Information

Why do BRAF mutations appear more in skin cutaneous melanoma?


When looking at the tissue expression of the BRAF protein it seems that BRAF is regularly expressed in almost all of the tissues. There is elevated expression in tissues like the Testis and the Parathyroid gland, but the expression in the Skin is about average. So how does it make sense that for example in tumor samples of skin cutaneous melanoma (TCGA-SKCM) the BRAF mutations are so dominant (about 50% of the cases contain a BRAF mutation)? And on the other hand, it occurs less frequently in other cancer types?


BRAF (Gene)

Emma Groves , . Alastair Greystoke , in Reference Module in Biomedical Sciences , 2020

Biology

The BRAF gene found on chromosome 7q34 is a 2949 base pair sequence of 18 exons encoding a 766 amino acid peptide ( Huang et al., 2013 ). The B-Raf protein is a key signaling molecule in the mitogen activated protein kinase (MAPK) signaling pathway involved in cell growth, proliferation and survival. BRAF gene mutations are oncogenic and mutated in

8% of all malignancies ( Davies et al., 2002 ), with higher incidence in melanoma (50%) ( Leonetti et al., 2018 ). Mutations in BRAF can render the RAS-RAF-MEK-ERK pathway constitutively activate and lead to uncontrolled cell proliferation and survival ( Wan et al., 2004 Khunger et al., 2018 ).

BRAF mutations can be categorized into V600E and non-V600E mutations. Within NSCLC, BRAF mutations are present in 1.5%–4% of cases approximately half of these are V600E mutations ( Weart et al., 2018 ). This mutation in BRAF is oncogenic and leads to a 500-fold increase in the kinase domain activity of B-Raf as compared with its wild type ( Wan et al., 2004 ), that continuously activates ERK, irrespective of RAS activation and ERK-dependent negative feedback ( Leonetti et al., 2018 ). Although a relatively small proportion of patients, a precision medicine approach to treatment for patients with BRAF V600E mutations has led to meaningful improvements in patient outcomes and quality of life when compared to traditional chemotherapy ( Leonetti et al., 2018 ).


Abstract

Analyzing the mutational load of driver mutations in melanoma could provide valuable information regarding its progression. We aimed at analyzing the heterogeneity of mutational load of BRAF V600E in biopsies of melanoma patients of different stages, and investigating its potential as a prognosis factor. Mutational load of BRAF V600E was analyzed by digital PCR in 78 biopsies of melanoma patients of different stages and 10 nevi. The BRAF V600E load was compared among biopsies of different stages. Results showed a great variability in the load of V600E (0%-81%). Interestingly, we observed a significant difference in the load of V600E between the early and late melanoma stages, in the sense of an inverse correlation between BRAF V600E mutational load and melanoma progression. In addition, a machine learning approach showed that the mutational load of BRAF V600E could be a good predictor of metastasis in stage II patients. Our results suggest that BRAF V600E is a promising biomarker of prognosis in stage II patients.

Citation: Sevilla A, Morales MC, Ezkurra PA, Rasero J, Velasco V, Cancho-Galan G, et al. (2020) BRAF V600E mutational load as a prognosis biomarker in malignant melanoma. PLoS ONE 15(3): e0230136. https://doi.org/10.1371/journal.pone.0230136

Editor: Suzie Chen, Rutgers University, UNITED STATES

Received: November 19, 2019 Accepted: February 21, 2020 Published: March 13, 2020

Copyright: © 2020 Sevilla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This research was supported by the Basque Government (grants ELKARTEK- KK2016-036 and KK2017-041 to MDB, grant IT1138-16 to SA and predoctoral fellowship PRE_2014_1_419 to AS), and by the University of the Basque Country (UPV/EHU) (grant GIU17/066). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: PCR, Polymerase chain reaction dPCR, Digital PCR AJCC, American Joint Committee on Cancer FFPE, Formalin fixed paraffin embedded IHC, Immunohistochemistry


What is the Sonic Hedgehog pathway?

