Mutational Heterogeneity in Melanoma: An Inconvenient Truth
2015; Elsevier BV; Volume: 135; Issue: 12 Linguagem: Inglês
10.1038/jid.2015.351
ISSN1523-1747
Autores Tópico(s)vaccines and immunoinformatics approaches
ResumoIdentification of oncogenic BRAF mutations in primary and metastatic melanomas supports a linear model of clonal evolution in cancer. Some mutational studies, however, have failed to identify BRAF mutations in metastatic tumors from patients with BRAFmutant primary melanomas. Using a combination of methods, Riveiro-Falkenbach et al. (2015) assert that technical issues, and not clonal heterogeneity, may explain prior discordant mutational results. Identification of oncogenic BRAF mutations in primary and metastatic melanomas supports a linear model of clonal evolution in cancer. Some mutational studies, however, have failed to identify BRAF mutations in metastatic tumors from patients with BRAFmutant primary melanomas. Using a combination of methods, Riveiro-Falkenbach et al. (2015) assert that technical issues, and not clonal heterogeneity, may explain prior discordant mutational results. Clonal evolution of cancers, proposed in 1976 by Nowell, 1976Nowell P.C. The clonal evolution of tumor cell populations.Science. 1976; 194: 23-28Crossref PubMed Scopus (4861) Google Scholar, states that malignant tumors are genetically unstable, producing clonal variants with mutations that are either favorable or unfavorable for cell survival. This linear evolution model predicts that metastatic and drug-resistant clones result from an accumulation of several favorable mutations, including the original initiating mutation. With this conceptual framework, a linear model of melanoma tumorigenesis, beginning with the development of a BRAFmutant nevus, followed by additional genetic and epigenetic alterations leads to primary melanoma and ultimately to BRAFmutant metastatic disease. This model makes sense for BRAFmutant melanoma. Although the findings of many studies are consistent with the model described above, several have failed to identify conservation of BRAF mutations in all tumor samples analyzed from individual patients (i.e., inter-tumor heterogeneity), results that challenge Nowell, 1976Nowell P.C. The clonal evolution of tumor cell populations.Science. 1976; 194: 23-28Crossref PubMed Scopus (4861) Google Scholar model (Table 1). For example, Saint-Jean et al., 2014Saint-Jean M. Quereux G. Nguyen J.M. et al.Is a single BRAF wild-type test sufficient to exclude melanoma patients from vemurafenib therapy?.J Investig Dermatol Symp Proc. 2014; 134: 1468-1470Abstract Full Text Full Text PDF Scopus (35) Google Scholar found inter-tumor heterogeneity in 10/74 (14%) patients with primary-metastatic tumor pairs and in 8/43 (19%) patients with more than 2 metastases. Colombino et al., 2012Colombino M. Capone M. Lissia A. et al.BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma.J Clin Oncol. 2012; 30: 2522-2529Crossref PubMed Scopus (367) Google Scholar found that of the 20/99 (20%) cases of inter-tumor heterogeneity, 8/20 (40%) had a mutant primary tumor but wild-type metastases, a finding inconsistent with Nowell’s model. Similarly, Heinzerling et al., 2013Heinzerling L. Baiter M. Kuhnapfel S. et al.Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations.Br J Cancer. 2013; 109: 2833-2841Crossref PubMed Scopus (81) Google Scholar identified 10/53 (19%) patients that had discordant BRAFV600E mutational results when multiple tumors were analyzed using pyrosequencing. Of those 10 patients, 4 exhibited mutant primary tumors with wild-type metastases (Heinzerling et al., 2013Heinzerling L. Baiter M. Kuhnapfel S. et al.Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations.Br J Cancer. 2013; 109: 2833-2841Crossref PubMed Scopus (81) Google Scholar). Using a BRAFV600E mutation-specific PCR assay we found that 2/18 (11%) patients had BRAFmutant primary tumors and BRAFwild-type metastases (Yancovitz et al., 2012Yancovitz M. Litterman A. Yoon J. et al.Intra- and inter-tumor heterogeneity of BRAF(V600E) mutations in primary and metastatic melanoma.PloS One. 2012; 7: e29336Crossref PubMed Scopus (240) Google Scholar). In addition, 5/19 (26%) patients with multiple metastases exhibited inter-tumor heterogeneity for the BRAF mutation. We also reported findings of intra-tumor heterogeneity. Using laser microdissection to analyze tumor cells isolated from separate regions of individual primary melanomas, we found that 6/9 (67%) primary melanomas demonstrated substantial variation in the relative abundance of BRAFV600E DNA in different tumor regions. Lin et al., 2011Lin J. Goto Y. Murata H. et al.Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression.Br J Cancer. 2011; 104: 464-468Crossref PubMed Scopus (107) Google Scholar also found evidence of intra-tumor heterogeneity in primary and metastatic tumors using several methods to identify BRAF mutations, including analysis of isolated single melanoma cells, mutation-specific PCR assays, or bacterial subcloning and sequencing. The most telling evidence of heterogeneity, however, comes from studies that found both NRAS and BRAF mutant alleles in multiple tumors from the same patient (prior to the advent of BRAF inhibitor therapy) or from the same tumor itself. Colombino et al., 2012Colombino M. Capone M. Lissia A. et al.BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma.J Clin Oncol. 2012; 30: 2522-2529Crossref PubMed Scopus (367) Google Scholar identified two patients with an NRASmutant primary melanoma and a BRAFmutant metastasis. Sensi et al., 2006Sensi M. Nicolini G. Petti C. et al.Mutually exclusive NRAS(Q61R) and BRAF(V600E) mutations at the single-cell level in the same human melanoma.Oncogene. 2006; 25: 3357-3364Crossref PubMed Scopus (144) Google Scholar isolated (via limited dilution cloning) separate BRAFV600E and NRASQ61R mutant melanoma cells from a short-term culture of an NRASmutant subcutaneous metastasis providing direct evidence that patient tumors may contain unique clonal subpopulations. Recently, Eriksson et al., 2015Eriksson H. Zebary A. Vassilaki I. et al.BRAFV600E protein expression in primary cutaneous malignant melanomas and paired metastases.JAMA Dermatology. 2015; 151: 410-416Crossref PubMed Scopus (25) Google Scholar identified 4 patients with BRAFV600E primary melanomas and BRAFwild-type or BRAFnon-V600E mutant metastases. Two of the BRAFwild-type metastases had NRAS mutations.Mutational heterogeneity, a feature of tumor evolution models, challenges current treatment strategies for melanoma.Table 1Selected publications describing tumor heterogeneityAuthorsSaint-Jean et al., 2014Saint-Jean M. Quereux G. Nguyen J.M. et al.Is a single BRAF wild-type test sufficient to exclude melanoma patients from vemurafenib therapy?.J Investig Dermatol Symp Proc. 2014; 134: 1468-1470Abstract Full Text Full Text PDF Scopus (35) Google ScholarColombino et al., 2012Colombino M. Capone M. Lissia A. et al.BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma.J Clin Oncol. 2012; 30: 2522-2529Crossref PubMed Scopus (367) Google ScholarHeinzerling et al., 2013Heinzerling L. Baiter M. Kuhnapfel S. et al.Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations.Br J Cancer. 2013; 109: 2833-2841Crossref PubMed Scopus (81) Google ScholarYancovitz et al., 2012Yancovitz M. Litterman A. Yoon J. et al.Intra- and inter-tumor heterogeneity of BRAF(V600E) mutations in primary and metastatic melanoma.PloS One. 2012; 7: e29336Crossref PubMed Scopus (240) Google ScholarBusam et al., 2013Busam K.J. Hedvat C. Pulitzer M. et al.Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions.Am J Surg Pathol. 2013; 37: 413-420Crossref PubMed Scopus (91) Google ScholarRiveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google ScholarDisease typeMelanomaMelanomaMelanomaMelanomaMelanomaMelanomaSamples analyzed• 278 patients• 367 tumors• 99 patients• 291 tumors (102 primaries paired with 165 metastases; 24 unpaired brain metastases)• 187 patients• 300 tumors• 73 patients• 112 tumors• 44 metastases (22 w/o BRAFV600E, 22 w/o BRAFV600E)• 20 primaries• 140 patients• 311 tumorsAnalysis tools• Sanger sequencing• Cobas• Therascreen BRAF RGQ assay• Sanger sequencing• Pyrosequencing• IHC• Sanger sequencing• MS-PCR• Laser microdissection• SNaPshot• IHC• Sequenom mass spectrometry• IHC• Cobas• Sanger sequencingPaired74 patients with multiple tumor samples53 patients with matched primary-single metastasis46 patients with primary-multiple metastases53 patients with multiple tumor samples18 patients with matched primary-metastases19 patients with multiple metastases1 patient with primary-metastatic tumor pair110 patients with primary+1 metastasis; 29 patients with primary+2 metastases; 1 patient with primary+3 