20.06.24 Techniques in tumour mutation testing Flashcards
1
Q
How do most cancers arise
A
Clonal expansion of a transformed cell through accumulation of serial somatic mutations (clonal evolution)
2
Q
Reasons for genetic analysis of tumours
A
- Diagnosis and classification
- Prognosis
- Prediction of response to therapeutic agents, therefore guiding treatment.
3
Q
Challenges of analysis of solid tumours
A
- Can be very heterogenous, with mutation status between patients with the same tumour and within an individual
- Tumour tissue is present on a background of normal tissue and % tumour present can be variable, therefore tumour enrichment required (macrodissection).
- Tumour tissue not always available (e.g. lung cancer diagnosis often based on cytology, leaving little material for molecular profiling)
- FFPE tissue is most commonly available material for testing but quality and quantity is variable. Promotes degradation of DNA and RNA
4
Q
Review of testing FFPE tissue
A
- Most common source of material available for testing
- Amount an quality of DNA varies (depends on length of time in formalin before processing, methods of proccessing- decalcification of bone samples)
- DNA extracted is fragmented, assays need to be designed with this in mind
- PCR artefacts are more abundant due to deamination of cytosine residue during formalin fixation. Can result in false positives
- Less likely to detect clinically relevant mutations if low % tumour
- Ability to detect low level mutations is dependent on technology used
- Tumour heterogenity can mean mutations may be present in only part of the tumour therefore the test result will depend on the section tested.
- Insufficient DNA or quality may mean that only a partial result or failed testing is possible
- Processing and testing is time and labour intensive and challenging to turn around in a clinically useful time frame.
5
Q
Fresh tissue testing
A
- 100k genome cancer, implemented fresh frozen tissue testing into the testing pathway. also investigated use of PAXgene media and shaken biopsy.
- Requires close working between clinical teams and histopathology.
6
Q
Cytogenetic testing of solid tumour samples
A
- FISH can detect clinically relevant rearrangement and copy number changes.
- Advantages= analysis of specific cell types/ separate clones, cost, speed and relatively small amount of material needed.
7
Q
Examples of FISH testing in solid cancer
A
- ALK, ROS1 rearrangement in NSCLC. To check for suitability for TKI therapy.
- HER2 amplification in Breast cancer. For suitability to Herceptin treatment
- MDM2 amplification to diagnose liposarcoma.
- EWSR1 rearrangement for diagnosis of Ewing sarcoma.
8
Q
Review of mutation analysis
A
- Sanger seq is rarely used as does not reliably detect low allele frequencies and is sensitive to contamination and poor DNA quality.
- qPCR and digital PCR are robust and sensitive.
- NGS panels. Can test multiple hot spot/genes in one assay.
- RT-PCR for fusion gene detection
9
Q
Review of NGS in solid tumour testing
A
- Large numbers of genetic mutations are known to contribute to tumorigenesis with each mutation playing a certain role (major or minor) in cancer initiation and progression.
- Provide comprehensive mutation profiles of cancer genomes, including various somatic mutation types
- WGS/WES on FFPE not yet in routine clinical practice as not reliable enough.
- Predominantly targeted panels for clinically actionable mutations.
- Bioinformatics is challenging
- germline testing may be required.
- Coverage and allele frequency is problematic in cases with low tumour content or poor sample preservation.
- Cost and TAT still too high
10
Q
Future of diagnostic tumour testing
A
NGS analysis that can test for both SNVs, CNVs and large structural rearranagements.
11
Q
Review of array CGH
A
- Whole genome investigation of tumour, to detect CNVs at a higher resolution than karyotyping
- Can be designed to have concentrate probes in regions of interest
- Limitations: can’t detect balanced rearrangements, different clones can’t be distinguished, technical challenges and data interpretation difficulties.
12
Q
Review of SNP array CGH
A
- Can detect CNVs, unbalanced translocations as well as UPD/LOH
- Acquired somatic UPD may lead to duplication of an activating somatic mutation or homozygosity for a disease probe minor allele present in the germline.
- UPD can result in increased/decreased gene expression due to the duplication of a particular methylation pattern
- Tumour paired germline samples needed to identify somatic events.
- Disadvantages: uneven distribution of SNPs throughout the genome resulting in variable coverage and resolution.
13
Q
Review of expression arrays or gene expression profiling (GEP)
A
- Expression arrays assay RNAs (or cDNAs) to obtain quantitative measure of corresponding transcripts. Results produce “gene signatures”
- Tumour samples are analysed against other samples or standards whose classification is known, under the same conditions.
- Can be used to identify prognostic or diagnostic groups.
- Gives information on alternative splicing in tumours
- Non coding transcriptome (non coding RNAs with functional relevance). Gives prognostic info and helps classify cancers. Due to their small size, less amenable to degradation in plasma and therefore useful biomarkers for liquid biopsies.
- Disadvantages of GEPs: FFPE RNA protocols are not optimised, high cost for routine clinical testing.
14
Q
Review of methylation arrays
A
- Create an epigenetic profile by looking at many methylation sites across genome. Compared to reference sample data.
- Bisulphite conversion prior to bead capture with sequence specific probes.
- Useful for CNS tumours, where diagnosis is difficult using histology, limited targeted tests or test results are conflicting.
- Also generates a copy number profile so can validate clinically relevant findings.
15
Q
Other techniques used in tumour testing
A
- Microsatellite instability: detected using a fluorescent PCR-based assay. MSI-H colorectal cancers may have less robust response to 5-FU therapy.
- Promoter hypermethylation (e.g. MLH1 and MGMT). Uses Bisulphite conversion and pyrosequencing. MLH1= CRC, MGMT= glioma. Methylation specific MLPA assays using methylation specific restriction enzyme Hha1.
