Haematological malignancies Flashcards
1
Q
What is cell culture?
A
Procedure used to grow cells under controlled conditions
2
Q
What is cell harvest?
A
Procedure used to collect cells from the specimen
3
Q
Of FISH and karyotype which is the specific and which is the global test?
A
FISH = specific Karyotype = global
4
Q
Which test has a higher sensitivity FISH or karyotype?
A
FISH
5
Q
Which test can be performed more quickly FISH or karyotype?
A
FISH
6
Q
Do you require metaphase or interphase for karyotype?
A
Metaphase spreads
7
Q
Do you require metaphase or interphase for FISH?
A
Can be performed on both
8
Q
What are the two cell lineages that can arise from haematopoeitic stem cells?
A
Myeloid and lymphoid
9
Q
Examples of cells in the myeloid lineage
A
Anything that’s not T cells, B cells or NK cells e.g. platelets, red blood cells (erythrocytes), granulocytes, basophills etc.
10
Q
Three reasons why we test for haematological malignancies?
A
Diagnosis
Prognosis
Monitoring
11
Q
Prognosis of complex karyotypes?
A
Bad
12
Q
Prognosis of balanced translocation?
A
Good
13
Q
In which haematological malignancies can BCR-ABL1 be found?
A
Most common in CML
ALL
AML
14
Q
What are blast cells?
A
Immature cells in either the myeloid or lymphoid lineage e.g. a myeloid blast can become all the myeloid cells but not lymphoid ones
15
Q
In leukaemias why is bone marrow a better choice of starting material than blood?
A
Because bone marrow will have more immature white blood cells than blood (in normal individuals blood shouldn’t contain immature white cells but in affected individuals it will)
16
Q
What is leukaemia?
A
Cancer of white blood cells where changes occur in the bone marrow
17
Q
Symptoms of leukaemia?
A
Fatigue, easy bruising, proneness to infection
18
Q
What does acute mean?
A
Occurs quickly
19
Q
What does chronic mean?
A
Occurs over a long period of time
20
Q
Different types of leukaemia?
A
AML (acute myeloid leukaemia)
ALL (acute lymphoblastic leukaemia)
CML (chronic myeloid leukaemia)
CLL (chronic lymphocytic leukaemia)
21
Q
What is more aggressive “lymphoblastic” or “lymphocytic”
A
Lymphoblastic because these blast cells are less differentiated and therefore more aggressive
22
Q
What is more aggressive ALL or CLL?
A
ALL
23
Q
What cells are overproduced in AML??
A
Myeloid cells
24
Q
Standard rearrangements in AML
A
t(15;17) PML;RARA
t(8;21) RUNX1T1;RUNX1
inv(16) CBFB-MYH11
All make chimeric fusion proteins
25
t(15;17) is characteristic of what?
APML - acute promyelocytic leukaemia
26
What treatment is APML sensitive to?
Retinoic acid and arsenic
27
Are APML and APL the same thing?
Yes
28
How are rearrangements in AML identified?
Can use karyotype, FISH, RT-PCR or multiplex PCR
29
If you have a suspected APML what analysis method would you use for a rapid analysis?
FISH or RT-PCR - can get result very quickly
30
If you have suspected AMPL should rapid analysis be performed?
Yes
31
If you have a cytogenetically normal AML what test could you use to look for mutations in FLT3 and NPM1?
Multiplex PCR - amplify up and look at size on gene marker. Controls run alongside with known size that these fragments should be
32
Prognosis of t(15;17) PML;RARA in AML
Favourable
33
Prognosis of t(8;21) RUNX1T1;RUNX1 in AML
Favourable
34
Prognosis of inv(16) CBFB-MYH11 in AML
Favourable
35
Prognosis of FLT3 internal tandem duplications in AML
Poor prognosis
36
Prognosis of NPM1 mutations in AML
Favourable
37
What would you use multiple PCR for in AML
To look for mutations in NPM1 and FLT3 if was cytogenetically normal
38
Why is APML worse than AML?
APML tend to have serious bleeding abnormalities due to low platelet count and clotting factors - at increased risk of bleeding in their brain
39
What is myelodysplastic syndrome (MDS)?
Affects the production of myeloid cells, causing cytopenias (reduction in mature blood cell)
40
How does MDS progress?
Can progress slowly but can rapidly convert into AML
41
Loss of Y or 15+ is problematic why?