metastasesInter- and intra-tumor heterogeneity observedInter-tumor heterogeneity found in 10/74 patients, including 8/43 patients with ≥2 metastasesInter-tumor heterogeneity detected in 20 cases2 cases carrying different mutations in paired samples (NRAS and BRAF)All other exhibiting mutant-wild-type mismatchesInter-tumor heterogeneity found in 10/53 patients who had multiple metastasesIntra-tumor heterogeneity shown using IHC analysisInter-tumor heterogeneity detected in 2/18 V600E primary-metastatic pairs and 5/19 V600E metastases-only pairsIntra-tumor heterogeneity found in 6/9 tumors using laser microdissectionIntra-tumor heterogeneity of BRAF expression in 6/22 V600E metastases and 2/7 V600E primariesInter-tumor heterogeneity not identified; only one patient with a paired sample analyzedInter-tumor heterogeneity not detectedIntra-tumor heterogeneity not detectedKey findings with respect to heterogeneityAnalysis of additional samples leads to greater probability of discordance between tumorsNRAS mutant primaries from 2 patients gave rise to BRAF mutant metastases providing significant evidence of inter-tumor heterogeneityInter- and intra-tumor heterogeneity with respect to BRAF mutational status and expressionRelative differences in abundance of BRAF mutant DNA in distinct microdissected regions of the same tumor sampleHigh sensitivity and specificity of VE1 suggests that focal staining of tumors may be evidence of heterogeneityIHC analysis shows that inter- and intra-tumor heterogeneity not present and that IHC analysis is more sensitive than DNA-based techniquesAbbreviation: IHC, immunohistochemistry.Several studies in melanoma have demonstrated intra- and inter-tumor heterogeneity using a wide range of techniques including cobas and IHC. The table below shows how the study by Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar compares with others. Open table in a new tab Abbreviation: IHC, immunohistochemistry. Several studies in melanoma have demonstrated intra- and inter-tumor heterogeneity using a wide range of techniques including cobas and IHC. The table below shows how the study by Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar compares with others. These reports demonstrating inter- and intra-tumor mutational heterogeneity can be accommodated in Nowell’s tumor evolution paradigm when one considers the modifications described in 1982 by Fidler and Hart (Fidler and Hart, 1982Fidler I.J. Hart I.R. Biological diversity in metastatic neoplasms: origins and implications.Science. 1982; 217: 998-1003Crossref PubMed Scopus (822) Google Scholar). They noted that primary tumors are heterogeneous, polyclonal neoplasms comprised of tumor cell subpopulations with varying biological characteristics, including metastatic potentials. As such, more than one subclone may have the ability to form metastases, which could explain the findings described above, assuming that BRAF mutations are not required for metastatic spread of melanoma. Further support for the polyclonality of human tumors is evidenced by the different organotropisms of metastatic disease, a prominent feature of melanoma and other malignancies (Figure 1). Currently, this polyclonal model of cancer best explains discordant inter- and intra-tumor mutational results and the organotropic characteristics of melanoma metastases. In the current issue, this concept is challenged by Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). They use immunohistochemistry (IHC) to compare expression of mutant BRAFV600E to DNA-based BRAFV600 mutation detection in paired primary and metastatic melanomas from 140 patients. The Ventana VE1 anti-BRAFV600E antibody (Ventana Medical Systems, Tucson, AZ) was used to conduct the IHC analysis. Two DNA-based methods were used: the cobas 4800 BRAFV600 Mutation Test, and Sanger sequencing. The cobas 4800 BRAFV600 Mutation Test is a Taqman-based real-time PCR mutation assay used to detect V600 mutations in formalin-fixed, paraffin-embedded human melanoma tissue. The test mainly detects V600E but is known to cross-react with V600K/D. This FDA-approved test is part of the standard of care for melanoma patients with advanced disease to determine eligibility for BRAF inhibitor therapy: patients with the mutation are eligible for treatment, whereas patients lacking the mutation are not. The manufacturer’s package insert states that the cobas 4800 has an over 95% positive detection rate in pre-clinical and clinical investigations. Additional validation studies demonstrate that the cobas 4800 test is able to detect more V600E mutations than Sanger sequencing, and that Sanger has more false-positive results than cobas when using an additional detection method to confirm results (Halait et al., 2012Halait H. Demartin K. Shah S. et al.Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.Diagn Mol Pathol. 2012; 21: 1-8Crossref PubMed Scopus (130) Google Scholar). Importantly, adequate tumor content is an essential requirement for any DNA-based mutational assay (Sensi et al., 2006Sensi M. Nicolini G. Petti C. et al.Mutually exclusive NRAS(Q61R) and BRAF(V600E) mutations at the single-cell level in the same human melanoma.Oncogene. 2006; 25: 3357-3364Crossref PubMed Scopus (144) Google Scholar). In tissue samples with 90% normal tissue), the chances of finding a mutation are diminished and the cobas test often fails to attain consistent results (Halait et al., 2012Halait H. Demartin K. Shah S. et al.Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.Diagn Mol Pathol. 2012; 21: 1-8Crossref PubMed Scopus (130) Google Scholar). In addition, there always exists the possibility of sampling error if cut sections are not representative of a genetically polyclonal tumor. The monoclonal BRAFV600E mutation-specific antibody, named VE1, binds to an 11-amino-acid sequence in the BRAFV600E mutant protein between amino acids 596–606 (Capper et al., 2011Capper D. Preusser M. Habel A. et al.Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.Acta Neuropathol. 2011; 122: 11-19Crossref PubMed Scopus (396) Google Scholar). Studies investigating VE1 have demonstrated the high sensitivity (80–97%) and specificity (98–100%) of the antibody to distinguish the V600E mutant protein from V600K/R/Q (Capper et al., 2011Capper D. Preusser M. Habel A. et al.Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.Acta Neuropathol. 2011; 122: 11-19Crossref PubMed Scopus (396) Google Scholar; Pearlstein et al., 2014Pearlstein M.V. Zedek D.C. Ollila D.W. et al.Validation of the VE1 immunostain for the BRAF V600E mutation in melanoma.J Cutan Pathol. 2014; 41: 724-732Crossref PubMed Scopus (43) Google Scholar). One of the values of IHC analysis is that normal tissue contamination, which can limit the sensitivity of DNA-based assays, is not a major concern. Quantifying the homogeneity of staining, however, can be highly subjective. There may be difficulties in identifying rare events in tissue sections (e.g., stained or unstained single cells), or distinguishing true staining from artifacts such as the presence of melanophages or necrotic tissue. Overall, Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) found that 52% of tumors had BRAFV600 mutations according to the cobas test. When analyzed for inter-tumor heterogeneity of BRAF mutations they found that 117/140 (83.6%) primary-metastatic pairs were concordant for the presence or absence of the BRAF mutation. They used IHC on a subset of the concordant pairs (n=61) and confirmed that those pairs were 100% concordant using VE1. When using IHC to test 23 cobas-discordant primary-metastatic tumor pairs, they found no evidence of discordance. They suggested that IHC is more sensitive than DNA-based mutation detection methods. There were several cases of positive VE1 staining in samples with <10% tumor content and Sanger sequencing and cobas testing yielded wild-type results. In addition, the high specificity of the VE1 antibody, evidenced by the lack of staining in all tumor samples harboring a V600K mutation, mediates concerns of possible false-positive results. Ultimately, Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) conclude that the cobas assay is less sensitive and accurate in calling mutant positive samples than either Sanger sequencing or IHC, and that the existence of tumor heterogeneity in published studies may be owing to technical issues rather than actual tumor heterogeneity. Although the VE1 antibody has proven to be a highly sensitive and V600E-specific reagent in several studies (Capper et al., 2011Capper D. Preusser M. Habel A. et al.Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.Acta Neuropathol. 2011; 122: 11-19Crossref PubMed Scopus (396) Google Scholar; Pearlstein et al., 2014Pearlstein M.V. Zedek D.C. Ollila D.W. et al.Validation of the VE1 immunostain for the BRAF V600E mutation in melanoma.J Cutan Pathol. 