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)
what IGH rearrangements are found in CLL?
~20% of cases have a rearrangement involving the IGH gene at 14q32
often associated with a more complex karyotype and poor prognosis
What are the common molecular findings in CLL?
IG gene is often rearraranged (50-60% shoe somatic hypermutation
TP53 and ATM may be mutated as well as deleted and have a poor prognosis
What is the diagnostic strategy in CLL
need to confirm that the pateint has CLL and not another disease that may look like CLL e.g. hairy cell leukemia or mantle cell lymphoma
- blood counts
- blood smear
- immunophenotype
cytogenetic testing in CLL?
cytogenetic testing is not required for diagnosis but can be useful for monitoring
Cells have a low division rate so can be hard to karyotype (even with the use of a mitogen e.g. PHA) therefore iFISH can be used.
lower abn rate by G-banding so not required. Most lab do iFISH for ATM and TP53 and may include T12 and del(13)(q14)
what are the treatments for CLL?
CLL is incurable
pateints are only treated at the onset of symptoms and relapse
treatment is a combination of chemotherapy and immunotherapy
what is myeloma?
a neoplasm of plasma B cells
terminally differentiated b cell which secrete a specific monoclonal ig
what is the presentation of Myeloma?
C- hyperCalcaemia
R- renal failure
A- anemia
B- bone disease
what are the recurrent genetic abnormalities found in myeloma?
Hyperdiploid (48-78 chromosome and favourable prognosis
non-hyperdiploid unfavorable prognosis typically includes an IGH (14q13) rearrangement - t(4;14) poor -t(11;14) good -t(14;16) poor
del13q- associated with t(4;14) poor prognosis
del (17)(p13) TP53- poor prognosis and late event associated with aggressive disease
loss of 1p or gain of 1q- poor prognosis
Cytogenetic diagnosis in Myeloma
iFISH
ELN recommend testing for t(4;14), t(14;16), del 17p13, 1p/1q
Like CLL there is a low proliferative rate so karyotype may fail to detect the abnormality
what is MDS?
a heterogeneous group of disorders characterised by an over expansion of myeliod cells in the BM
hematopoeisis and dysplasia or 1 or more myeloid lineages is the hall mark of MDS
there is a high risk of transformation to AML
what distinguishes MDS from AML?
in MDS <20% blast cells and in AML there are >20%
what are the symptoms of MDS?
anemia, thrombocytopenia, neutropenia
What is the incidence of MDS?
incidence increases with age
average age of diagnosis is 70yrs
there is a male predominance
What are the causes of MDS?
de novo, smoking, viral, Trisomy 21, Fanconi anemia, exposure to benzene, exposure to cytotoxic chemotherapy e.g. alkylating agents.
what is the treatment for MDS?
chemotherpay, transfusion and BM stimulation
limited role for allogenic HSCT but is the only curative therapy
what is the prognostic scoring system used for MDS?
IPSS-R
What cytogenetic abnormalities are associated with a very good prognosis in MDS?
-Y (may also be age related. presence in >75% of cells indicates it is a marker of clonal disease)
dell (11q)
What cytogenetic abnormalities are associated with a good prognosis in MDS?
normal karyotype
del(5q)- low risk of progression to AML
del (12p)- loss of ETV6
del(20q) common in myeloid malignancies and low risk of transformation to AML
2 abnormalities including del(5q)
What cytogenetic abnormalities are associated with a intermediate prognosis in MDS?
del(7q)
+8
+9
i(17)(q10)- loss of TP53
What cytogenetic abnormalities are associated with a poor prognosis in MDS?
-7
inv(3), del(3q), t(3q)
complex 3 abn
What cytogenetic abnormalities are associated with a very poor prognosis in MDS?
complex >3abn
high risk of transformation to AML
What molecular mutations in MDS are associated with a poor prognosis?
ETV6 TP53 RUNX1 E2H2 ASXL1
hypermethylation is also associated with a poor prognosis but is not currently tested for or treated with hypomethylating agents
How many patients will transform from MDS to AML?
approx 1/3 of patients due to the gain of a driver mutation resulting in proliferation of a sub population which cannot mature and differentiate normally.
