L3 cancer genetics - detecting mutations

what are driver mutations

Mutations that provide a growth advantage to cancer cells and are selected for during cancer evolution.

• contribute to oncogenesis

• occur in ‘cancer genes’ - selective for hyperproliferation of cells

what are passenger mutations

Mutations that occur in cancer cells (during growth of cancer) but do not contribute to cancer progression.

• not selected for (random)

• no effect on oncogenesis

difference between driver and passenger mutations

• drivers give the cloned cell a selective advantage and contribute to oncogenesis

• passengers have no effect/contribution to oncogenesis

• cancer cells have few drivers, lots of passengers

how do driver mutations increase the chance of more mutations?

• mutated cells have higher growth rate, so divide faster - more cells, higher risk of mutation

• mutations in DNA repair genes destabilise the genome

what is meant by ‘cancer genome’

the genetic makeup of the tumour, and the mutations (germline + somatic) that lead to cancer

cancer genome sequencing

detecting somatic mutations in the tumour to identify genes responsible for initiation, development, and progression of tumour

compare to database of known germline genetic variation of population (take biopsy, compare sequence with non-cancerous sequence) to ensure it is somatic and not a germline mutation

Sanger sequencing

chain-termination by dideoxynucleotide

reads one DNA fragment at a time

1. 4 reactions each terminating at a different base

2. small amount of ddNTP - termination only occurs occasionally

3. results in strands of all lengths

4. strands are separated on a gel

5. sequence read from 5’→3’ from the bottom up

used in human genome project

next gen sequencing

• reads millions of DNA fragments simultaneously - high volume, low cost

• Library Preparation: DNA/RNA is fragmented, and reversible terminators are added to the ends.

• Cluster Generation (Amplification): Fragments bind to a flow cell surface, creating millions of identical copies (clusters).

• Sequencing by Synthesis: Fluorescently labeled nucleotides are added one by one; each addition is captured as an image, revealing the sequence.

• Data Analysis: Computational tools convert light signals into nucleotide sequences, aligning them to a reference genome. 

• can be carried out on whole genome, exomes, or gene panels

illumina sequencing

arrayCGH

human mutation rates

germline mutation rate = ~70 new mutations in each diploid genome

somatic mutations = ~x20

most mutations are neutral (functionally silent)

tumour clonality

cancer cells are clones descending from a common ancestor cell characterised by one or more somatic driver mutations

mutations can be fully clonal - founder mutation found in all cells

or sub clonal - secondary mutations present in a portion of cells

single cell sequencing

whole genome amplification and sequencing of DNA from a single cell to track the initial mutation events that lead to cancer

challenges of single cell sequencing (technical issues)

• isolating rare cells is very difficult - might be missed

• very little DNA that you’re working with

• false negative results - bias resulting in uneven sequencing

• false positive results - single bp errors by polymerase

• allelic dropout - one allele in a heterozygous mutation is not amplified - results in genotype that appears homozygous

circulating tumour cells (CTCs)

Fragments of DNA shed by tumours into the bloodstream, contributing to metastasis. can be present in low-levels in non-metastatic cancers

can be sequenced by SCS

cancer evolution

cancer starts off clonal and then become heterogenous (different clones with different genotypes) depending on selective pressures of their microenvironment

not linear, branched, dynamic, like darwinian evolution

sub clones acquire new genetic and epigenetic changes

risks of chemotherapy

chemotherapy acts as a selective bottleneck - the fittest sub clones survive and dominate the tumour

relapsed malignancies are therefore usually more aggressive and treatment-resistant

challenges of tumour sequenecing

• must determine somatic mutations specific to the cancer - must match it with normal tissue

• limited DNA from biopsies

• biopsies are fixed in formalin and sent to pathologist - can fragment and alter DNA

• tumour may be contaminated with germline DNA

• germline may be contaminated with tumour DNA

• difficult to distinguish between driver and passenger mutations

• different sub clones may have different mutations

• might need to sequence more tumour cells to detect mutations in all subclones

• tumours are ever-evolving

• may not pick up aneuploidy

challenges of genome sequencing

• difficult to interpret mutations of ‘uncertain significance’

• may be mutations in new genes

• we don’t know what ALL genes do - we don’t know what much of non-coding regions do

• possibility of ‘incidental findings’

  • non-paternity

  • mutation for a late onset disease

  • carrier for another disease

• translating genetic findings into clinically useful info

cell-free circulating tumour DNA (ctDNA)

cell-free DNA found in plasma that is released from normal cells + tumours by apoptosis

liquid biopsies

cell-free ctDNA and CTCs obtained from blood samples to provide a non-invasive method for cancer/ relapse detection and treatment monitoring

cause of relapses?

mutations in subclones during evolution of tumour due to selective pressures

temporary effects of therapies are almost always followed by a relapse due to drug-resistance

stresses importance of using combination of therapies