Often occurs in elderly people with no haematological disease but may also be markers of neoplastic myeloid clones (MDS/AML)
42
What is CML?
Myeloproliferative neoplasm (MPN)
43
What cells are overproduced in CML?
Myeloid cells
44
CML is well managed in what phase?
Chronic phase
45
What are the three phases of CML?
Chronic
Aggressive
Blast
46
What happens when CML switches into "blast crisis"?
It resembles acute leukaemia which is difficult to treat
47
Testing available for CML diagnosis
Karyotype
FISH
RT-PCR
48
Translocation found in almost all cases of CML
t(9;22)(q34;q11) on derivative 22 (Philadelphia chromosome)
49
Does t(9;22) in CML create a chimeric fusion protein?
Yes, BCR-ABL1. This has enhanced tyrosine kinase activity resulting in increased proliferation
50
Drug treatment that can be used in individuals with Ph+ CML
Imatinib (TKI)
51
In terms of monitoring what haematological malignancies can we monitor for?
Can do for all, but in Glasgow only do for CML (two transcripts) all other monitoring is sent down south
52
Does "blast crisis" in CML have a high mortality rate?
Yes
53
When does "blast crisis" occur in CML
When >30% of cells in blood/bone marrow are immature blood cells (blast cells)
54
When is MRD done?
When the patient is in remission
55
When is monitoring done?
At certain time periods after the patient has received a diagnosis and while they are receiving treatment
56
How is monitoring performed for CML?
RT-PCR to determine transcript and then QRT-PCR to determine level
57
Imatinib resistance can be primary or secondary, explain
Primary - failure to achieve a response
| Secondary - acquired resistance
58
What are the three myeloid disease categories?
MDS
MPN
Myeloid leukaemia
59
Examples of myeloproliferative neoplasms
CML
Polycythaemia vera
essential thrombocythaemia
60
Genes mutations generally seen in MPN
JAK2, CALR, MPL
61
What cells are overproduced in ALL
Lymphoid cells
62
What age group is ALL mostly seen in
Young children
63
Is ALL generally a T-cell proliferation or a B-cell
B-cell
64
Translocation seen in 25% of ALL cases
t(12;21) ETV6;RUNX1
65
What is the prognosis for t(12;21) in ALL
Favourable
66
In ALL if children are diagnosed early whats the cure rate
Above 90%, if diagnosed early respond well
67
Is BCR-ABL found in ALL?
Yes, found in adults (11-30%) and in children (2-4%) has a poor prognosis in both
68
Does ALL have a high mortality rate in adults?
Yes
69
Poor prognosis abnormalities in ALL children
```
iAMP21
t(17;19)
MLL translocations
low hypodiploidy
t(9;22)
```
70
Poor prognosis abnormalities in ALL adults
iAMP21
MLL translocations
low hypodiploidy
t(9;22)
71
What cells are overproduced in CLL
Lymphoid cells (generally B)
72
Does CLL affect old or young
Most common in old
73
How does CLL progress?
1/3 regress, 1/3 don't progress and 1/3 progress to acute leukaemia
74
Good prognostic abnormality in CLL
-13q4
75
Intermediate prognostic abnormality in CLL
Trisomy 12
76
Poor prognostic abnormalities in CLL
-17p (or mutation of TP53)
| Deletion of segment of ATM (chr11)
77
What is lymphoma?
Cancer of white blood cells where changes occur in the lymph nodes and other lymphoid tissues (spleen, GALT)
78
Symptoms of lymphoma
Lumps, night sweats, fever
79
If individuals said to have a Ph+ variant translocation in CML what does this mean
They have a translocation involving chromosomes 9;22 and other chromosome(s)
80
What percentage of individuals with CML have a Ph+ variant translocation
5-10%
81
What referrals come from haematology?
Leaukaemias and myelomas
82
What do haematology base their ?diagnosis on?
Patient demographics
Symptoms
Full blood count
What cells look like on blood film
83
Is FLT3 a good marker for monitoring of AML?
No because it can disappear when patient is in remission
84
What are good markers for AML monitoring?
NPM1 and the fusions
85
What leukaemias are classed as urgent referrals?
Diagnostic acute leukaemia and CML
86
What is the time frame for urgent diagnostic acute leukaemia and CML results to go out
14 calendar days - could do rapid test by FISH/PCR in three working days and then karyotype within the 14
87
Routine referrals for the leukaemias turn around time
21 days
88
What is myeloma?