What are the diagnostic strategies in MDS?
chromosome analysis (not FISH) is gold standard for identifying the IPSS-R prognostic groups
SNP arrays can detect UPD (present in 20%) but cannot distinguish different clones
NGS can be used to screen for variants in recurrently mutated genes and inform on diagnosis and prognosis
differential diagnosis can be difficult and need to distinguish MDS from reactive forms of cytopenia or dysplasia
Treatment options for MDS
FDA has approved 3 drugs azacitidine- hypomethylating drug lenalidomide decitabine all used for ,lower risk groups
for high risk groups allo HSCT is the only cure
what is lymphoma?
malignancy of lymphocytes (b and T cells) which accumulate in the lymph nodes which from tumours due to inhibition of apoptosis and uncontrolled cell growth
5th most common cancer in the UK
what organs are affected by lymphoma
malignancy of B and T cells in lymph nodes. cells may also spill over into non lymphoid tissue (usually spleen)
What is are the hallmaarks of Hodgkins lymphoma?
Presence of reed sternberg cells is a hallmark
Epstein barr virus is present in >50% of cases but its role in pathogenesis is unclear
What are the symptoms of HL?
painless enlargement of superficial lymph nodes in neck, fever, weight loss and sweating
good prognosis with >80% 5 yr survival
what is are the genetics and immunophenotype of HL?
IGH (14q32) rearrangements are common bu no other recurrent abn
CD45-, CD20-, CD19+, CD30+
Describe non-hodgkins lymphoma
clonal lymphoid tumours
heterogeneous disease affecting B-cells, T-cells and NK cells.
cytogenetic abnormalities can be used to inform of diagnosis and prognosis e.g. (14;18) IGH-BCL2 in follicular lymphoma and t(11;14) cyclin D-IGN in mantle cell lymphoma
what distinguished hodgkins and non-hodgkins lymphoma?
Hodgkins lymphoma is characterised by the presence of reed sternberg cells. if no present it is a non-hodgkins lymphoma
What is the recurrent genetic rearrangement in Burkitts lymphoma?
t(8;14)(q24;q32)
constitutive activation of MYC by juxtaposition of IGH regulatory region
What is the treatment for Burkitts lymphoma?
high intensity treatment targettin proliferating cells
cyclophosphamide, methotrexate, etoposide
very aggressive but can be cured with correct treatment and commonly associated with immunodeficiency e.g. AIDs
What is the recurrent genetic rearrangement in follicular lymphoma?
t(14;18)(q32;q21)
BCL2 over expression due to juxtaposition to IGH
genrally indolent but may progress to hihgh grade lymphoma
What is the treatment for Burkitts lymphoma?
Rituximab- chimeric monoclonal Ab against CD20 surface Ag on B-cells
(disease originates in B-cell germinal centre)
What is the recurrent genetic rearrangement in Mantle cell lymphoma?
t(11;14)(q13;q32)
Juxtaposition of IGH and CCND1= CCND1 over expression and accelerated passsge through G1 so cell divide before they are mature
most aggressive small cell lmphoma
Use of karyotype in diagnosis of solid tumours e.g. lymphoma
recommended where fresh tissue is available
Genome wide
can detect all rearrangement partners and different clones
some rearrangements may require FISH to distinguish
culturing is relatively labor intensive
Use of FISH in diagnosis of solid tumours e.g. lymphoma
Does not require dividing cells so can be used on FFPE tissue
can detect cryptic rearrangements
targeted so cannot detect novel rearrangement partners- unless breakapart probes are used but characterization of the abn present will require further testing
Rapid, robust, cheap and can easily score a hig number of cells
Use of Array CGH in diagnosis of solid tumours e.g. lymphoma
does not require dividing cells
genome wide- non targeted
can only detect unbalanced rearrangements
Use of SNP array in diagnosis of solid tumours e.g. lymphoma
can detect LOH and UPD
need to test germline and tumour DNA to distinguish acquired and constitutional abn
cannot detect balanced rearrangements
Use of PCR in diagnosis of solid tumours e.g. lymphoma
used for clonality analysis of B and T cell receptors
can detect some recurrent rearangements but is complicated by the diversity of breakpoints in lymphoma
qRT-PCR is not widely used in lymphoma as only a minority of rearrangements produce novel fusion proteins
what is rhabdomyosarcoma?
Sarcoma of the connective tissue (skeletal muscle progenitors)
most common pediatric soft tissue sarcoma
2 types- alveolar and embryonal
what is embryonal rhabdomyosarcoma?