Some medications used to treat advanced basal cell carcinoma affect a cell signaling pathway called Sonic Hedgehog. (Yes, like the video game.) 6

Before you were born, the Sonic Hedgehog pathway had an important role in the development of your organs and making sure the right side of your body matches your left. This pathway is not active in most healthy adult tissue, but skin is an exception. 7 In skin, Sonic Hedgehog maintains the supply of stem cells. Stem cells are flexible cells that can develop into many different cell types. The Sonic Hedgehog pathway has a role in the development of your hair follicles and glands and it helps to control skin growth.

Key players in the Sonic Hedgehog pathway are two receptors called PTCH1 (“Patched”) and SMO (“Smoothened”). 7 Figure 1 illustrates how the Sonic Hedgehog pathway works. (Please note: This is a simplified explanation. Although some details are missing, it illustrates the basic way that the receptors interact with each other.) You can imagine that PTCH1 works like a door, holding SMO in a little box. The Sonic Hedgehog protein is a key to open the box (Figure 2). When Sonic Hedgehog protein opens up the PTCH1 door, SMO is free to start working (Figure 3). It starts sending signals that cause cells to grow and survive.

Figure 1. PTCH1 and SMO signaling

PTCH1 is a receptor in the cell membrane. Without the Sonic Hedgehog protein (SHH), PTCH1 blocks SMO, another receptor, from sending any signals. When the SHH protein binds to PTCH1, the SMO travels to the cell surface. SMO begins sending signals that lead to cell growth and survival.

Figure 2. Sonic Hedgehog protein binding

PTCH1 is known as a “tumor suppressor gene.” By keeping SMO inactive, PTCH1 acts as an “off switch” for the process that causes cells to divide and survive. 5 But PTCH1 is mutated in about 60% of BCC tumors. 8 When PTCH1 becomes mutated, the “off switch” no longer works, and SMO signals freely. The result is uncontrolled cellular growth. Two medications, Erivedge® (vismodegib) and Odomzo® (sonidegib), work by blocking the SMO receptor.

Figure 3. SMO mutation

SMO is known as an “proto-oncogene” or a type of gene that causes cells to grow, divide, and stay alive. 5,9 Certain mutations to SMO cause it to stay “on” all the time, leading to cancer development. SMO is mutated in 10-20% of BCC. 15 When SMO is mutated, the medications that block SMO may not work. 10


The Biology of Advanced Melanoma

Transcript: Keith T. Flaherty, MD: Hello, and thank you for joining us today for this OncLive Peer Exchange Panel Discussion on the Treatment of Advanced Melanoma and Basal Cell Carcinoma. In the last 5 years, there have been FDA approvals for 12 new agents and combination therapies for patients with advanced melanoma. This is only the tip of the iceberg, with extensive research still ongoing in the field. With this explosive progress, there is a lot to learn about optimal use of newly developed agents and emerging agents. This OncLive Peer Exchange will focus on personalized approaches to treatment as well as looking at schedules, dosing, and sequencing of novel agents.

I’m Dr. Keith Flaherty. I’m the director of the Termeer Center for Targeted Therapy at the Massachusetts General Hospital in Boston, Massachusetts, and professor of medicine at Harvard Medical School. Joining me today are Dr. Georgina Long, professor of melanoma medical oncology and translational research at the Melanoma Institute of Australia, the University of Sydney Dr. Jason Luke, assistant professor of medicine and in the Melanoma and Developmental Therapeutics Clinics at the University of Chicago Dr. Jeff Weber, the deputy director of the Laura and Isaac Perlmutter Cancer Center and professor of medicine at NYU Langone Medical Center in New York and Dr. Jonathan Zager, a professor of surgery and the director of regional therapies at Moffitt Cancer Center in Tampa, Florida. Thank you all for joining us. Let’s begin.

So, to the distinguished panel today, I thought it would be useful before diving into the therapies to take a little bit of a look at the biology of melanoma and how that sets us up for thinking about the application of targeted therapy, immune therapy, the same. Again, saving the therapies for a moment, maybe Georgina, just give the audience a little bit of a sense of how it is that we think about the important genetic subgroups or subpopulations within melanoma.