When changes occur in activated plasma cells (B-cells that make antibody)
89
Symptoms of myeloma?
Bone fractures, kidney injury
90
What is the commonest haematological malignancy?
Myeloma
91
Is myeloma curable?
It is treatable but not curable
92
Genetic testing offered for myeloma
FISH
93
Genetic testing offered for lymphoma
FISH
| Sometimes B/T cell clonality
94
Starting material for leukaemia
Preferably bone marrow but can use blood
95
Starting material for lymphoma
FFPE FISH slides
96
Starting material for myeloma
Preferably bone marrow but can use blood
97
Where do referrals for lymphoma come from?
Pathology
98
Where do referrals for myeloma come from?
Haematology
99
Where do referrals for leukaemia come from?
Haematology
100
Poor prognosis karyotypes in myeloma
t(4;14)
-13q
t(11;14)
Deletion of TP53
101
When do we offer chimerism testing?
For leukaemia bone marrow transplantation patients
102
What is a chimera?
A single organism composed of cells with distinct genotypes
103
When are bone marrow transplantation's carried on in individuals with leukaemia
When other treatments have failed
If have very poor prognosis
In kids with ALL
104
What does chimerism testing monitor?
Engraftment
105
What does 100% chimerism mean?
No recipient cells can be found
106
How is chimerism testing performed?
Chemo to get rid of malignant cells, QF-PCR to look at microsatellites from donor and recipient to see if recipient cells have engrafted in donor
107
What can you do if leukaemia returns after transplant?
Give T cells from donor which attack leukaemia cells
108
What is MYC and where is it located?
Proto-oncogene, chromosome 8, role in cell cycle progression and apoptosis
109
What type of haematological malignancy is MYC generally mutated in?
Lymphoma
110
Different types of FISH probes available and what they detect?
```
Enumeration probes (copy number)
Break apart probe (gene rearrangement)
Dual fusion (specific translocations)
```
111
Why do we try and diagnose type of lymphoma someone has?
Different types are treated differently depending on how aggressive they are.
112
Different classes of lymphoma
Hodgkins and Non-Hodgkins lymphoma
113
Difference between Hodgkins and Non-Hodgkins lymphoma
Hodgkins always have Reed Sternberg cells (B lymphocytes), while Non-Hodgkins can be B or T cell
114
Characteristics of Burkitt lymphoma
highly aggressive B cell lymphoma, fast growing and high grade, rare. Most common abnormality t(8;14) MYC;IGH
115
Characteristics of Follicular lymphoma
2nd most common NHL, B cell, most common abnormality t(14;18) IGH;BCL2. Sometimes IGH;BCL6
116
What is the most common type of NHL
Diffuse large B cell (aggressive), no specific abnormality associated
117
Most common BCR-ABL1 transcripts
e13a2 and e14a2 (MAJOR transcripts) produce 210kDa protein and e1a2 (MINOR transcript) produce 190kDa protein
118
Most common BCR-ABL1 transcript in CML
Ones that produce 210kDa protein
119
Most common BCR-ABL1 transcript in ALL
One that prodcues 190kDa protein
120
What mitogen can be used to stimulate B cells
PMA
121
What mitogen can be used to stimulate T cells
PHA
122
Do clinicians prefer ISCN or words
They prefer words describing how many chromosomes youve got
123
What does hypodiploid mean?
35-46 chromosomes
124
What does hyperdiploid mean?
47-57 chromosomes
125
What does pseudodiploid mean?
46 chromosomes (abnormal)
126
What does near triploid mean?
Number close to 69 (3n)
127
In ISCN what is // used for
To represent a chimera - separate cells from host and donor
128
In ISCN what is [*] used for
To detail the number of cells in that cell line
129
In ISCN are normal cells presented at beginning or end?
End
130
Three different types of karyotype
Normal
Abnormal
Complex
131
Categories of acquired genetic disease
Formation of chimeric fusion protein
Gain or amplification of gene or gene product
Deletion/loss of function of a gene
132
If you see a +8 what do you think it is
A myeloid disorder (MDS or AML)
133
What is the tumour suppressor affected in retinoblastoma
RB1
134
Deletion of chromosome 5 is common in what type of disease?
MDS
135
What is the percentage for no response in monitoring?
96-100%
136
What is the percentage for minimal response in monitoring?
66-95%
137
What is the percentage for minor response in monitoring?