70-80% of RMS in children (manily <5yrs)
no recurrent chromosomal rearrangements
associated with allelic loss of imprinted region at 11p15.5- associated with BWS results in being iodisimic for pat 11p and loss of mat 11p = over expression of IGF2
What are the advantages and disadvantages of performing G-banded chromosome analysis in a soft tissue sarcoma?
Advantages:
- whole genome scan and non-targeted so can identify novel and recurrent fusions
- can distinguish different clones
- culturing can be faster than prepping FFPE
Disadvantages:
- requires fresh tissue (may not always be available)
- tissue degrades quickly which means necrotic tissue may be present which can compromise growth
- poor quality preps may mean the not all fusions can be differentiated
- labour intensive
- risk of cultural artefact- need to confirm clonality of an abnormality
What are the advantages and disadvantages of performing FISH analysis in a soft tissue sarcoma?
Advantages:
- FISH can be performed on FFPE tissue so no risk of degradation and can test archived samples, + easily sent to different labs, can be tested retrospectively if new targeted drugs become available
- low level of tumour cells can be detected against a high background of normal with careful dissection and a histopathologist clearly marking the regions of tumour on the slide
- rapid results in a few hours
Disadvantages:
- requires accurately marked H&E stained slide to ensure that tumour tissue is analysed
-cell morphology and layering can affect analysis (need to screen more cells than G-banding due to risk of artefactual staining or results due to overlaid cells)
rapid result
-targeted and only specific rearrangements can be detected
-break apart probes can detect all rearrangements affecting one gene but not the partner
what are the issues with FFPE samples?
Formalin crosslinks DNA and proteins which prevent degradation of the sample by blocking the action of proteinases, DNAses and RNAses but this also affects the quality of DNA for downstream processing. The longer a sample is in formalin the greater the extent of the crosslinking.
what are the advantages and disadvantages of using RT-PCR for chromosome analysis in soft tissue samples?
can detect the fusion transcripts of recurrent rearrangements- fusion needs to be expressed and need to know breakpoints.
extract RNA–> RT-PCR to cDNA-> PCR with primers targeted to the fusion gene of interest.
Advantages:
- small amount of tissue required
- RNA can be extracted form FFPE but is better quality from fresh frozen tissue. 100K project has included investigating the feasibility of fresh frozen rumour tissue for genetic analysis in a routine diagnostic pathway
- can identify variant fusion partners (but only if suspected)
Disadvantages:
- low level of tumour in a sample against a high background of normal can make to fusion hard to detect
- RNA is less stable than DNA and degrades quicker which can give false results.
what are CNS tumours?
CNS tumours include tumours of the brain and spinal cord/. Gliomas are the most common (70%)
- account for 25% of childhood cancers and are less common in adults
what is the difference between high and low grade tumours?
not truly distinguished by being malignant or not as a significant amount of mortality is due to the growth of the tumour and its affecting on the surrounding e.g. brain
Grade 1 & 2 - low grade, slow growing, relatively contained and unlikely to metastasise
Grade 3 & 4- high grade, aggressive, ‘malignant’- usually require chemotherapy or radiotherapy
What medulloblastoma?
Pediatric primary malignant brain tumour. Invasive embryonal tumour of the cerebellum that frequently metastasizes.
30% dies in 2 years and remaining suffer long term side effects due to treatment
What are the molecular and cytogenetic changes in medulloblastoma?
i(`17)(q10) in ~ 50% loss of T53
+7
amplification MYC and MYCN in tumors with double minutes (poor outcome)
- tumours with wingless and SHH activation have a favorable activation
what familial genetic conditions are associated with an increased risk of brain tumours?
NF1- neurofibromatoses
- NF1 associated with gliomas and astrocytomas
- NF2 associated with vestibular schwanomas
TSC1/TSC2= tubersous sclerosis = increased risk of benign tumours in brain and elsewhere
TP53: li Fraumeni + increased risk of multiple early onset tumours in brain, beast, soft tissue
VHL: von hippel lindau syndrome
- hemangioblastomas of the brain or spinal cord
45,X
increased risk of childhood brain tumours especially meniglioma.
what are malignant round cell tumours?
give examples
Small, round , relatively undifferentiated cells which arise from the primitive embryonic cells
- common type of pediatric tumour
examples: Ewing sarcoma Wilms Tumours neuroblastoma Rhadbomyosarcoma medulloblastoma etc
due to the primitive undifferentiated nature of the cells diagnosis required IHC, immunophenotyping, RT-PCR and FISH
What is Ewing sarcoma
bone tumour
Ewing sarcoma or PNET (primitive neuroectodermal tumour) is the 2nd most common cancer in children (after rhabdomyosarcoma)
- tumours arise in the bone and primitive neuroectodermal tissue
- aggressive and 15%have already metastasised at the time of diagnosis
What are the characteristic genetic changes in EWS?
t(11;22)(q24;q12) is present in 785% and results in a fusions between EWS gene on 22q with Fli1 on 11q. fusion gene acts as a transcription factor and results in dysregulation of key oncogenic genes.