Georgina Long, BSc, MBBS: Sure. What we really know and understand very well are these driver mutations which we see commonly in melanoma. And that would be mutated BRAF, particularly V600, NRAS mutations, and in alterations in NF1. However, having said that, we know from the extensive research and the recent data from the TCGA (The Cancer Genome Atlas) that there are many mutations and aberrations in melanoma, particularly cutaneous melanoma, which is the most common of the melanoma subtypes. And how this relates to what’s happening with genes being expressed, the immune system, and the microenvironment, it’s still something we’re working out. But in terms of how we approach it therapeutically, we think in terms of that big group of BRAF, NRAS, NF1, and then CK, of course, but there are many aberrations. And we know that cutaneous melanoma is highly, highly mutated and has a strong UVC (ultraviolet C) signature in general. The proportions are important as well. About 40% have a BRAF mutation in cutaneous melanoma.

Keith T. Flaherty, MD: And in your practice, how would you quote this? The NRAS mutant population for which new therapies may be emerging, how many patients, what percentage?

Georgina Long, BSc, MBBS: In Australia it’s around 20% to 25%. We know NRAS is associated with some chronic sun damage and highly mutated state. So, in Australia it’s around 25%. But if we looked across the world, it’s probably around the 20% mark.

Keith T. Flaherty, MD: Yes. Jeff, melanoma has long been favored by the tumor immunology field as a tumor type in which to investigate therapies. Now, of course, it’s the first home of many of the recently developed therapies. What is it about the biology of melanoma, the immunobiology of melanoma, that sets it apart from other cancers?

Jeffrey S. Weber, MD: Well, I think there are two things, Keith. One is that it is a tumor of transformed melanocytes. And melanocytes, as part of their physiologic processes, tend to export things to the surface. They express antigens or molecules at the surface that potentially could be recognized by the immune system. Perhaps more importantly, it appears almost amazingly that the mutational load in a tumor determines whether or not new epitopes, new molecules that could be recognized by the immune system, could arise. So, as Georgina was saying, if you look at the average mutational load, melanoma is the number one of all tumors that’s a median of about 600 or 700 individual nonsynonymous mutations—meaning a mutation present in the DNA of the tumor that’s not present in the DNA of the normal cell, and it’s not a so-called SNP, a single nucleotype variant. They would have the potential to then encode a peptide, a fragment of a protein that could be recognized by the immune system. Because of all those mutations—even though I guess that’s bad news for the transformed state of the cell—a lot of mutations mean the cells grow out of control and make cancers. It contains within it the potential seed of its own destruction so that it could be recognized by the immune system.

Keith T. Flaherty, MD: So, Jason, the past few years there’s been this theme developing a deep look at tumor cells and their genetic makeup, and then over here the constituents of the tumor microenvironment—immune cells in the environment. But there’s more and more insights, including from your group, that suggest there’s actually an interplay. Like the tumor cell actually essentially interacts with the microenvironment, probably in its development most importantly. Maybe a couple thoughts about merging evidence along those lines about what it is about tumor cells and target pathways that otherwise relate to this immunobiology.

Jason J. Luke, MD: Absolutely. What we’ve been finding is that there appear to be, actually, tumor-intrinsic mechanisms of immune exclusion. Meaning molecular signaling pathways that are perhaps not recurrently mutated in a way that we have thought of, like BRAF and NRAS, but may influence whether or not an immune infiltrate can enter the tumor. And we’ve been finding, particularly around beta-catenin, signaling was the first of these, but now P10 has been identified. Some other molecular signaling pathways that people have heard of that perhaps weren’t considered quite so important may be part of our future consideration when developing combination immunotherapy approaches. So, we’re going to get into it, and BRAF inhibitor is very effective, immunotherapy very effective, and there’s going to be an intersection. But perhaps there’s going to be a role for targeted therapy drug development to enhance immunotherapy beyond just going after those driver mutations in the future.