36-65%
138
What is the percentage for partial response in monitoring?
1-35%
139
What is the percentage for complete response in monitoring?
o%
140
For CML once found abnormality monitor using what kind of test
QRT-PCR (but they will send cytogenetic sample at intervals because you can get other abnormalities cropping up in Ph- cells)
141
Name some abnormalities in the poor prognosis risk group for AML
-5
-7
del(5q)
complex abnormality
142
In AML what is an abnormal karyotype defined as?
4 abnormalities or more
143
Is high hyperdiploidy a good or bad prognosis in ALL
Good
144
What is iAMP21
>5 copies of RUNX1 = amp = iAMP21
145
Does pyrosequencing look at short or long fragments?
Short
146
If you have a sex mismatch donor and recipient for a bone marrow transplant what other testing could you do other than QF-PCR for microsatellite analysis
FISH for X/Y
147
Screening for those with BRCA1/2 mutation
Mamography or MRI
148
Screening for those with mutations in MMR genes/APC/MUTYH
Colonoscopy
149
Factors that increase risk of breast/ovarian cancer
```
Increasing age
Higher body mass index
Late menopause
Pill
Breast density
```
150
Ethical issues of testing someone for breast cancer
Issues regarding health insurance, employment
Confidentiality - will have to ask about other family members etc
If do have a mutation, concerns regarding child bearing issues
151
Down syndrome is a risk factor for what haematological malignancy?
Acute leukaemia
152
Environmental risk factors of cancer?
Radiation
Chemotherapy and radiotherapy
Epstein Barr Virus - Burkitt's lymphoma
153
What is a major cytogenetic response in CML?
More than 65% Ph-ve
154
What is a complete cytogenetic response in CML?
100% Ph-ve
155
What FISH tests do we offer for myeloma?
TP53
IGH-FGFR3
IGH-MAF
CDKN2C
156
Do they use interphase or metaphase FISH for haematological specimens?
Both interphase and metaphase
157
Do specimens require culturing to get metaphases?
Yes
158
Do specimens require culturing to get interphases?
No, can get interphases from a straight harvest
159
For say ?BCR-ABL/PML-RARA samples what kind of FISH would be done on these fast turn around samples?
Interphase FISH because would get a straight harvest, no culturing
160
For haematological malignancies when would you use interphase FISH
If you were looking for a standard rearrangement for which probes were available/fast turn around
161
For haematological malignancies when would you use metaphase FISH
If you saw something unusual (say 3 way t) on a karyotype and you wanted to know location of it/needed help in working out breakpoints would do metaphase FISH
162
If you saw a marker chromosome what would you do?
If experienced staff have an idea what it is FISH for that chromosome. If hard to tell consider standard rearrangements for that diagnosis or ?. If have no idea just report as "marker1/2/3" in ISCN
163
Prognosis of unbalanced translocation
Depends on the level of imbalance
164
Why is ISCN important?
As an international standard it enables cytogeneticists across the world to understand the exact nature of the chromosome constitution in an individual
165
Sibling donors for bone marrow transplant - what considerations need to be taken in analysis of engraftment (QF-PCR of markers)
May struggle to get enough informative markers because siblings share 50% of genes
166
What % of donor cells need to be present for bone marrow transplant to have worked?
90%
167
If you see an abnormality on karyotype of an individual with ?leukaemia/myeloma, what do you need to consider about this abnormality
Is it clonal? Can you see it in almost every abnormal cell?
168
In which myeloproliferative neoplasm do you find JAK2 variants? And are these acquired or inherited?
Polycythemia vera, and they are acquired
169
Most common JAK2 variants in PV?
c.1849G>T, p.(Val617Phe) 90-95% cases
| Variants in JAK2 exon 12 2-4% cases
170
How is JAK2 analysis performed?
Allele specific PCR for c.1849G>T variant and exon 12 deletion, followed by GeneMarker for analysis. For the deletion you get an extra peak 3 bp smaller than the WT and for the variant will get a peak in the "positive" bin
171
Are there sanger sequencing best practice guidelines available?
Yes
172
What is "improving outcomes guidance"?
Guidelines put in place by NICE, looks at haematological cancers and how we can use things like integrated reporting, MDTs, staffing levels and facilities to improve care for these patients
173
What does NICE stand for?
National Institute for Health and Care Excellence
174
What does NICE do?
Provides national guidance and advice to improve health and social care