- also may have a variant fusion between EWS-ERG (also a transcription factor)
diagnosis by FISH of FFPE tissue, G-banding of fresh tumour, RT-PCR
What is Wilms Tumour?
Malignancy of the kidney and most common rnal cancer in children
what are the genetics associated with Wilms tumour?
- WT1 germline variants (zn finger transcription facotr involved in normal kidney development)
- 11p15.5 epigenetic chages- BWS (pat UPD15 rsults in increased expression of paternally derived growth promotogin gene IGF2 and loss of maternally derived growth suppressor genes)
- contiguous deletion including WT1 and PAX6 on 11p is associated with WAGR syndrome (Wilms tumour, aniridia, genital anomalies and MR)
- germline muts in WT1 exon 8 & 9 is associated wuith Deny-Drash = early onset renal failure, intersex disorders, and high risk of developing Wilms tumour
Diagnosed by variant and dosage analysis of WT1, microarray, methylation studies of 11p15.5
What is retinoblastoma
- Malignancy of the embryonic neural retina due to mutations of RB1 gene at 13q14 (TSG involved in cell cycle progression)
- Most common in <5yrs but can occur at any age
can be bilateral (usually familial) or unilateral (usually sporadic) - First TSG discovered and investigations into RB led to the development of Knudsons (2 hit) hypothesis
- AD inheritance and >90% penetrance, LOH of WT allele found in 60% of tumours
- familial RB also associated with increased risk of other tumours
- germline carriers are screened from 3 yrs
- characteristic finding is white iris
- in sporadic cases there is later onset and no risk of other tumours
How is RB diagnosed?
- sequencing to identify germline mutations. NGS can detect mosaic mutations
- detection of second hit requires a tumour sample which is not always available
- MLPA to detect large whole exon or whole gene deletions
- array can be useful if larger contiguous deletion is suspected (other syndromic features also present)
- LOH can be detected by comparative genotyping of polymorphic loci flanking the RB1 gene in germline sample compared to tumour
What is the treatment for RB?
- chemotherapy and radiotherapy
with eearly diagnosis 99% of patients with intraoccular RB survive and 90% keep vision in at least 1 eye
what is neuroblastoma?
3rd most common solid tissue malignancy of infancy
- though to arise from neurla crest cell, commonly originates in the adrenal glad and can metastasise to other tissues.
mainly sporadic ~1% familial and associated with ALK activating mutations and PHOX2B
what are the main genomic profiles in NB
- Near triploidy - 55%
associated with a good prognosis and spontaneous regression in infants - di/tetraploid
poor prognosis
structural rearrangements also often present
- MYCN amp (2p24.1) common - associated with severe disease but not thought ot cause and often found with 17q gain or del 1p36
- 11q23 also associated with poor prognosis and is found in cases without MYCN gain
What genetic testing is performed for NB?
international NB risk group recommends testing ploidy, MYCN and 11p23 for prognosis
iFISH for 1p36, MYCN, 17q, 11p23 (will also inform on ploidy)
Karyotype
MLPA
Treatment for NB
depends on risk group. Options are chemotherapy, radiotherapy or myeloablative chemo followed by autologous stem cell transplant
what is EWS?
Ewing sarcoma is the most common bone tumour. tumours may also develop in soft tissue
histologically undifferentiated round cells expressing CD22
what is the characteristic rearangement in EWS?
85% have t(11;22)(q24;q12.2)- FLI1-EWSR1
c-terminal DNA binding domain of FLI1 and N-terminal transcriptional activation domain of EWSR1= oncogenesis through misregulation of transcription
15% have t(21;22) EWS-ERG fusion
What is the mechanism of tumourigenesis in EWS?
the EWSR1-FLI1 (and variant fusions) target the transcriptional activation domain to a wide range of genes resulting in aberrant transcription
what are the challenges of genetic analysis in solid tissue samples
- access- biposy may not always be possibel e.g. lung cancer
- heterogeneous and mutation staus can differ between patients with the same tumour or within an individual
tumour is present against a background of normal cells so need to enrich for tumour - macrodissection or marked H&E stained slides - FFPE is the most commonly available tissue but formalin fixation crosslinks DNA and RNA affecting the quality.
