Molecular Characterization of Advanced-Stage Melanomas in Clinical Practice Using a Laboratory-Developed Next-Generation Sequencing Panel

Cutaneous melanoma is one of the most lethal tumors among skin cancers, characterized by complex genetic and molecular alterations that result in uncontrolled cell proliferation and metastatic spread. Next-generation sequencing (NGS) enables the simultaneous examination of numerous genes, making this molecular technique essential for melanoma diagnosis, prognostic stratification, and therapy planning. Herein, we present the experience with our laboratory-designed NGS panel for the routine assessment of advanced-stage melanoma. A total of 260 specimens of advanced-stage melanomas were evaluated utilizing a laboratory-developed multi-gene NGS panel, which allowed the investigation of 229 amplicons in 25 oncogene/oncosuppressor genes. The NGS panel proved to be a reliable tool, failing to produce results in only 1.2% of the samples tested. BRAF and TERT were the two more commonly altered genes in 44.0% and 59.9% of samples, respectively. In 59.3% of the mutated cases, at least two concomitant variants were detected. In eight cases, both primary lesion and metastatic disease were analyzed by NGS. In all specimens (8/8, 100%), a perfect concordance in variants harbored by the primary and recurrence lesions was observed. Finally, this study described the validity of a laboratory-developed multi-gene NGS panel built specifically for advanced-stage melanomas in ordinary clinical practice.


Introduction
Cutaneous melanoma is one of the most lethal tumors among skin cancers, and its incidence is rising worldwide.It is caused by a complex interplay of genetic and epigenetic alterations that drive its initiation, development, and metastasis.These changes frequently impair essential signaling networks that regulate cell growth, proliferation, differentiation, and survival.The vast majority of melanomas are sporadic, and one of the more common molecular alterations is the mutation of the BRAF gene.
BRAF.One of the most well-known genetic alterations in melanoma is the mutation of the BRAF gene, specifically the p.V600E mutation, which occurs in around 50% of cases.BRAF is a serine/threonine kinase protein involved in the MAP kinase pathway, which controls cell growth and proliferation.The most common variants are BRAF class I mutations (mainly BRAF p.V600E, followed by BRAF p.V600K), which are almost exclusively induced by the oncogenic/pathogenetic key role of UV radiation [1].However, other non-V600 BRAF variants may be found in advanced-stage melanoma [1][2][3].These variations in the BRAF gene may cause constitutive protein activation, which results in uncontrolled cell proliferation and tumor formation [3,4].
TERT.Telomerase reverse transcriptase (TERT) mutations are key players in melanoma genesis and progression.TERT activity is typically carefully regulated, but changes can disrupt this control, resulting in unregulated cell proliferation and cancer development.Recurrent mutations in the promoter of the TERT gene were initially detected in melanoma and then in various additional cancer types [5][6][7].TERT promoter mutations are the most common type of TERT alterations in melanoma, accounting for up to 65% of cutaneous melanoma [1,8].These mutations increase TERT expression, which helps cancer cells grow and survive.Mutations in the promoter region of TERT are related to reduced disease-free survival, increased tumor recurrence, and an increased rate of metastasis in melanoma [8][9][10][11].
NRAS.Mutations in the NRAS gene are found in about 15-20% of melanomas [1,12].Hot-spot mutations in the RAS genes are generally at the Q61 codon and less frequently in G12 or G13 [1].Mutations in RAS genes cause the constitutive activation of the MAPK pathway, resulting in increased cellular proliferation, survival, and resistance to apoptosis [13].Inhibiting proteins farther down the RAS pathway, such as MEK and ERK, can indirectly prevent the carcinogenic signals triggered by RAS mutations [13].
Other common genetic alterations observed in melanoma are the loss of function of the CDKN2A tumor suppressor gene, TP53 mutations/deletions (thus resulting in the loss of heterozygosis) but with a lower frequency and a lower magnitude effect compared to other solid tumors, and KIT mutations mainly detected in acral and mucosal melanoma [1,12,[14][15][16].
Next-generation sequencing (NGS) enables the simultaneous sequencing of numerous genes with a very high depth of coverage.Given the ongoing discovery of molecules as potential targets or molecules that are accountable for treatment resistance mechanisms, using a classic single-gene approach is becoming challenging.The adoption of multi-gene panels is now essential for the molecular investigation of solid tumors, including melanoma.According to the ESMO guidelines, "If the tumour is BRAF wild-type (WT) at the V600 locus (class I BRAF mutant), sequencing the loci of the other known minor BRAF mutations (class II and class III BRAF mutant) to confirm WT status and testing for NRAS and c-kit mutations are recommended [II, C] [. ..].Alternatively, a clinically validated next-generation sequencing panel covering all key oncogenic drivers is increasingly being carried out" [17,18].
The present study aims to disclose a laboratory-designed multi-gene panel that allows for assessing advanced-stage melanoma in routine clinical practice.