2014; 41: 724-732Crossref PubMed Scopus (43) Google Scholar) including Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), their conclusions regarding the existence (or lack thereof) of inter-tumor heterogeneity needs to be interpreted cautiously. Close inspection of their results reveals that among the 23 patients with cobas-discordant mutational results, 5 patients had V600K mutant tumors. These cases should not be included among the discordant cases as the cobas assay is not primarily designed to detect this mutation. Besides, these cases were concordant using Sanger sequencing. In addition, 1 patient had two primary melanomas, of which the thicker one was not tested via cobas, and it was determined to be mutationally concordant with its paired metastasis using Sanger sequencing. Finally, there were 2 cases where the cobas test was repeated and subsequently yielded results concordant with other samples from that patient. Removing these 8 cases from the analysis leaves 15 cobas-discordant cases. Fortunately, Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) included Sanger sequencing as a second DNA-based mutation detection method. When analyzing the tumor sets for inter-tumor heterogeneity using Sanger sequencing, only 3/15 (20%) cases had discordant mutational results. In all three of the discordant cases, the discordant, wild-type samples contained ≤15% tumor content prior to macrodissection. The authors note that all samples were macrodissected prior to DNA extraction; however, enriching samples with very low tumor content using macrodissection can be technically challenging as this is performed on unstained sections. It may still result in low (or no) tumor content in the dissected sample if not carefully performed. If we consider VE1 IHC to be the most sensitive V600E mutation detection method employed in this study, then their results suggest that Sanger sequencing is more sensitive than the FDA-approved cobas assay. This finding contradicts previous reports that demonstrated that the cobas assay is more sensitive than Sanger sequencing (Halait et al., 2012Halait H. Demartin K. Shah S. et al.Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.Diagn Mol Pathol. 2012; 21: 1-8Crossref PubMed Scopus (130) Google Scholar). This raises concerns about their cobas assay results. If we consider their Sanger sequencing results to be the more accurate DNA-based mutation method in this study, we find a lack of inter-tumor heterogeneity among this sample set. The authors, therefore, cannot conclude that prior reports of inter-tumor heterogeneity—of which several were based on Sanger sequencing or other methodologies using tumor rich samples—are solely due to artifact. With respect to intra-tumor heterogeneity, Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) reported homogenous staining of all 137 positively stained tumor samples, and they assert that their results provide evidence that intra-tumoral heterogeneity does not exist. They suggest that artifacts and necrotic tissue may have a role in the heterogeneous results reported by others. These results, in fact, contradict the findings of other groups including Busam et al., 2013Busam K.J. Hedvat C. Pulitzer M. et al.Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions.Am J Surg Pathol. 2013; 37: 413-420Crossref PubMed Scopus (91) Google Scholar, Heinzerling et al., 2013Heinzerling L. Baiter M. Kuhnapfel S. et al.Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations.Br J Cancer. 2013; 109: 2833-2841Crossref PubMed Scopus (81) Google Scholar and Eriksson et al., 2015Eriksson H. Zebary A. Vassilaki I. et al.BRAFV600E protein expression in primary cutaneous malignant melanomas and paired metastases.JAMA Dermatology. 2015; 151: 410-416Crossref PubMed Scopus (25) Google Scholar who demonstrated heterogeneous staining using the same VE1 antibody. Busam et al., 2013Busam K.J. Hedvat C. Pulitzer M. et al.Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions.Am J Surg Pathol. 2013; 37: 413-420Crossref PubMed Scopus (91) Google Scholar found that 2/10 (20%) superficial spreading melanomas exhibited focal staining of a subpopulation within the tumor suggestive of intra-tumoral heterogeneity. In addition, they found that 6/22 (27%) BRAFmutant metastases had both BRAFV600E-immunopositive and -immunonegative populations within the tumor (Busam et al., 2013Busam K.J. Hedvat C. Pulitzer M. et al.Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions.Am J Surg Pathol. 2013; 37: 413-420Crossref PubMed Scopus (91) Google Scholar). Heinzerling et al., 2013Heinzerling L. Baiter M. Kuhnapfel S. et al.Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations.Br J Cancer. 2013; 109: 2833-2841Crossref PubMed Scopus (81) Google Scholar found strong VE1 staining in one part of a mutation-positive lymph node whereas adjacent tumor cells did not react with the antibody. Eriksson et al., 2015Eriksson H. Zebary A. Vassilaki I. et al.BRAFV600E protein expression in primary cutaneous malignant melanomas and paired metastases.JAMA Dermatology. 2015; 151: 410-416Crossref PubMed Scopus (25) Google Scholar described heterogeneous staining within 10/200 (5%) tumors, most of which were primary melanomas. Clearly, the conclusion that all heterogeneity is due to artifact or technical issues must be explored in future studies. Altogether, the findings by Riveiro-Falkenbach et al. (Riveiro-Falkenbach et al., 2015Riveiro-Falkenbach E. Villanueva C.A. Garrido M.C. et al.Intra- and inter-tumoral homogeneity of BRAFV600E mutations in melanoma tumors.J Invest Dermatol. 2015; 135Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) support the potential use of the VE1 antibody as a triage tool to identify the V600E mutation. IHC negative results, however, still require DNA testing to rule out V600K and other rare V600 mutations, as patients with those less common BRAF mutations may still benefit from BRAF inhibitor therapy (Klein et al., 2013Klein O. Clements A. Menzies A.M. et al.BRAF inhibitor activity in V600R metastatic melanoma.Eur J Cancer. 2013; 49: 1073-1079Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Ultimately, heterogeneity with respect to BRAF mutations is part of a larger story when considering polyclonal variations in gene expression, or analysis of additional mutations to identify tumor subclones. Several studies in breast, prostate, pancreatic, and renal cancers have contributed to the growing body of evidence for tumor evolution leading to biological diversity and heterogeneity. Recent studies in melanoma using next-generation sequencing methods have made similar findings. Ding et al., 2014Ding L. Kim M. Kanchi K.L. et al.Clonal architectures and driver mutations in metastatic melanomas.PloS One. 2014; 9: e111153Crossref PubMed Scopus (58) Google Scholar identified tumor subclones in 11/15 melanomas, as well as evolutionary mutation relationships between four anatomically separate metastases in two different patients. They also showed that mixed treatment responses were associated with different subclonal populations possessing (or not) a BRAF resistance gene mutation. Other studies analyzing tumors removed from patients who progressed on BRAF inhibitor therapy have also revealed mutational tumor heterogeneity. Van Allen et al., 2014Van Allen E.M. Wagle N. Sucker A. et al.The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma.Cancer Discov. 2014; 4: 94-109Crossref PubMed Google Scholar found multiple resistance gene alterations in a single metastatic tumor providing evidence of continued divergent evolution that supports Fidler and Hart, 1982Fidler I.J. Hart I.R. Biological diversity in metastatic neoplasms: origins and implications.Science. 1982; 217: 998-1003Crossref PubMed Scopus (822) Google Scholar polyclonal model. Shi et al., 2014Shi H. Hugo W. Kong X. et al.Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.Cancer Discov. 2014; 4: 80-93Crossref PubMed Scopus (731) Google Scholar analyzed 100 metastatic tumors from 44 patients and found that 9/44 (20%) patients had ≥2 resistance mechanisms in their tumors, and 13/16 (81%) patients with multiple tumors had multiple mechanisms of resistance. With advanced methodologies in molecular pathology, the polyclonal nature of malignant neoplasms including melanoma is becoming more apparent. Although this phenomenon may challenge our current ability to cure patients with melanoma, additional investigations into the mechanisms driving clonal diversity may lead to greater understanding of the molecular pathogenesis of various melanoma subtypes and the development of more effective therapies.
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