What are the technical and logistical challenges of FFPE?
- variable amount and quality of DNA- degradation of DNA is correlated to the amount of time the tissue is in formalin
-decalcification of bone tumour samples makes the DNA unsuitable for analysis as it is highly fragmented - PCR artefacts are common due to deamintation of cytosine during fixations- this can result in false positive results
- only a small amount of tumour may be present in a section reducing the chance of detecting low level mutations
processing is labour intensive and so can be challenging to turn around results in a clinically useful TAT - EGFR testing in lung cancer needs to be reported in 7 to 14 days to direct treatment options
the sensitivity of mutation detection is dependent on the technique used.
what are the alternatives to FFPE?
100K project cancer stream looked at using alternatives to FFPE for tumour processing as it is not ideal for downstream genetic testing.
implimented fresh frozen tissue into the testing pathway as a proof of principle and to determine the benefit and requirement of processing tissue in this way.
implementation of new technique needs collaboration between clinicians, histopathologists and diagnostic labs.
Give examples of |FISH testing of solid tumours to for diagnosis and treatment stratification
ALK/ROS1 to test for rearangements in NSCLC to identify patients suitable for TKI therapy
HER2 amplification in breast cancer for herceptin treatment
MDM2 amplification for diagnosis of liposarcoma
EWSR1 rearrangement to diagnose ewings sarcoma
What are the methods of mutation analysis in solid tissues?
clinically significant point mutations are detected by RT-PCR, ddPCR or NGS (all have high sensitivity and can detect low level mutations.
sanger seq is avoided and it cannot reliably detect low level mutations and is sensitive to contamination
Give examples of clinically useful genetic tests for treatment of solid tumours
CRC- test for RAS - cetuximab, pantimumab
malignant melanoma- BRAF V600E - verurafenib, drabafenib, tramafenib
NSCLC- EGFR- gertifinib, erlotinib, afatinib
Describe the use of NGS in genetic testing of solid tumours
- increasingly being applied to FFPE tissue
- currently targeted panels containing clinically relevant genes are primarily used
- majority of the information provided by WES/WGS cannot be used clinically.
- need to enrich for DNA of interest. methods includeL pull-down, long range PCR, multiplex amplicon seq
- commercial kits and panel are available e.g sureSeq Solid tumour panel (OGT) and TruSeq amplicon (illumina)
- NGS in research informs on molecular characterization if tumours and leads to prognostic information and the development of new therapies.
What are the advantages of NGS in solid tumours?
- can detect frequent and rare mutations as well as (theoretically) detect CNVs, indels and structural variation to give the most comprehensive mutation profiles of cancer genomes. this is likely to be the future of testing
- with high coverage the sensitivity is also high and can detect low level mutations
what are the disadvantages if NGS in solid tumours?
- NGS on FFPE tissue is still under development due to the poor quality of DNA obtained
coverage and allele frequency is problematic in cases with low tumour content of poor sample preservation - high cost for slow TAT (needs to be quick for diagnosis and treatment decisions)
Describe the use of arrayCGH in solid tumour testing
- cannot detect balanced rearrangements
- can detect CNVs at a high resolution than karyotype
- cannot distinguish clones
Describe the use of SNA arrays in solid tumour testing
- can detect LOH and UPD as well as CNVs and gene amp
- acquired somatic UPD/LOH can be a common second hit in oncogenesis
- need to compare normal and tumour tissue to identify acquired and germline mutations
- there is uneven SNP density across the genome resulting in uneven probe coverage
- there are various commercial kits for haem malignancies and solid tumours e.g, OGT cytosure haematological cancer and SNP and illumina cancer SNP panel
How are gene expression arrays used in solid tumours
can detect qualitative transcript expression by testing RNA or cDNA to give a gene expression profile of tumour and can be compared to WT tissue.
can also detect alternate splicing patters in tumour and expression of non-coding RNAs e.g. miRNAs
miRANs are small and lees likely to degrade making them a useful biomarker
Issue is that RNA protocols from FFPE is not optimised so ideally need fresh frozen tissue
cost is high for routine diagnostics
How are methylation arrays used in solid tumours
e.g. illumina 500K epi array
targets 550 methylation sites across the genome and serial sampling can give real time view of methylation changes during tumour development
what are the advantages and disadvantages of testing FFPE samples
PROS
presence of tumour is known and can be staiend for, marked on slides to enusre it is tested
easily stored at room temp and last for a long time
CONS
poor quality DNA
requires specialist histopathologist for micro dissection and staining
requires and invasive biopsy procedure and may not always be possible
tumour sampled from a single time point
what are the advantages and disadvantages of using circulating tumour cells?