Materials and Methods
A total of 260 cases of advanced-stage melanomas were analyzed for routine practice at the Molecular Pathology Laboratory of IRCCS Policilinico di S.Orsola in Bologna, Italy, from January 2021 to December 2023.All samples analyzed were extracted from formalinfixed and paraffin-embedded (FFPE) histological blocks.Briefly, DNA was extracted from 2 to 3 10 µm thick sections, according to the selection performed by a pathologist on the last Hematoxylin and Eosin (H/E) slide.DNA was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

NGS Analysis
The next-generation sequencing (NGS) analysis was performed using a multi-gene panel developed in the Molecular Pathology Laboratory of IRCCS Policlinico di S.Orsola [19].
Of the 113 BRAF-mutated cases, 79 (69.9%) harbored at least one variant in another gene, mainly TERT (72 of 79 cases-91.1%).NRAS was mutated with other genes in 51 out of 76 (67.1%) mutated cases, mainly TERT (50 of 51-98.0%).In three cases, concomitant BRAF/NRAS mutations were observed, but in all these cases, the BRAF variant was a Diagnostics 2024, 14, 800 6 of 11 class III mutation, which is known to have low activity compared to the BRAF wild type (Supplementary Table S2).Of the 28 cases with the TP53 mutation, 22 (78.6%) had at least one other mutation and the combination TP53-TERT was detected in 12 of these 22 (54.5%)samples.Interestingly, all cases harboring IDH1 (n = 7) or CTNNB1 (n = 6) variants had a mutation in at least one other gene.As regards IDH1, 6 out of 7 mutations (85.7%) were concomitant with TERT variants, and all 6 mutated CTNNB1 cases (100%) were also mutated in the TERT promoter.

Primary and Metastatic Lesions
In eight cases, both primary and metastatic lesions were analyzed by NGS.In all specimens, we detected a perfect concordance in variants harbored by the primary and recurrence lesions (Table 6).Three out of the eight samples harbored a BRAF p.Val600 variant together with a mutation in the TERT promoter region; three samples had an NRAS p.Glu61Arg mutation and a TERT promoter region mutation; one sample had a BRAF and IDH1 mutation; and one case harbored four different variants in BRAF, NRAS, TERT, and TP53 genes (Table 6).Intriguingly, all but one sample had a TERT promoter mutation concomitant with other pathogenic variants.All these specimens were considered only one time in the whole cohort of 260 cases.