Targeted Therapies

Therapies targeting BRAF V600E and c-KIT mutations offer an alternative systemic option in eligible patients with metastatic disease. BRAF inhibitors in combination with MEK inhibitors have shown improved survival in patients with advanced cutaneous melanoma, but whether the same response is seen in patients with mucosal melanoma is not known. 2,23 As noted previously, c-KIT mutations are more prevalent in AMM than BRAF V600E mutations. Although data in mucosal melanomas are primarily limited to case reports, other studies in the broader advanced melanoma population with confirmed KIT mutations have shown modest response to KIT inhibition. 24-26 In a phase II open-label trial of patients with advanced melanoma (unresectable or metastatic) with a known KIT mutation treated with imatinib, a durable response was seen in 16% of patients. 25 Another phase II trial in patients with metastatic melanoma harboring KIT mutations demonstrated partial or stable disease in 54% of patients who were treated, with overall survival significantly longer than for those who did not respond. 26 Responses in both of these trials were limited to tumors with specific KIT mutations on exons 11 and 13.


Acknowledgments

Funding for the study was provided by the National Comprehensive Cancer Network (NCCN)‘s Oncology Research Program (ORP) from general research support provided by Novartis Pharmaceuticals Corporations (formerly GlaxoSmithKline, LLC). Drug supply was provided by Novartis. D.B.J. was supported by the American Society of Clinical Oncology and NCCN Career Development Awards during conduct of the study, and by National Institutes of Health (NIH) National Cancer Institute grant K23 CA204726. C.A.N. was supported by NIH National Cancer Institute grant F32 CA254070.


RESULTS

D-HPLC was used to screen for BRAF and NRAS mutations in cases of UM and CM because this method is highly sensitive for a variety of mutation types, including point mutations, insertions, and deletions (40) . As illustrated in Fig. 1 ⇓ , D-HPLC readily detected the presence of the most common BRAF mutation reported in melanoma, V599E. The sensitivity of the system for detecting this mutation was assessed by serially diluting pure V599E amplimer with pure wild-type exon 15 amplimer (see “Materials and Methods”). The V599E mutation was detectable down to a level of ∼20%, which is equivalent to 40% tumor DNA, assuming heterozygosity for the allele (Fig. 1) ⇓ . Because all of the tumor samples included in the study were highly enriched in tumor cells (>90%), this was considered an adequate level of sensitivity.

D-HPLC elution profiles of BRAF exon 15 amplimers. Progressive dilutions of V599E amplimer with wild-type amplimer reveal a distinct heteroduplex peak at concentrations above 15% V599E allele.

Because D-HPLC profiling is dependent on heteroduplex formation between wild-type and mutant amplimers, it can miss mutations that are hemizygous or homozygous, such as the BRAF V599E mutation present in the melanoma cell line SK-MEL-28. Two approaches were used to exclude such mutations among the melanoma tissue samples that were studied. First, BRAF exon 15 amplimers from 12 UMs and 12 CMs with wild-type D-HPLC profiles were annealed with SK-MEL-28 amplimer and then were reanalyzed. All of the 24 samples showed the appropriate mutant profile, indicating that the tumor DNA was indeed negative for the V599E mutation. Second, direct DNA sequencing was performed on another 10 UMs and 10 CMs with wild-type D-HPLC profiles, and no mutations were detected. Thus, hemi/homozygosity for BRAF mutations was not detected in the melanoma tissue samples.

Among 44 CMs that were analyzed (10 primary, 34 metastatic), 16 had mutations in BRAF exon 15 (14 metastatic, 2 primary Table 1 ⇓ ). The frequency of BRAF mutations was not significantly different between primary and metastatic melanomas (P = 0.70156, Fisher’s exact test). No BRAF exon 11 mutations were detected among seven CMs that were wild-type for exon 15. UMs (61 primary, 1 secondary) were uniformly negative for mutations in BRAF exon 15 (0 of 62 Table 1 ⇓ ). Nine UMs screened for exon 11 mutations were likewise negative (0 of 9).

Frequency of B-RAF exon 15 mutations in tested tumors

Two independent cases of metastatic CM harbored a novel L596Q missense mutation (Fig. 2A) ⇓ . A novel, in-frame deletion/insertion mutation, VKSRWK599–604D, was also discovered in one primary CM (Fig. 2B) ⇓ . In all cases, the novel mutations were confirmed by reextraction of DNA from the original melanoma sample, followed by repeat amplification, D-HPLC screening, and direct sequencing.

