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I&I SC2 > Molecular diagnostics III > Flashcards

Molecular diagnostics III Flashcards

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1
Q

What are the three use cases of immunodiagnostics throughout the disease course of lymphoproliferations?

A
  • Diagnosis -> tumor clone vs. immune response
  • Prognosis -> heterogeneity - different outcomes
  • Monitoring -> evaluation of therapy effect
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2
Q

Which specific code involved in lymphoproliferative disease can be used to diagnose, prognose and monitor therapy effect?

A

Unique DNA-code of the receptors of lymphocytes

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3
Q

How does it come to be that every lymphocyte receptor has an unique receptor? (3)

A
  • VDJ-recombination
  • n-nucleotide addition/deletion
  • p-nucleotide addition/deletion
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4
Q

Where does the diversity in receptors occur?

A

Mainly in junction region = CDR3

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5
Q

In case of lymphoproliferation seen in lymph nodes or blood, lymph nodes can be investigated to determine whether this increase in lymphocytes is due to … (2)

A
  • Physiological immune response
  • Malignant process
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6
Q

Which kind of testing is used to determine the cause of lymphoproliferation?

A

Clonality testing

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7
Q

What are the results of clonality testing of lymphoid tumors/immune responses?

A

Lymphoid tumors: all cells have identical Ig/Tr-genes (monoclonal)
Immune responses: multiple different markers

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8
Q

How does a PCR-based analysis of Ig/TR rearrangement work? (2)

A
  • Filtering out non-lymphoid cells
  • PCR fragment analysis
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9
Q

How do you filter out non-lymphoid cells in a PCR-based analysis of Ig/TR rearrangement?

A

Ig/Tr genes are far apart -> no primers span this distance -> no PCR products

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10
Q

Lymphoproliferations: What does the PCR fragment analysis investigate? How?

A

Length diversity in coupling area -> using fluorescent primers

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11
Q

Lymphoproliferations: How do fluorescent primers determine length diversity in coupling area?

A

Different fluorescent intensities can be used to determine fragment length

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12
Q

Lymphoproliferations: monoclonal/polyclonal situations lead to a normally distributed curve of coupling area lengths (no signal fragment is dominant)

A

Polyclonal

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13
Q

Lymphoproliferations: monoclonal/polyclonal situations lead to a single peak because one fragment is dominant over the others?

A

Monoclonal

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14
Q

Lymphoproliferations: What makes fragment analysis complicated? (2)

A
  • VDJ recombinations -> grouped into 7 families -> multiplex settings needed
  • Somatic hypermutation
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15
Q

Lymphoproliferations: What is multiplexing?

A

Multiple primers at both sides

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16
Q

Lymphoproliferations: Why is it not necessary to use a primer for every different VDJ-combination?

A

7 families -> contain regions in which the DNA sequence is very similar

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17
Q

Lymphoproliferations: What ensures that all fragments will be covered by the assay?

A

Addition of probes for all IGVH-families

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18
Q

Lymphoproliferations: Why do B cells add extra complexity to PCR analyses?

A

Somatic hypermutation -> cause primer misannealing

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19
Q

Lymphoproliferations: How do you compensate for primer misannealing?

A

Extra primers at different locations -> reduce complete non-binding

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20
Q

Lymphoproliferations: How can we make immunodiagnostics on B cells more reliable? This leads to?

A

Investigate both heavy- and light chain –> higher sensitivity

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21
Q

Lymphoproliferations: PCR-based fragment analysis has a lower/higher resolution than NGS-based techniques

A

Lower

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22
Q

Lymphoproliferations: Why do NGS-based techniques allow for higher resolution than PCR-based fragment analysis?

A

Allows for analysis of all sequences

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23
Q

Lymphoproliferations: Which two NGS assays can be used to investigate lymphoproliferations?

A
  • Amplicon PCR amplification
  • DNA-based capture assay
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24
Q

Lymphoproliferations: Describe the amplicon PCR amplification workflow (2)

