Week 2B - minimal residual disease

Question 1

what does early detection of residual cells allow

Answer 1

clinical intervention with the aim of reversing the proliferation of resistant leukaemic cells

Question 2

what is minimal residual disease

Answer 2

the name given to small numbers of leukaemic cells that remain in the patient during treatment

Question 3

what is the sensitivity of MRD assays

Answer 3

> 1 x 10^4

i.e. detection of 1 malignant cell in 10,000 normal ones

Question 4

what can chromosomal translocation result in

Answer 4

leukaemia

Question 5

what does chromosomal translocation generate

Answer 5

fusion gene coding for an oncoprotein

Question 6

what does chromosomal translocation target

Answer 6

junctional breakpoints

Question 7

what does RT-PCR detect

Answer 7

RNA from viable cells - target genes expressed that are likely to have a functional role in cellular prolifertaion

Question 8

what is BCR-ABL1

Answer 8

the product of the chromosome translocation associated of Chronic Myeloid Leukaemia

Question 9

what do BCR-ABL1 RNA transcripts remain

Answer 9

detectable after disease cells can no longer be seen down the microscope - complete cytogenetic remission - CCyR

Question 10

what is the only tool capable of monitoring responses after patients reach CCyR

Answer 10

RT-qPCR

Question 11

how are low levels of minimal residual diseased achieved

Answer 11

during therapy call for a sensitive monitoring assay for reliable detection and quantitation

Question 12

what does the amount of specific mRNA reflect

Answer 12

the amount of protein being expressed

Question 13

what is the methodology for BCR-ABL-1 transcript quantification

Answer 13

1. EDTA peripheral blood
2. mRNA extraction
3. reverse transcription (cDNA synthesis)
4. TaqMan real time quantitative PCR (qPCR) for ABL and BCR-ABL1

Question 14

how can RNA -> DNA be amplified

Answer 14

polymerae chainreaction

Question 15

how does PCR work

Answer 15

1. RNA is extracted from patients blood and converted to double stranded cDNA
2. cDNA can be used in conventional PCR
3. Many copies of gene-specific template made – giving great sensitivity

Question 16

how does TaqMan real time qPCR work

Answer 16

1. probe binds to target cDNA of interest
2. quencher close to dye, stopping flourescnece
3. generation of complementary strand releases dye from its quencher
4. amount of fluorescence is proportional to amount of template

Question 17

how can amount of cDNA be measured

Answer 17

through titration against a standard curve

Question 18

what is ABL gene fragment used for

Answer 18

a house keeping gene

so we can assess the amount and quality of RNA derived from the sample

Question 19

what does the BCR-ABL1 fusion gene provide

Answer 19

a unique biomarker for diagnosis and monitoring response to treatment

Question 20

how can continued molecular monitoring be used

Answer 20

• to assess whether a patient with CML is making progress toward a desired response to therapy.
• Ensure that a patient’s treatment is optimized over time

Question 21

what do B and T-clonal recombinations in leukaemia generate

Answer 21

patient-specific DNA length and sequences - potential molecular markers for detection and quantification of leukemic cells among normal lymphocytes in remission samples

Question 22

what do primers do in Allele Specific Oligonucleotide PCR (ASO-PCR)

Answer 22

anneal to a unique patient specific Ig sequence.

Question 23

is B and T cell rearrangements specific

Answer 23

yes - 1x10^-4 and -5

fully quantitative

Question 24

what is high levels of MRD at end of induction therapy associated with

Answer 24

high risk of relapse

Question 25

what is considered the most widely applicable and sensitive methodology for MRD

Answer 25

qPCR using allele specific oligonucleotide PCR

Question 26

how is MRD results categorised

Answer 26

positive negative intermediate

Question 27

what is MRD positive

Answer 27

MRD detectable ≥ 1 × 10-4 with either of 2 ASO primers (level of disease corrected to control gene will be reported)

Question 28

what is MRD negative

Answer 28

no MRD detectable by 2 markers, one with a quantitative range of 10-4.

Question 29

what is MRD intermediate

Answer 29

MRD detectable but <10-4.
– MRD not detected but sensitivity of assay is low (<10-3).
– MRD not detected by one available marker

Question 30

what happens in single nucleotide polymorphism (SNP)

Answer 30

we count PCR amplicons in a binary fashion, so can be used a quantitatively

Question 31

what is the droplet system

Answer 31

routinely count 20,000 droplets, giving high enough sensitivity for MRD analysis

Question 32

what do STR markers track

Answer 32

low level
disease in haematological stem cell transplant patients

Question 33

what is next generation sequencing’s potential in MRD

Answer 33

Over 10 years it has become cost effective to use NGS in NHS Laboratory services.

It offers a chance to get much more out of every pathological samples.

Question 34

what does the NGS methodology detect

Answer 34

variants at very low prevalence – the ‘variant allele frequency’(VAF) - so can be very sensitive

Question 35

how is targeted NGS panels good

Answer 35

because several different key genes can be effectively covered for MRD

Question 36

what else can occur if targeted NGS is performed

Answer 36

identification of new clones

treatment can be modified

clinicians can be one step ahead of cancer