Discussion
To date, the molecular characterization of metastatic melanomas for predictive purposes primarily relied on the assessment of BRAF mutations for the use of BRAF inhibitors.However, the genetic changes that distinguish these cancers extend beyond the single BRAF p.V600 mutation.
From a technical standpoint, the evaluation of the BRAF mutation alone can be accomplished using "single-marker" approaches, such as real-time or pyrosequencing, which allow the mutation to be studied quickly and affordably.However, if one wants to characterize a larger number of molecular markers in addition to BRAF, such approaches become less cost-effective and are difficult to use in everyday practice due to the multiplicity of tests required for proper characterization.The advent of NGS in molecular diagnostics enabled us to integrate multigene analysis with great analytical sensitivity.
In recent years, a large number of multi-gene panels have become commercially available.However, these panels contain a large number of targets and are typically intended for specific tumors or genes.Creating custom/laboratory-developed multi-gene panels enables the selection of targets based on the demands of the medical community, as supplied by the molecular laboratory.These panels allow for the optimization of the number of specimens that can be evaluated in a single run, reducing expenses.
The use of multi-gene NGS panels enables the characterization of various genomic areas while maximizing time and costs.Furthermore, using lab-developed panels allows for the "design" of the panel to be based on clinical needs, incorporating markers such as TERT or TP53 that may not be available in commercial "targeted" panels.
Although the ESMO guidelines for the use of NGS in patients with metastatic cancers do not include melanoma between the advanced neoplasms in which NGS is recommended, it is also true that these guidelines "strongly recommends that clinical research centers perform multigene sequencing as part of their missions to accelerate cancer research and drug development through clinical trials, provide access to innovation to patients and to collect data" [20].Furthermore, the ESMO guidelines for the characterization of the diagnosis and treatment of melanomas suggest not to be limited to the single analysis of the BRAF V600 locus.In tumors that do not carry this type of mutation, the molecular analysis has to be extended not only to less common BRAF variants but also to other genes, such as NRAS and KIT [18].The NGS multigene approach allows the simultaneous analysis of these potential hot spots, which is a preferable approach compared to sequential analysis (V600 WT → other BRAF variants not present → analysis of genes other than BRAF); also, in light of the data obtained in this study in which BRAF V600 mutations are commonly found together with TERT promoter variants, it suggests that non-class I BRAF mutations could coexist with variants in other driver genes.
The panel we created for the characterization of melanomas with gene alterations is consistent with what has been described in the literature.TERT was shown to be the most frequently mutant marker, particularly when combined BRAF.The NGS panel was also demonstrated to be reliable, failing to produce results in only 1.2% of the samples tested.
Although BRAF is one of the most common mutations, our findings show that relying just on BRAF to characterize advanced melanomas is quite limiting.Identifying novel prognostic markers and therapeutic targets is greatly needed, as well as tailored characterization approaches, to detect patients at high risk of disease recurrence [21,22].
In addition to the aforementioned TERT, numerous mutations have been reported in NRAS, TP53, and IDH1, KIT, albeit at a lower frequency.Intriguingly, in the majority of analyzed samples (52.7%), more than one mutation was detected.The more frequent matching was between TERT and BRAF in 72 out of 127 analyzed samples (56.7%).The combination of TERT promoter mutations and BRAF p.V600E is expected to provide a strong genetic basis for tumor aggressiveness [23].Furthermore, TERT mutations are being studied as possible therapeutic targets due to their role in melanoma progression.Strategies include developing drugs that directly inhibit the TERT function or target the mechanisms that induce TERT alterations [24].
BRAF and NRAS variants were confirmed to be mutually exclusive, except for RAS and BRAF class III variants.These latter variants are known to have low activity compared to the BRAF wild type and cannot directly phosphorylate MEK [3].In fact, it has been previously described in the literature that BRAF class III variants may co-occur with RAS-activating mutations [17,25].
Interestingly, in all eight cases in which both primary and recurrence lesions were analyzed, a perfect match in the molecular status of the two specimens was observed, as reported in the literature [26,27].Even if, in our cohort, the primary and metastatic samples showed the same molecular structure, the analysis of other molecular markers could be performed to understand whether variants that are not present in the primary lesion may be acquired in the metastasis.
In those cases that do not harbor any variants in the analyzed genes, other alterations may be present in markers other than those covered by the panel.In fact, although the data demonstrate that this panel was able to identify mutations in almost all advanced melanoma, in 10% of these, no alterations were identified.In these samples, it might be worth investigating other markers, such as using a panel for fusion genes (e.g., ALK fusions) [28] or a Comprehensive Genomic Profiling (CGP) panel [29,30], allowing the identification of mutations in uncommonly altered genes.With our panel, the primary and metastatic samples showed the same molecular structure; however, the extension to other markers could also be performed to understand whether there are any acquired variants in the metastases that are not present in the primary lesion.
We then provide the validation of a laboratory-developed, custom-designed multigene NGS panel.Using this laboratory-developed panel, we were able to analyze multiple types of cancers in the same run.This laboratory-developed NGS panel was designed to cover the diagnostic/prognostic/predictive clinical needs not only for melanomas but also for other tumors, such as CRCs (colorectal carcinomas), thyroid nodules, pancreatic lesions, gliomas, and GISTs (gastrointestinal stromal tumors) [19].Because this panel is intended for the key gene targets of all the tumors stated above, it may be utilized to analyze several tumor types in a single run, reducing the turn-around time and NGS costs.In comparison to existing/commercial NGS multi-gene panels, the optimized selection of genes and the possibility of analyzing the relevant markers in different tumor types enables a high number of specimens to be analyzed in each run.This versatility is not possible with commercially available multi-gene panels that are dedicated to the in-depth analysis of specific tumors, whereas commercially available comprehensive multi-gene panels include a large number of targets, limiting the number of samples that can be analyzed in the same run.Pooling

Figure 1 .
Figure 1.Frequency of mutations in the analyzed cohort.WT: wild type; NE: Not able to evaluate.

Figure 1 .
Figure 1.Frequency of mutations in the analyzed cohort.WT: wild type; NE: Not able to evaluate.

Table 1 .
Frequency of altered genes in the analyzed cohort and comparison with data obtained from TCGA.

Detected Frequency in Present Study (Lab-Developed NGS Panel) (n = 260) Frequency in TCGA (Whole-Exome Sequencing) [1] *
* Only advanced-stage melanomas analyzed in the TCGA study, at n = 115, were considered for the comparison.^ Performed by Sanger sequencing.NA: data not available.

Table 2 .
BRAF variants in the analyzed cohort.
ACMG: American College of Medical Genetics and Genomics; P: pathogenic; LP: likely pathogenic.

Table 2 .
BRAF variants in the analyzed cohort.
ACMG: American College of Medical Genetics and Genomics; P: pathogenic; LP: likely pathogenic.

Table 3 .
NRAS variants in the analyzed cohort.
ACMG: American College of Medical Genetics and Genomics; P: pathogenic; LP: likely pathogenic.

Table 4 .
TERT variants in the analyzed cohort.
ACMG: American College of Medical Genetics and Genomics; P: pathogenic; LP: likely pathogenic.

Table 5 .
Genes more frequently found mutated in combination with other genes.

Table 6 .
Cases in which both primary and metastatic lesions were analyzed.