptosis by TP53 in response to stress makes the cells more susceptible to apoptosis
intermediate prognosis if detected with a FLT3-ITD
what are the different diagnostic techniques used for AML?
Morphology- 20% blasts in BM or PB
Cyto- presence of a diagnostic rearangement = t(15;17)(q21;q24), t(8;22)(q22;q22) or inv/t(16)(p13;q22)
molecular- RT-PCR for cytpo rearrangement (defining the breakpoints is important for MRD). Work-up should also include testing for NPM1, FLT3-ITD, CEBPA and RUNX1 mutations
What cell lineage does ALL originate from?
Lymphoid cells in the BM
B-cells (85%) and T-cells (15%)
B and T cell are ALL are morphologically indistinguishable so immunopheonotyping is used
what is the incidence of ALL
accounts fo 75% olf all childhood leukemias and 6% of adult leukemias
What are the symptoms of ALL?
fatigue, easy bruising, fever, joint/bone pain, splenomegaly, hepatomegaly, weightloss, swollen lymph nodes
anemia, neutropenia, leucocytosis, thrombocytopenia
What recurrent cytogenetic abnormalities are associated with a good prognosis in ALL
- High hyperdiploidy (51-65 chromosomes)
adult and childhood ALL
commonly gains of 4,6,10,14,17,18,21,& X
Important to distinguish from doubled up near haploid (4 copies of gained chr) which has poor prognosis - t(12;21)(p13;q22) ETV6 RUNX1
childhood ALL
cryptic rearrangement detected by FISH or RT-PCR - IGH rearrangemtns incl 9p
What recurrent cytogenetic abnormalities are associated with an intermediate prognosis in ALL
TCF3 rearrangements
t(1;19) intermediate prognosis in adult and child ALL
must distinguish from t(17;19) which has a poor prognosis
What recurrent cytogenetic abnormalities are associated with an poor prognosis in ALL
- t(9;22)(q34;q11) BCR-ABL (usually minor breakpoint p190)
- KMT2A (MLL) rearrangements
t(4;11)(q21;q23) has poor prognosis, unclear for other rearrangement partners - complex karyotype (>4 abn) adult ALL only
- hypodiploidy (<44)/ low hypodiplpoidy (30/39)/ near haploidy (23-29)
- TCF3 rearrangment t(17;19)
- iAMP21 > 5 copies of RUNX1 (3 extrac copies on 1 chr 21)
What is the principle of diagnostic testing in adult ALL?
>25 yrs
need to test fro: ploidy complex karyotype t(9;22)(q34;q11) t(4;11)(q21;q23)
Considerations for G-banding in ALL?
high chance of apoptosis of cells in culture so need at least 2 preps, 1 of which is only cultured for 24hrs to have a good chance of identifying the abnormal cell line
need to distinguish high hyperdiploidy from doubles up near haploidy as they have different prognosis
What is the diagnostic strategy in childhood ALL?
1-25 yrs
4 mandatory FISH tests. Howver as detected imbalances are mutually exclusive, once one has been detected there is no need to perfrom the remaining FISH tests.
- t(12;21)(p13;q22) good prognosis 2R1G1F
will also detect high hyperdiploidy (good prognosis) 4R2G
and iAMP21 (poor prognosis) - 1R1G1Rcluster - MLL (t4;11) poor prognosis
- t(9;22)(q34;q11) poor prognosis
- TCF3- need to distinguish t(1;19) intermediate from t(17;19) poor prognosis
What are the TATs for ALL cytogenetics
3 days for rapid preliminary result
14 days for urgent (new diagnosis/ ? relapse)
21 days for routine follow-up
Describe molecular testing in ALL?
no mandatory molecular tests
RT-PCR can be used to test for rearrangements and is useful to define the breakpoints so that it can be used for MRD
qPCR/ flow cyotmotery can be used to test for IGH and TCR rearrangements
Follow up testing in ALL
chromosome analysis is remission is not mandatory but can be useful to look for a secondary abnormality in relapse cases
RT-PCR for recurrent rearrangements
MRD by RT-PCR for the clonal TCR or IGH rearrangement
IGH rearrangements in ALL
B and T cells express antigen receptors on thei cell surface and undergo gene rearrangement to enable them to display receptors for the full range of epitopes.
rearranged genes can be detected bu multiplex PCR. In a normal population of B or T cells there will be a range or rearrangements present. In a malignant population their will be clonal expression of a single receptor rearrangement
what are immunoglobulins composed of?
composed of heavy chaisn (IGH) and light chains either kapp at 2p12 or lambda at 22q11
how are functional immunoglobulins produced
functional immunoglobulins are produced by complex rearrangements of the heavy and light chains followed by class switching and receptor editing. This increases variation so that the full repertoire of epitopes can be identified.
what cell lineage is affected in CLL (chronic lymphocytic leukemia)
CLL is a chronic mature B-cell neoplasm.
mature non-functional B cell accumulate in the BM and blood prevent hematopoeisis and resulting in cytopenia
what is the incidence of CLL?
CLL is the most common leukemia in the western world (2-6 per 100,000)
What ate the symptoms of CLL?
usually asymptomatic and patients are diagnosed by a routine blood test.
if symptoms are present they may include fatigue, hemolytic anemia, splenomegaly, lymphadenopathy and infections
What are the recurrent cytogenetic abnormalities in CLL? and their prognosis
Isolated del 13q - favourable Trisomy 12- intermediate (early clonal driver) del 17p13 (TP53)- poor dell 11q23 (ATM)- poor (implies progressive disease)
