In this study we demonstrate that histopathological features of the primary tumor, many of which have been part of the WHO classification, provide substantial information on the mutation status of an important melanoma gene, BRAF. We found that phenotypic features such as increased upward scatter and nest formation of intraepidermal melanocytes; thickening of the involved epidermis; sharp demarcation from the surrounding skin; as well as the presence of a larger, rounder, and more pigmented tumor cells were distinguishing features of melanomas with BRAF mutation. This finding suggests that further research will reveal additional phenotypic associations with other genetic factors, including both germ line constitution of the patient and the somatic alterations in the tumor. In general it is not to be expected that single morphological parameters will be associated with individual genetic factors since it is likely that there will be interactions, or partial or functional redundancy, among them.
Results of genotype-phenotype studies can be applied in two directions. Starting with the genetics, the resulting phenotypes may allow generation of hypotheses concerning the function of a genetic factor. For example, our observation of increased melanin pigment in the tumor cells in melanomas with BRAF
mutations is in line with recent reports of cooperation between BRAF
and MITF in melanoma development. MITF is an important transcriptional regulator of pigmentation that is amplified in a subset of melanomas and cooperates with BRAF
in transforming immortalized human melanocytes [27
]. A link between BRAF
and pigmentation is further supported by a recent independent observation that BRAF
-mutant melanomas are also more pigmented macroscopically [28
]. Our study also identified strong associations between upward scatter of intraepidermal melanocytes and formation of nests and BRAF
mutation. Upward scatter is unlikely to be entirely attributable to BRAF
mutations, as scatter is typically absent in nevi, many of which have BRAF
], suggesting that other genes cooperating with BRAF
may be involved. By contrast, many nevi also show melanocytes arranged in nests and are sharply circumscribed, features that are also more frequent in BRAF
-mutant melanomas. Studies to determine which of the features found here to be associated with BRAF
mutant melanomas show similar genotype-phenotype associations in nevi are currently underway.
In the other direction, phenotypic characteristics that are conventionally assessed in the clinic can be used to develop a practical classification system in which the classes are genetically homogeneous, and thus more likely to provide information relevant to patient management. Such a system may provide valuable, outcome-related information from its classes even before the genetic factors of each class are completely understood. Such a classification system is likely to have an important clinical impact. To achieve this goal the practicability and interobserver reproducibility of the criteria that we defined need to be assessed in a clinical setting and the classification algorithms validated on independent cohorts of cases. Such efforts have been initiated and shown good to excellent interobserver agreements in scoring the features used in this study among expert pathologists (to be published separately).
Among the few variables in our study that overlapped with data recorded in large patient registries, age was the variable that best predicted BRAF
status. A strong association between patient age at diagnosis and BRAF
mutation status has been reported previously by us and others [11
]. Using the age cutoff of 55 y determined in our experimental cohort, we found a significant difference in disease-specific and relapse-free (unpublished data) survival in an independent clinical cohort of 4,785 patients. Several previous studies including our own [11
] have not found significant survival differences between melanomas with and without BRAF
mutation. However, the numbers of patients in these studies are comparably small, and this simple cutoff may be reflecting currently unknown genetic effects that interact with or substitute for BRAF
mutations, and thus reveal significantly different clinical behavior. Future studies will be required to better understand the factors involved in this survival difference, hopefully resulting in improved ability to predict the future course of a particular tumor.
The presence of NRAS
mutations was not strongly associated with the phenotypic measurements we performed. In the univariate analysis the majority of the OR point estimates for association with NRAS
mutation are close to one, and so if true associations are present we would expect them to be much weaker than for BRAF
. Multivariate analyses did not significantly enhance the prediction power. This dissimilarity could reflect differences in signaling between BRAF
, with NRAS
having more pleiotropic effects. Larger numbers of cases and additional knowledge of equivalent genetic alterations in melanomas without BRAF
mutations are required to evaluate whether some of the features used here and/or others can further increase the discrimination between genetically defined subtypes. We have recently described activation of KIT by mutation and/or copy number increases in mucosal, acral, and CSD melanomas [13
]. Too little tissue or DNA was left from the current cohort of cases to test for associations between KIT aberrations and any of our features.
Disease classification evolves from descriptions of a combination of symptoms (syndromes), to more refined definitions that integrate underlying causes. The increase in knowledge of underlying causative genetic alterations in melanoma offers an opportunity to reassess the syndromic classification scheme that emerged from the Sydney classification [3
] and its revision [4
] into the current WHO classification. The genotype-phenotype correlations discovered in our study testify to the intuition of the originators of the morphological classification scheme. The observation that simple combinations of features could predict BRAF
mutation status independent of, and even within WHO subtypes, indicates that the morphological criteria we used can be employed to refine the existing classification, providing an opportunity to identify disease subsets that are genetically more homogenous. Our result represents but one step towards improved classification, and we expect it to be modified in light of future discoveries.
In the distant future when genetic knowledge is complete and targeted therapeutic options are numerous, the appropriate clinical work up of a patient is likely to be quite different from current practice. While initial diagnosis and aspects of staging will require histopathology, more subtle distinctions will employ molecular analysis. Reaching this goal requires following a complex, uncertain path of incremental advances built on new discoveries and continuous refinement of perceived relationships among disparate types of knowledge. Here we have investigated the relationship of elementary histopathological characteristics of melanomas to the mutation status of two genes, BRAF and NRAS. A strong relationship was found for the first but not for the second. As more studies are performed, additional strong genotype-phenotype relationships may be found, more subtle effects of genetic interactions and complementation may be revealed, and consequences of these factors for clinical behavior of individual tumors may be elucidated. As this process unfolds, phenotypic characteristics will become progressively more useful as surrogates for missing genetic knowledge. Thus development of a melanoma classification system that combines analysis of known genetic factors with histopathology may produce a clinically powerful method for managing individual patients and guiding research in the immediate future.