Mutational signatures are characteristic combinations of mutation types arising from specific mutagenesis processes such as DNA replication infidelity, exogenous and endogenous genotoxin exposures, defective DNA repair pathways, and DNA enzymatic editing.
The term is used for two distinct concepts, often conflated: mutagen signatures and tumor signatures. Its original use, mutagen signature, referred to a pattern of mutations made in the laboratory by a known mutagen and not made by other mutagens – unique to the mutagen as a human signature is unique to the signer. Uniqueness allows the mutagen to be deduced from a cell's mutations Later, the phrase referred to a pattern of mutations characteristic of a tumor type, although usually not unique to the tumor type nor to a mutagen. If a tumor mutational signature matches a unique mutagen mutational signature, it is valid to deduce the carcinogen exposure or mutagenesis process that occurred in the patient's distant past. Increasingly refined tumor signatures are becoming assignable to mutagen signatures.
Deciphering mutational signatures in cancer provides insight into the biological mechanisms involved in carcinogenesis and normal somatic mutagenesis. Mutational signatures have shown their applicability in cancer treatment and cancer prevention. Advances in the fields of oncogenomics have enabled the development and use of molecularly targeted therapy, but such therapies historically focused on inhibition of oncogenic drivers (e.g. EGFR gain-of-function mutation and EGFR inhibitor treatment in colorectal cancer). More recently, mutational signatures profiling has proven successful in guiding oncological management and use of targeted therapies (e.g. immunotherapy in mismatch repair deficient of diverse cancer types, platinum and PARP inhibitor to exploit synthetic lethality in homologous recombination deficient breast cancer).
General concepts
Mechanisms – overview
DNA replication infidelity DNA proofreading is the process by which DNA polymerase excises an incorrectly incorporated nucleotide via exonuclease enzymatic reaction. Inability of DNA polymerase to correct these replication errors leads to progressive accumulation of mutations through successive cell mitosis. Genotoxins Endogenous cellular (e.g. spontaneous 5-methylcytosine deamination leads to C>T transition (genetics)) mutations (see DNA damage (naturally occurring)) Exogenous/carcinogens Ultraviolet radiation: UVB radiation causes direct DNA damage and is a known risk factor for skin cancer (e.g. melanoma) Alkylating antineoplastic agents: This group of chemotherapy agents adds alkyl group to DNA, which causes crosslinking of DNA and interferes with DNA replication and DNA repair. Cancer cells are most impacted because of their high mitosis rate. Tobacco: Tobacco contains several carcinogens which are harmful to DNA, including polycyclic aromatic hydrocarbons, acrolein, nitrosamines, cyanide and others (see health effects of tobacco) DNA repair deficiency Homologous recombination deficiency (HRD): DNA double-strand break requires homologous recombination mechanism for accurate repair of breakpoints. DNA mismatch repair (MMR) deficiency: The mismatch repair machinery recognizes and repairs erroneous base pair insertion, deletion or mis-incorporation. Enzymatic DNA editing Cytidine deaminase enzymes: This family of enzymes are part of the innate immune system and are involved in the control of retroviruses and transposons elements (including endogenous retroviruses). These enzymes (cytidine deaminase/CDA, activation-induced cytidine deaminase and APOBEC protein family) actively cause cytidine deamination and therefore introduce C>T transition (genetics) mutations.
Genomic data
Cancer mutational signatures analyses require genomic data from cancer genome sequencing with paired-normal DNA sequencing in order to create the tumor mutation catalog (mutation types and counts) of a specific tumor. Different types of mutations (e.g. single nucleotide variants, indels, structural variants) can be used individually or in combination to model mutational signatures in cancer.
Types of mutations: base substitutions
There are six classes of base substitution: C>A, C>G, C>T, T>A, T>C, T>G. The G>T substitution is considered equivalent to the C>A substitution because it is not possible to differentiate on which DNA strand (forward or reverse) the substitution initially occurred. Both the C>A and G>T substitutions are therefore counted as part of the "C>A" class. For the same reason the G>C, G>A, A>T, A>G and A>C mutations are counted as part of the "C>G", "C>T", "T>A", "T>C" and "T>G" classes respectively.
Taking the information from the 5' and 3' adjacent bases (also called flanking base pairs or trinucleotide context) lead to 96 possible mutation types (e.g. A[C>A]A, A[C>A]T, etc.). The mutation catalog of a tumor is created by categorizing each single nucleotide variant (SNV) (synonyms: base-pair substitution or substitution point mutation) in one of the 96 mutation types and counting the total number of substitutions for each of these 96 mutation types (see figure).