A
  • Multiplex expansion of IGH-and IGK regions
  • Sequencing expanded products
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25
Amplicon PCR amplification: how can a result suggest clonality?
One peak clearly stands out from the curve
26
Amplicon PCR amplification: If there are a lot of different sequences represented in a curve (even though there is a clear peak that stands out), what does this suggest?
No clonal proliferation
27
Lymphoproliferations: Describe the DNA-based capture assay workflow
Probes pulldown ROI from total genomic information
28
DNA-based capture assay: Probes can target what? (4)
- Rearrangements - Translocations - Copy no. alterations - Mutations (indells)
29
DNA-based capture assay: What is meant with probes being used to target rearrangements?
Simultaneous reporting of clonality at all Ig/TR loci
30
DNA-based capture assay: Probes can/can't be specific for all different V-, D-, and J-genes
Can
31
DNA-based capture assay: What is the advantage of using probes to target translocations?
Detecting previously unknown translocation partners
32
DNA-based capture assay: What does using a probe to target copy number alterations allow for?
Clonality detection in difficult B-cells cases exhibiting somatic hypermutation
33
DNA-based capture assay: Adding mutations as an addition method does not help/help in detecting clonality
Help
34
What is meant with the heterogeneity in lymphoproliferations?
Specific subgroups have different prognosis/require different treatment
35
Lymphoproliferations: which techniques are available for prognosis? (2)
- Molecular immunodiagnostics - Cytogenetics
36
Lymphoproliferations: What is analyzed using molecular immunodiagnostics?
Somatic mutation analysis of rearranged Ig-genes
37
Lymphoproliferations: Which sequencing techniques are used in molecular immunodiagnostics? (2)
- Sanger - NGS-based immune repertoire sequencing
38
Lymphoproliferations: What is analyzed using cytogenetics? (2)
- Chromosome abberations/translocation/deletions - Mutations/SNVs
39
Which physiological mutations are studied in CLL? Which technique?
IGHV-genes -> Sanger
40
Prognosis: Why are mutations in IGHV-genes studied in CLL?
They give information about whether the origin cell has been through SMH or not -> gives big difference in prognosis
41
What is the percentage identical IGHV in U-CLL?
> 98%
42
Lymphoproliferations: Patients with U-CLL have a better/worse prognosis than patients with M-CLL
Worse
43
Lymphoproliferations: Describe the process of sequencing IGHV mutations (3)
- Clonal rearrangement is amplified - V-gene almost fully sequenced - Blast sequence in IMGT database
44
Lymphoproliferations: How is the clonal rearrangement amplified in IGHV mutation sequencing?
Using IGH FR1 or leader multiplex PCR
45
Lymphoproliferations: What is determined in the blast of IGHV mutation sequences? (2)
- V-, D- and J-gene usage - Total no. mutations in sequence compared to reference sequence
46
Lymphoproliferations: when do you consider a IGHV sequence non-mutated?
If more or the same as 98% is similar to the reference sequence
47
Lymphoproliferations: What factor can also impact CLL prognosis (sequencing)?
Amino acid sequence of the junctional region
48
Lymphoproliferations: what is CLL monitoring aimed at?
Evaluation of therapy effect
49
Lymphoproliferations: What is the main monitoring analysis?
Minimal residual disease analysis
50
Minimal residual disease analysis: Which patient data is used for this analysis?
Patient-specific Ig/TCR-rearrangements
51
Minimal residual disease analysis: What do patient-specific Ig/TCR-rearrangements allow for?
High-resolution monitoring
52
Which techniques are used to monitor CLL? (2)
- Real-time quantitative PCR - NGS-based MRD monitoring
53
What is the common principle in both real-time quantitive PCR and NGS-based MRD monitoring?
Clone sequencing upon diagnosis
54
CLL monitoring: What are the steps of the PCR-based process? (3)
- Identification of targets at diagnosis - Testing patient-specific RQ-PCR - Analysis of MRD during follow up using RQ-PCR
55
CLL monitoring: In over 95% of CLL patients, at least two ... markers are found through sequencing
Ig/TR markers
56
CLL monitoring: What does identification of targets at diagnosis allow for...?
Design of a patient-specific primer
57
What region is also called the DNA fingerprint of a leukemic cell?
Junctional region
58
PCR-based process CLL monitoring: On what are sensitivity tests performed?
Dilutions
59
CLL monitoring: Primers are designed and tested in silico for...(3)
- Hairpin formation (internal looping) - Duplicate/dimer formation - Aspecific binding
60
What is MRD?
Minimal residual disease -> remaining leukaemic cells after cancer treatment
61
MRD analysis: how is the standard curve obtained?
Serial dilution of diagnostic sample
62
MRD analysis: what does the yellow line show?
CT-value measured in follow-up samples
63
Monitoring allows for detection of slowing in the decrease of leukaemic cells. What can be done if such a decrease is spotted?
Therapy can be adapted to mitigate this higher risk of relapse
64
CLL monitoring: What are validation normalization reagents used for in NGS-based monitoring techniques?
To trace back the number of reads of a leukaemic sequence to the actual no. of cells
65
CLL monitoring: How can validation normalization be performed?
Central in tube quality/quantification control
66
CLL monitoring: NGS-based MRD detection allows/doesn't allow for deeper sampling than PCR-based techniques
Allows
67
What is the most polymorphic gene locus of the human genome?
HLA
68
Genetic variation in MHC II is mainly in the ... chains?
B-chains
69
HLA: Which class II genes show some variation in the a-chain?
DP and DQ
70
HLA: Which class II gene shows only variation in the B-chain?
DR
71
True or false: polymorphisms contribute to amino acid substitutions in the peptide binding cleft of the HLA molecule
True
72
What does diversity of HLA within the population lead to?
Diversity of adaptive immune response
73
What are reasons to perform HLA-typing? (3)
- Transplant rejection - Predisposition for certain (auto-) immune diseases - Adverse reactions to medication
74
Which HLA-genes need to be matched for a kidney transplant? (3)
- HLA-A - HLA-B - HLA-DRB1
75
True or false: For hematopoietic stem cell transplantation, matching of all HLA genes is required
True
76
Do you need to perform HLA matching for all solid organs (except kidneys)?
No
77
Why is a HLA matching test not performed during solid organ transplant (except kidney)?
No time in the transplantation setting for solid organs
78
HLA can typed through.. (2)
- Serological techniques -> using antibodies - Molecular techniques -> mostly PCR/sequencing
79
What is a problem in serological HLA typing?
No. of HLA alleles is far higher than the amount of serological HLA specificities
80
On what principle is HLA typing mostly based?
Lysis
81
Cells that need to be HLA-typed are incubated with... (2)
- Serum containing known anti-HLA antibodies, OR - Specific monoclonal anti-HLA antibodies
82

I&I SC2 (13 decks)

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