Tumour Immunology

Question 1

What are the two key concepts in tumour immunology?

Answer 1

Immunosurveillance and Immunoediting

Immunosurveillance - recognition of tumour neoantigens on the surface of tumour cells by the immune system.

Immuniediting occurs in three phases
Phase 1- Elimination phase where tumour cells are neutralised by natural killer (NK), CD4+ and CD8+ cells.
Phase 2 - Equilibrium between immune cells and tumour cells.
Phase 3 - Escape phase where the immune system is unable to destroy the tumour cell - corresponding to the appearance of a clinically detectable tumour

Question 2

What is tumour mutational burden (TMB)?

Answer 2

TMB refers to the number of somatic gene mutations present in a tumour. It is defined as the number of mutations per megabase of DNA analysed. This varies across tumour types

Question 3

What tumour features can impact TMB?

Answer 3

• Variations across different cancer types in levels of TMB
• Tumour heterogeneity
• Primary/Metastatic - Primary tumours have higher heterogeneity compared to metastatic (higher TMB)

Question 4

How can TMB influence the effect of the immune system in recognising tumour cells?

Answer 4

Cancers with low TMB have fewer mutations and therefore decreases the chance that one will activate the immune system

Cancer cells with high TMB have more mutations, increasing the chance that one will activate the immune system

Question 5

What treatment type has TMB been considered as a proposed biomarker for and how do they work?

Answer 5

Immune Checkpoint Inhibitors (ICIs) such as CTLA-4 inhibitors, PD1 inhibitors and PDL1 inhibitors.

One mechanism that tumours use to survive is to increase immune checkpoint molecules that can help to suppress antitumor immune responses. ICIs reinvigorate antitumor immune responses by interrupting co-inhibitory signaling pathways and promote immune-mediated elimination of tumor cells.

Question 6

Why is TMB a marker for ICI response?

Answer 6

Tumours with high TMB had a higher number of neoantigens and therefore can identify patients who would in turn benefit from ICI therapy.

Question 7

What are the high/low thresholds or TMB and what response is seen in patients treated with ICIs?

Answer 7

TMB-High - >20mutations/Mb, response 58%

TMB-Low - <20mutations/Mb, response 20%

Question 8

What methods can be used to detect TMB?

Answer 8

Calculation of TMB is generally carried out using WGS, WES or panel-based approachess. Research has favoured WGS/WES whereas clinical applications have more often utilised panels due to a lower sequencing cost, lower DNA inpur requirement and a shorter turnaround time

Question 9

What are the primary factors which influence how TMB is calculated?

Answer 9

1) Tumour cell content & sequencing coverage
Quality of TMB data is dependant on greater tumour content and sequencing coverage. Targeted panels allow for sequencing to a greater depth than WGS/WES enabling a higher sensitivity when the tumour content is low (<10%) and can detect variants down to lower frequencies.

2) Pre analytical/processing factors
Tissue is normally fixed in formaldehyde (FFPE) which can lead to significant noise in NGS and impact TMB calculations

3) Sequencing strategy and size of assay
As the size of a panel becomes smaller. the uncertainty associated with TMB becomes greater with the coefficient of variation rapidly increasing when the panel size is less than 1Mb

4) Bioinformatics pipelines
Pipeline filtering can differ between analysis methodologies. Most will filter out synonymous and germline variants as they are unlikely to be involved in creating neoantigens however this is not standardised. Lack of matched tumour-germline analysis in standard clinical practice means germline variants cannot be filtered out. Choice of variant caller (and its sensitivity) can impact how TMB calculated

5)TMB thresholds
Not yet standardised. Different studies have utilised different cut offs for high TMB