Tumor mutation catalog
Once the mutation catalog (e.g. counts for each of the 96 mutation types) of a tumor is obtained, there are two approaches to decipher the contributions of different mutational signatures to tumor genomic landscape:
The mutation catalog of the tumor is compared to a reference mutation catalogue, or mutational signatures reference dataset, such as the 21 Signatures of Mutational Processes in Human Cancer from the Catalogue of Somatic Mutation In Cancer (COSMIC) database. De novo mutational signatures modelling can be accomplished using statistical methods such as non-negative matrix factorization to identify potential novel mutational processes.
Identifying the contributions of diverse mutational signatures to carcinogenesis provides insight into tumor biology and can offer opportunities for targeted therapy.
Types of mutations: indels
Signature 3, seen in homologous recombination (HR) deficient tumour, is associated with increased burden of large indels (up to 50 nucleotides) with overlapping microhomology at the breakpoints. In such tumors, DNA double-strand breaks are repaired by the imprecise repair mechanisms of non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) instead of high fidelity HR repair.
Signature 6, seen in tumors with microsatellite instability, also features enrichment of 1bp indels in nucleotide repeat regions.
Types of mutations: structural variants
Homologous recombination deficiency leads to Signature 3 substitution pattern, but also to increase burden of structural variants. In the absence of homologous recombination, non-homologous end joining leads to large structural variants such as chromosomal translocations, chromosomal inversions and copy number variants.
Mutational signatures
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Age-related mutagenesis
Signature 1 features a predominance of C>T transition (genetics) in the Np[C>T]G trinucleotide contexts and correlates with the age of patient at time of cancer diagnosis. The underlying proposed biological mechanism is the spontaneous deamination of 5-methylcytosine.
Signature 5 has a predominance of T>C substitutions in the ApTpN trinucleotide context with transcriptional strand bias.
Homologous recombination deficiency
Signature 3 displays high mutation counts of multiple mutation classes and is associated with germline and somatic (biology) BRCA1 and BRCA2 mutations in several cancer types (e.g. breast, pancreatic, ovarian, prostate). This signature results from DNA double-strand break repair deficiency (or homologous recombination deficiency). Signature 3 is associated with high burden of indels with microhomology at the breakpoints.
APOBEC enzymes
APOBEC3 family of cytidine deaminase enzymes respond to viral infections by editing viral genome, but the enzymatic activity of APOBEC3A and APOBEC3B has also been found to cause unwanted host genome editing and may even participate to oncogenesis in human papillomavirus-related cancers.
Signature 2 and Signature 13 are enriched for C>T and C>G substitutions and are thought to arise from cytidine deaminase activity of the AID/APOBEC enzymes family.
A germline deletion polymorphism involving APOBEC3A and APOBEC3B is associated with high burden of Signature 2 and Signature 13 mutations. This polymorphism is considered to be of moderate penetrance (two-fold above background risk) for breast cancer risk. The exact roles and mechanisms underlying APOBEC-mediated genome editing are not yet fully delineated, but activation-induced cytidine deaminase(AID)/APOBEC complex is thought to be involved in host immune response to viral infections and lipid metabolism.
Both Signature 2 and Signature 13 feature cytosine to uracil substitutions due to cytidine deaminases. Signature 2 has a higher proportion of C[T>C]N substitutions and Signature 13 a higher proportion of T[C>G]N substitutions. APOBEC3A and APOBEC3B-mediated mutagenesis preferentially involve the lagging DNA strand during replication.
Mismatch repair deficiency
Four COSMIC mutational signatures have been associated with DNA mismatch repair deficiency and found in tumors with microsatellite instability: Signature 6, 15, 20 and 26. Loss of function MLH1, MSH2, MSH6 or PMS2 genes cause defective DNA mismatch repair.
DNA proofreading
Signature 10 has a transcriptional bias and is enriched for C>A substitutions in the TpCpT context as well as T>G substitutions in the TpTpTp context. Signature 10 is associated with altered function of DNA polymerase epsilon, which result in deficient DNA proofreading activity. Both germline and somatic POLE (gene) exonuclease domain mutations are associated with Signature 10.
Base excision repair
Somatic enrichment for transversion mutations (G:C>T:A) has been associated with base excision repair (BER) deficiency and linked to defective MUTYH, a DNA glycosylase, in colorectal cancer. Direct DNA oxidation damage leads to the creation of 8-Oxoguanine, which if remains un-repaired, will lead to incorporation of adenine instead of cytosine during DNA replication. MUTYH encodes the adenine glycosylase enzyme which excise the mismatched adenine from 8-Oxoguanine:adenine base pairing, therefore enabling DNA repair mechanisms involving OGG1 (Oxoguanine glycosylase) and NUDT1 (Nudix hydrolase 1, also known as MTH1, MutT homolog 1) to remove the damaged 8-Oxoguanine.
Exposures to exogenous genotoxins
Selected exogenous genotoxins/carcinogens and their mutagen-induced DNA damage and repair mechanisms have been linked to specific molecular signatures.
Ultraviolet radiation (UV)