A, BRAF L596Q mutation. The double-peaked elution profile detected on D-HPLC analysis at denaturing temperature (56.3°C, left) corresponded with the point mutation found on direct DNA sequencing (right). B, BRAF deletion/insertion mutation. D-HPLC elution profile at the nondenaturing temperature (50.0°C) revealed three distinct peaks (left). Direct sequencing of the amplicon confirmed a VKSRWS599–605D deletion/insertion (right).

The overall frequency of BRAF exon 15 mutations in our CM samples (36.4% Table 1 ⇓ ) was lower than that reported by Davies et al. (28) , who used a heteroduplex detection assay based on capillary electrophoresis. Concerned that methodological differences might be a factor, we examined a series of gliomas and detected BRAF exon 15 mutations at a higher frequency (5.6% Table 1 ⇓ ) than observed by Davies et al. (0 of 15 Ref. 28 ). It was also higher than that recently reported by Chan et al. (1 of 166 Ref. 41 ). These findings indicate that the sensitivity of D-HPLC is at least comparable with, if not better than, other published methodologies.

Previous reports have indicated that NRAS exon 2 mutations are present in a subset of melanomas that lack a BRAF mutation (28 , 32) . We screened for NRAS exon 2 mutations in 19 CM samples that were wild-type for BRAF exon 15 and found one point mutation [1 (5.3%) of 19 Fig. 3 ⇓ ] . No NRAS exon 2 mutations were detected among eight CMs positive for a BRAF mutation (0 of 8). UM samples were completely negative for mutations of NRAS exon 2 (0 of 47). NRAS exon 1 mutations were not observed among the samples of CM (0 of 21) and UM (0 of 22) tested.

NRAS exon 2 point mutation. A double-peaked elution profile detected on D-HPLC analysis at denaturing temperature (59.3°C, left) corresponded with the point mutation Q61R found on direct DNA sequencing (right).


Future Perspectives

Cancer therapy has evolved dramatically over the last decade. We have advanced strategies to gain local control for most tumours however, the metastases are still challenging, and their presence will in most cases kill the patient. While we have identified numerous of new biomarkers and molecular events in melanoma with the use of high-throughput “omics” technology, we still do not know the function of most of them. As the amount of data increases, we also discover that cancer is not as “simple” as it was thought to be for a long time. Pathways are not simply turned on or off in a binary fashion, but they interact in complex networks. Thus, functional studies confirming some of the numerous potential biomarkers reported in the literature are highly needed.

With regard to conjunctival melanocytic lesions, we still classify the lesions mostly based on histopathology, which is not always accurate. We do not fully understand the pathway from benign melanocyte to premalignant lesion and further on to metastatic melanoma. Genetic studies investigating the relationship between different conjunctival melanocytic lesions are urgently needed, as the conjunctival melanoma phenotype may genetically be the result of different genotypes depending on whether the lesion is sun-induced or shares pathogenetic factors with mucosal melanomas at other locations. Additionally, a deeper understanding of these pathways would clinically benefit patients referred with a primary acquired melanosis, as genetics would tell whether a lesion is premalignant or benign.

Furthermore, while genetic methods are becoming increasingly more sensitive, a complete genetic evaluation of all cases in our Danish database would be of great interest. Such a study would give a complete view of mutations in a complete conjunctival melanoma population. This could be done with a panel covering a broad variety of melanoma-associated genes. In our study, we found several cases where no obvious driver mutation was found. Such cases should be further evaluated using whole-exome sequencing, which in most cases is possible to perform despite formalin fixation.

In our study, we were not able to detect a difference between sun-exposed and sun-shielded conjunctival melanoma at the RNA level, probably due to a small sample size. As our results indicated a difference, this should be evaluated with a proper sample size in a similar study using NanoString. Furthermore, gene expression analysis similar to those applied in this thesis could be used to build prognostic models of mucosal and conjunctival melanoma.

Staging systems are not available for mucosal melanomas in general, and these would be of particular interest to develop. Our studies have shown deregulations of H2AFX, and measuring the expression of this gene could be of use in such staging systems.