PROS
extracted from PB so no need for invasive biopsy
can be taken several times to enable study of the tumour evolution
CONS
short half life
uncertain how much tumour DNA is present in the sample
low level of ctDNA = risk of false -ve
describe the use of ddPCR in solid tumour samples
- very high sensitivity ?10-3
- can be used for ctDNA
- low error rate and fast to analyse without the need for - complex bioinformatics like NGS
- can ID SNVs, CNVs, and SVs
- can only detect the targeted mutation so wil not detect novel variants
what are the benefits of MRD monitoring?
- determine the effectiveness of treatment and does determination
- relapse stratification after induction to allow optimal treatment
- detect the risk of impending relapse for early treatment
- spare toxicity, ADR and expense in patients that are not benefiting from the treatment.
FISH for MRD
- can be used for cases where a cytogenetic abnormality was detected at diagnosis
- low sensitivity
- breakapart and fusion probes have lower sensitivity than single copy number probes for gains and losses
Quantitative RT-PCR for MRD
detect expression of the mutant transcript- e.g. fusion protein by pCR of cDNA. need to determine rearrangement breakpoints to be used for MRD
PCR amplification is measured during the exponential phase to enable relative quantification against a housekeeping gene (usually ABL1)
e.g. BCR-ABL1- to monitro molecular response in CML. CMR when >4 log reduction (undetectable)
Extensive optimization and standardization of MRD monitoring by QF-PCR has been achieved in the EuroMD consortium
Quantitative PCR for MRD
less popular than RT-PCR as measures DNA so not expression of abnormal gene product and as genes will still have introns the products can be larger reducing the PCR efficiency.
How is FLT3-ITD measured for MRD?
preferential amp of the shorter WT transcript than the longer mutated transcript reduces the sensitivity of standard PCR.
Tandem dup PCR is used- uses a pair of primer but they are orientated in the opposite direction to standard PCR, therefore only allowing amp when the dup is present.
How is immunological profile used for MRD?
leukemic cells express cell surface proteins which can be detected using fluorescently labelled Abs or flow cytometry
a specific immunophenotype can be present at diagnosis but relapsed disease may not present with the same immunophenotype due to evolution
QF-PCR for MRD (chimerism)
QF-PCR can be used to detect highly polymorphic markers for chimerism monitoring.
How are BM transplants monitored?
This is to measure the success of the engraftment and detect impending relapse by the re-emergence of recipient cells.
low sensitivity so not a true MRD method
sex mismatched BMT= X.Y FISH, amelopgenin SRY PCR
sex matched= PCR using microsatellite markers.
NGS technologies for MRD monitoring
may offer and MRD method whihc is independent of th leukemic clone that was present at diagnosis and can cope with clonal evolution or the emergence of new clones
recent reports suggest that it is more sepcific for relapse prediction than MRD by qRT-PCR
standards for NGS based MRD monitoring in the future must be further defined in the future including but not limited to:
- at what time points during treatment NGS should be performed
- which targets should be included in the NGS panel
Describe MRD monitoring in ALL
- most adult ALL cases do not have a specific cytogenetic abnormalitly.
- MRD is primarily by flow cytometry- widely applicable, fast and reliable
in T-cell ALL flow cytometry identifies the immunophenotypic features of immature T-cells
in B-cell ALL leukemic cells are differentiated by the presence of aberrantly expressed cell surface markers. - TCR rearrangements are present in approx 90% of T-cell ALL and are unique to each ALL so require identification at diagnosis
- can test for gene fusions if they are present at diagnosis e.g BRC-ABL1, ETV^-RUNX1, MLL-AFF1. Not stanardised for MRD (can use karyotype, FISH or RT-PCR)
MRD monitoring in AML?
cytogenetic abnormalities detected at presentation can diagnose AML and stratify patients into favourbale, intermediate and unfavourable prognostic groups.
