*5.3. Tumour Mutational Burden as Biomarkers*

In different clinical studies, a high tumour mutation burden (TMB) has been associated with better response rates and improved survival of patients treated with immunotherapies such as ICI. Therefore, TMB is beginning to be used as a biomarker of response to these immunotherapy agents [318].

TMB is the total number of somatic mutations in a defined region of a tumour genome and varies by tumour type and among patients [319–321].

The mutational load of a tumour contributes to its immunogenicity. Tumours that have high TMB, such as melanoma and lung cancers, are thought to be more likely to express neoantigens and induce a stronger immune response after treatment with ICI [322]. Highly mutated tumours ("hot" tumours) have a histological immune signature of depleted immunosuppressive cells and high expression of immune inhibitory molecules. Less mutated tumours ("cold" tumours) have amplified immunosuppressive cells, negative regulation of MHC molecules, and low expression of immune inhibitory molecules. The adaptive immune response is very accurate in predicting patient outcome [294], so it is important to identify whether the presence of effector T cells in MSD is related to antigen-specific T cells [152,323]. For some tumours, this parameter may be a suitable clinical biomarker for making immunotherapy treatment decisions [324,325]. STM is a quantifiable measure of the number of mutations in a tumour, which is an advantage over neoantigens, since not all mutations result in immunogenic neoantigens and it is difficult to determine which mutations may induce these neoantigens, thus new techniques and strategies are required to discover new TAAs as biomarkers [156,320].

Neo-antigens are currently more easily identified by complete exome sequencing. The sequencing of new generation tumours allows the identification of mutations and, using computer algorithms, the identification of mutated peptides that bind to MHC molecules, which helps in the choice of targets to improve the response of T cells [294].

Antigenic peptides are the result of abnormal transcription, translation of alternative open reading frames or post-translational modifications. This diversity of peptides also involves the mechanism of peptide splicing by the proteasome [326]. A variety of human leukocyte antigens are involved in the processing of antigenic peptides [156]. O-glycosylation of cancer-associated aberrant proteins can modify antigenic processing and the immune response [327], and phosphopeptides associated with MHC class I are targets of memory immunity. Phosphopeptide-specific immunity has an important role in tumour recognition and control [294,328].

TMB was first determined using complete exome sequencing, but this method is expensive and has a long response time, so panel-specific sequencing is now used. The implementation of TMB implies a solid clinical and analytical validation. In addition, bioinformatic analysis is also important for its successful implementation in the clinic, since the measurement of BTM is based on new generation sequencing (NGS) techniques [329].

For ICI therapy, expression of PD-L1 correlates with an increased response to therapy and may be a predictor [330]. In contrast, not all patients who express PD-L1 respond well to ICI treatment [331]. Therefore, other MSD factors, such as LIL, also play an important role [332].

In the study by Yu-Pei Chen et al., it was proposed to classify the different types of tumour microenvironments according to the expression of PD-L1 and the presence or absence of LILs, in order to design appropriate combination immunotherapies for cancer [322,333,334]. This study attempted to establish a classification model based on analysis of mRNA expression of PD-L1 and CD8A, and evaluated the applicability of this classification to predict response to ICI treatment, i.e., its correlation with mutation load and number of neoantigens, using RNA-seq [322].

PD-L1 positive MSDs with LILs were generally associated with high BMT or number of neoantigens in multiple tumours, so these cancers would benefit from anti-PD-1/PD-L1 therapies, as these tumours have evidence of pre-existing intra-tumoral T cells being inactivated by compromised PD-L1 [322,334]. In contrast, MSDs that have low expression of PD-L1 and little infiltration of LILs will have a worse prognosis, as no immune reaction is detected. Combination therapy to attract LILs to MSDs, along with ICI, may be a good option in these cases [335]. For MSDs with high expression of PD-L1 but low infiltration of LILs, radiotherapy- mediated immunogenic cell death to release antigens and induce T-cell responses, together with ani-PD-1/PD-L1 inhibitors, may also be a beneficial approach [322,336].

In recent years, studies evaluating TMB as a predictive marker have increased significantly for the response to ICI, demonstrating the importance of this approach in selecting patients to benefit from immunotherapy.