Prognostic factors would aid planning a personalized follow-up for each conjunctival melanoma patient. Until now, neither genetics nor miRNAs have proven to be robust prognostic markers of the metastatic potential of conjunctival melanoma, and studies utilizing gene expression (potential with the use of NanoString), methylation assays, or other omics, such as proteomics, must be performed.

We have noted alterations in several immunological pathways in conjunctival melanoma. A deeper understanding of the immune system and its relationship to the development of conjunctival melanoma and mucosal melanoma would be beneficial in the future treatment of metastases, and the studies included in this thesis will hopefully encourage such studies. There are numerous immunological targets of various agents, and it could be of great interest to test these agents in conjunctival and mucosal melanomas. Targets such as LAG3, TIM3, and OX40 are of particular interest in this matter, and studies investigating these targets in mucosal melanoma and conjunctival melanoma must be performed. Hopefully, checkpoint inhibitors are just the first small indication of the powers that can be revealed by turning on the patient’s own immune system in cancer therapy. This investigation may encourage such studies.


RNA-protein network may explain why melanoma grows more

The molecular mechanism underlining melanoma cell growth. Credit: Nobuhiro Takahashi

With five-year survival rates being around 30 percent for patients with distant metastatic disease, cutaneous melanoma is the leading cause of skin cancer-related deaths. The major causes of the low survival rate for melanoma patients are the limited number of options for patients lacking the BRAF mutation and the intrinsic and acquired resistance to existing therapies. It is therefore essential to develop new therapeutic strategies to eradicate resistant cells and/or target patients irrespective of their driver mutations.

A collaboration led by scientists from KU Leuven, Belgium, with Tokyo University of Agriculture and Technology (TUAT), Japan, revealed a new way to fight melanoma. They report that a melanoma-specific long non-coding RNA, named SAMMSON, interacts with the protein CARF to properly coordinate protein synthesis in both the cytosol and mitochondria of melanoma cells. This mechanism ensures the maintenance of proteostasis during cell growth, thus avoiding the induction of cell death.

"We identified a long non-coding RNA named SAMMSON expressed in the vast majority (>90 percent) of melanoma patients and never detected in normal adult tissues," said co-senior author Dr. Eleonora Leucci, LKI Leuven Cancer Institute, KU Leuven. "In melanoma, SAMMSON is essential for proper localization of the protein p32 into the mitochondria, where it is essential for ribosome biogenesis and protein synthesis."

"We reported that protein CARF presents in the nucleoplasm, indirectly reduces the ribosome synthesis in the nucleolus through directly binding to another protein XRN2," said co-author Dr. Hideaki Ishikawa, Department of Agriculture, TUAT, Tokyo, Japan. "In the nucleolus, XRN2 acts as an enzyme that increases the ribosome synthesis. But its activity and localization are regulated by CARF whose binding keeps XRN2 away from the nucleolus."

These independent observations where connected later by first author MSc Roberto Vendramin who found that SAMMSON binds to CARF. This observation triggered the international collaboration that led to a work published in the journal Nature Structural & Molecular Biology.

"We found that in melanoma, a portion of CARF is sequestered into the cytoplasm where it interacts with p32 in a SAMMSON-dependent manner" said Dr. Eleonora Leucci.

"Released from the CARF binding, XRN2 translocates from the nucleoplasm to the nucleolus where it promotes ribosome biogenesis. Concertedly the complex CARF-SAMMSON-p32, moves to the mitochondria and boosts the mitochondrial ribosome synthesis."

"We concluded that, in melanoma cells, SAMMSON increases the ribosome synthesis concertedly in the nucleolus and mitochondria," said Dr. Leucci. "Cells have a mechanism to sense the abnormality if the ribosome synthesis is altered in either the nucleolus or mitochondria, and induce cell death. However, when this process is altered in both compartments the cell is unable to recognize the insult. Therefore, by hacking concertedly both ribosome biogenesis machinery, SAMMSON makes the cell insensitive to the damage and promotes melanoma cell growth"

Hence, a substance that inhibits SAMMSON or/and CARF is expected to be a new therapeutic reagent for melanoma.