- fusion gene monitoring e.g. PML-RARA can be carried out by qRT-PCR
the British MRC15 trial showed that a 3 log reduction in t(8;21) after completion of therapy was associated with a much lower risk of relapse than patient who do not achieve this level - FLT3-ITD monitoring by tandem PCR
_WT1 over expression (overexpressed in 90% of AML in PB and BM at diagnosis) ELN study found that the magnitude of the WT1 log reduction is an independent predictor of relapse
flow cytometry- >90% have an immunophenotype suitable for monitoring at diagnosis
What is MPN?
myeloproliferative neoplasm (from myeloid lineage) The high number of mature cells (erythrocytes, granulocytes, platelets) in the BM as normal proliferation and maturation is maintained differentiates MPN form MDS
10-19% balst in the BM signifies accelerated disease and >201% is sufficient to diagnose transformation to AML
what are the symptoms of MPN?
splenomegaly and hepatomegaly are common
Give 3 examples of MPNs
poycythemia vera
Primary myelofibrosis
Essential thrombocythaemia
describe Polcythemia Vera
MPN charactersed by increased RBC production
nearly all pateints have a somatic GOF mutation in JAK2 e.g V617F
results in a proliferation of the erythroid lineage, garnulocytes and megakaryocytes
median survival is 10 yrs
most patients die from thrombosis
~20% progress to AML
Describe essential thrombocythaemia
MPN
primarily involves the megakaryocytic lineage and results in sustained thrombosis in BM
50% are asymptomatic and are discovered due to a high platelet count at a routine blood test
50% have JAK2 V617F, 5-10% have a ctyogenetic abnormality (+8, abn 9q, del 20q)
Describe primary myelofibrosis
MPN
proliferation of megakaryocytes and garnulocytes in BM resulting in the deposition of fribrous connective tissue and extra medullary hematopoesis
Role of JAK2 in MPN
JAK2 encodes a non-receptor tyrosine kinase which is involved in the JAK/STAT signalling pathway and is a signal transducer for the MAPK and PI3K pathways
mutated in >90% of PV and 50% of ET and MF. Diagnostic of MPN but its absence does not exclude a diagnosis
p. Val617Phe (V617F) is a GOF mutation which releases autoinhibition and results in constitutive activation
- can be used for MRD
- can see LOH for 9p
- familial predisposition to MPN- specific haplotypes preferentially acquire V671F
Role of MPL in MPN
MPL gene encodes thrombopoeitin which regulates differentiation of megakaryocytes and platelets
constructively activates the JAK/STAT, PI3K and MAPK pathways
may be found with JAK2 mutations
3-4% of ET and 4-8& of MF (not seen in PV)
Role of CALR in MPN
CALR plays a role in transcriptional regulation
mutations occur in exon 9n and 80% are 52bpdel of 5bp insertion
60-80% of ET and MF cases negative for JAK2 and MPL mutations
How is classic MPN diagnosed?
cytogenetics is not required for diagnosis but can be useful to exclude alt diagnose e.g CML, AML, MDA
no specific abnormality confrims liklihood of transformation to AML but abn of 5q, 7 and 17p are common
cases with unexplained eosiniphilia should be screened for PDGFRa rearangements
MPN pathways should always include testing for JAK2 V617F in ET, MP and PV
- it is associated with a high risk of thrombosis and thombotic effects in ET and MF
What treatments are available for MPN
JAK2 inhibitor ruxolitinib is FDA and NICE approved for treatment of myelofibrosis
What is a carcinoma?
cancer of epithelial cells
what is an adenocarcinoma?
carcinoma with glandular origin
Describe VHL is respect to renal cancer?
VHL gene mutations are the most connon genetic alterations in renal cell carcinoma
associated with AD LOF mutations
clinical features: increased risk of clear cell carcinoma
phaeochromocytoma
hamngioblastoma
renal cysts
What other genes are associated with an increased risk of Renal cell carcinoma
- VHL LOF mutations
- MET GOF mutations
- FH mutations (part of Krebs cycyle)
- TSC1/TSC2 LOF mutations
- SDHB,C & D - mutations in genes that form the succinate dehydrogenase complex are associated with hereditary paraganglioma/phaeochormocytoma = bening tumours in the paraganglia
(only develop type 1 or 2 if the mutation is paternally inherited) - chromosome 3 translocations- multiple cancer genes are located on 3p including VHL, PBRM1, BAP1 and SETD2
(all AD)
