4. Impact of Heterogeneity on Treatment Responses
The overall survival rate for metastatic melanoma patients has significantly improved owing to the development and advancement in targeted therapies and immunotherapies
[103]. However, there is a large proportion of patients who do not benefit from these new treatments due to differential responses resulting from tumour heterogeneity. Resistance to BRAFi treatment is often associated with reactivation of the MAPK pathway through mechanisms such as
RAS mutation,
BRAF amplification and alternative splicing
[104]. For example, a patient developed resistance to BRAFi treatment after subpopulations in the tumour switched from
NRASWT/BRAFV600E to
NRASG13R/BRAFV600E, thereby reactivating the MAPK pathway
[42]. In a rare case of transplacental transmission of metastatic melanoma with
BRAFV600E mutation, the mother was refractory to BRAFi treatment, whereas the baby was responsive to therapy
[105]. Genomic analyses showed that there were at least two related but distinct subpopulations causing resistance, which later led to relapse. Notably, single-cell analysis of a panel of 16 melanoma cell lines and a primary cell line (epidermal melanocyte) found the status of markers, such as MITF, AXL and NGFR, impacted the BRAF/MEKi treatment
[106]. AXL
high cells were more resistant to various treatments in comparison to MITF
high or NGFR
high cells. Resistant AXL
high cells exhibited partial MAPK pathway inhibition with high Ki67 expression, while both MITF
high and NGFR
high cells demonstrated considerable inhibition of the MAPK pathway, suggesting that the MAPK pathway is crucial for AXL
high cells but not MITF
high and NGFR
high cells. Co-treatment of BRAF/MEKi with different epigenetic modulators in the cells revealed selective sensitivities presented by different populations of cells
[106]. Several slow-cycling subpopulations, such as MITF
low/NF-κB
high/AXL
high, are also correlated to reactivation of the MAPK pathway
[75][76]. Importantly, elevated expression of AXL was observed following BRAFi and MEKi (either single or combination) treatments in patient-derived xenografts (PDXs)
[77]. To better understand the mechanisms behind resistance, Shaffer and colleagues compared the expression of resistance markers in pre-resistant cells and BRAFi-treated cells
[50]. Interestingly, the number of resistance markers expressed increased from 72 in the pre-resistant cells to 600 (of the 1456 genes tested) in the post-drug-treatment cells by transcriptome analysis. Only 33 differentially accessible sites were discovered by comparing the non-resistant and pre-resistant cells versus more than 10,000 differentially accessible sites between pre- and post-drug-treatment using ATAC-seq, suggesting minute variations between the non-resistant and pre-resistant cells, and that the treatment induced phenotype switching resistance. Yet, whether the 33 differentially accessible sites between non-resistant and pre-resistant cells are sufficient to provoke a remarkable effect in drug resistance is arguable. In another study, resistance-conferring genetic alterations were evidenced in PDXs established from treatment-naïve patient samples, suggesting the existence of intrinsic resistant populations in melanoma patients
[107]. Nevertheless, acquired resistance was observed in some PDXs too, indicating variabilities in causations of resistance to treatments. Meanwhile, other slow-cycling populations, such as JARID1B
high cells, CD133+, ABCB5+ and NGFR+ cells, have also been implicated in resistance to BRAFi and other anti-cancer drugs
[49][83][85][108][109]. Furthermore, the PIK3CA
E545K mutation was found to confer resistance to combined MEKi + CDK4i in a melanoma patient
[110]. Nonetheless, aberrant expression of methyltransferases in melanomas could affect the sensitivity of melanoma cells to chemotherapy
[111].
In addition, not only did pre-existing NGFR
high melanoma cells show reduced sensitivity to the combination of BRAFi and MEKi but the cells were also resistant to antigen-specific cytotoxic CD8+ T cells
[112]. Consistent with this finding, patients whose tumours are NGFR
high were not responsive to anti-PD-1 and/or a combination of anti-PD-1 and anti-CTLA-4 therapy
[112]. Furthermore, acquired
JAK1 and
JAK2 loss-of-function mutations were found in tumours after patients developed resistance to anti-PD-1 therapy
[113]. The mutations were discovered by comparing the whole-exome sequencing data of tumour samples before treatment and after relapse. The cells with
JAK1 mutations were resistant to interferon α, β and γ-induced growth arrest, while the cells with
JAK2 mutations were insensitive to interferon γ-induced growth arrest. The same study also revealed the mutation of beta-2-microglobulin (B2M) in another patient’s baseline and matched relapse samples, where the loss of B2M reduced the immune-cell recognition of cancer cells. Nonetheless, there are ambiguous responses to immunotherapy due to tumour heterogeneity. For instance, data from a mouse study suggested that tumours with a low level of intratumoural heterogeneity may be more immunogenic and, hence, have an improved overall survival rate
[102]. Lin and colleagues found that tumours with high heterogeneity were associated with decreased immune cell infiltration, immune response activation and overall patient survival
[101]. In contrast, a positive correlation was observed between high mutational load and enhanced T cell infiltration in patient samples
[100], indicating that tumours with a high mutational load may be more immunogenic. Despite the inconsistencies observed, it is possible that the identity of the genes mutated is potentially more pertinent in determining the treatment response regardless of the amount of mutational load
[114].
Altogether, studies have shown that drug treatment could trigger secondary mutations in tumour cells, which lead to resistance. It is also evident that some tumour cells harbour pre-existing resistance, conferring genetic properties that reduce sensitivities to treatments and later lead to treatment resistance. The heterogeneous tumour environment similarly contributes to immunotherapy treatment resistance. The development of immunotherapy has greatly transformed the treatment for advanced melanoma and dramatically increased the overall survival rate for melanoma patients. However, there are still nearly 40% of advanced melanoma patients that do not respond initially to immunotherapies, with adverse side effects observed in 60% of the patients
[115]. The reason why some patients respond well while others do not respond at all is still unclear, although it is clear that heterogeneity at the tumour and immune levels plays an important role.
5. Conclusions and Implications
Heterogeneity within melanoma was initially described by Norris, an attentive general practitioner, in 1820
[116]. Since that time, the researchers have gained significantly more discerning information regarding melanoma development and tumour heterogeneity, particularly at the molecular level. However, many aspects of the development and role of heterogeneity remain unclear. For example, the true identity of each of the subpopulations within the patient tumour remains to be clarified. Further, the specific roles of communication between different subpopulations within the tumour and the various biological components within the tumour microenvironment (ECM and immune cells) are not well characterised. As more subpopulations develop in the tumour, the more challenges patients must face, such as tumour growth, metastasis, efficacy of the therapeutic treatments and, finally, resistance to treatment. These open questions must serve as a reminder that much more needs to be done in order to fill in the missing pieces and truly understand the role of heterogeneity in melanoma metastasis and treatment resistance in order to effectively tackle the disease. Effectively targeting the tumour heterogeneity in melanoma could lead to better therapeutic options and better outcomes for patients.