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Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, SwedenDepartment of Clinical Pathology, Skåne University Hospital, Lund, Sweden
Cutaneous malignant melanoma (CMM) is one of the most common cancers in the Western world, with a rapidly increasing incidence. Clinically, CMM is a highly heterogeneous disease that is currently staged according to the American Joint Committee on Cancer (AJCC) system, which is based on the thickness of the tumor, ulceration status, mitotic rate, and node involvement (
), and their virtually mutually exclusive occurrence with NRAS mutations, tumors are today molecularly subgrouped by BRAF and NRAS mutation status. This mutation status is believed to correlate with histological and clinical tumor phenotypes (
). Molecular classification of CMM based on gene expression profiles was recently described, identifying four distinct phenotypes (proliferative, high-immune, normal-like, and pigmentation) independent of BRAF or NRAS mutations and associated with patient outcome independent of other prognostic factors (
). The most common high-penetrance melanoma susceptibility gene is CDKN2A, located at chromosome band 9p21. Distinct CDKN2A founder mutations exist, including the Swedish p.R112_L113insR and the Dutch c.225-243del19 mutations (
In this study, we aimed to determine whether primary melanomas from CDKN2A mutation carriers exhibit a distinct gene expression signature. To this end we collected formalin-fixed paraffin-embedded primary CMMs from five centers in Europe and Australia, including 237 sporadic cases and 43 tumors from CDKN2A carriers, with the addition of 6 normal skin and 11 nevus specimens (n=297 in total) (Table 1). Among CDKN2A carriers, 31 out of 43 CDKN2A carriers harbored the Swedish founder mutation p.R112_L113insR and five harbored the Dutch founder mutation c.225-243del19. The sporadic CMMs consisted of samples from a consecutive Swedish cohort (thickness >0.8 mm to ensure sufficient DNA and RNA, n=225) collected at the Department of Pathology, Lund University, as previously described (
), and 12 sporadic cases from Brisbane, Leiden, and Stockholm. From the 297 samples RNA and DNA were extracted, and global gene expression data were obtained using the WG-DASL technique as described (
). p16 nuclear and PTEN immunohistochemical (IHC) analysis was performed on 13 CDKN2A and 152 sporadic cases, respectively (Supplementary Information online). The study was approved by the Ethics Review Boards of each participating center according to the Declaration of Helsinki guidelines.
Table 1Clinicopathological and molecular features of sporadic melanomas and melanomas from CDKN2A mutation carriers
) and Clark’s level of invasion and Breslow thickness (borderline significant) differed between the CDKN2A and sporadic cohorts (Table 1). Moreover, 54% and 9% of CDKN2A carriers had mutations in BRAF and NRAS, respectively, compared with 29% and 18% in sporadic cases (P=0.03). However, this difference in mutation frequency between the groups was lost when adjusting for thickness and age at onset, representing features previously associated with BRAF mutation (
reporting no difference in BRAF and NRAS mutation frequencies between CDKN2A carriers and matched sporadic cases.
To investigate whether CDKN2A mutation carriers displayed gene expression patterns different from sporadic CMM, we applied principal component analysis (PCA) to the expression data from the 297-sample cohort, finding that CDKN2A carriers were firmly intermingled with sporadic cases (Figure 1a). In addition, PCA showed that the main variation in gene expression in CMM was related to specific clinicopathological factors (tumor thickness and histology) and gene expression phenotypes, whereas CDKN2A mutation status contributed little to the overall variation (Supplementary Figure 1 online). To further investigate the molecular landscape of melanomas from CDKN2A carriers, we classified the 280 familial and sporadic tumors according to our reported gene expression phenotypes (
). Here, we found overall similar frequencies of gene expression phenotypes in the CDKN2A and sporadic cohorts, but with a slightly higher frequency of normal-like melanomas in the CDKN2A cohort (P=0.02, Table 1, Figure 1b). However, after adjustment for thickness and age at onset, this statistical difference was lost. Finally, we searched for differentially expressed genes between CDKN2A carriers and sporadic CMMs. On the basis of significance analysis of microarrays (SAM) only seven genes showed a significant difference (q-value=0) (Figure 1c). Notably, the maximum fold change of these genes was <1.4, suggesting very subtle changes not inconsistent with random observations. Moreover, to determine the validity of the differences in these seven genes, an independent CMM cohort including CDKN2A mutation carriers needs to be analyzed. Although gene expression differences stratified on somatic CDKN2A mutation status have been observed in other tumor types (
Diffuse large B-cell lymphomas with CDKN2A deletion have a distinct gene expression signature and a poor prognosis under R-CHOP treatment: a GELA study.
), our gene expression analyses suggest that CMMs from CDKN2A germline mutation carriers do not constitute a distinct gene expression subtype and that only subtle, if any, gene expression differences characterize melanomas from CDKN2A carriers.
Figure 1Gene expression landscape of melanomas from CDKN2A mutation carriers compared with sporadic melanomas. (a) Principal component analysis (PCA) 3D-plot highlighting the gene expression profile similarities between primary melanomas from CDKN2A carriers (black dots) and sporadic cases (yellow dots). mRNA levels for 7,752 genes in 297 samples, including 17 normal skin and nevi (light-gray dots), are included in the analysis. (b) Classification of primary melanomas in the four gene expression phenotypes based on 270 genes described by
. Melanoma tumors (n=280) are ordered according to gene expression phenotype, CDKN2A mutation status, and centroid correlation coefficient. Melanomas from CDKN2A mutation carriers (n=43) are denoted by black bars. (c) Genes differentially expressed between melanomas from CDKN2A carriers and sporadic cases. Fold change is unlogged and is relative to the CDKN2A carriers; that is, STAT4 is downregulated in melanomas from carriers as compared with sporadic cases. (d) Comparison of molecular and clinical features in different subsets of melanomas.
Results from the gene expression analyses are further corroborated by p16 nuclear IHC analysis of 152 sporadic cases from our cohort, wherein 125 demonstrated a complete loss of p16 nuclear staining (Table 1 and Figure 1d). This suggests that loss of nuclear p16 protein is an early event in melanomagenesis and may partly explain why melanomas from CDKN2A carriers are molecularly almost indistinguishable from sporadic cases. Moreover, in melanoma tumors the MAPK and the PI3K pathways are commonly altered (
). PTEN, a regulator of the PI3K pathway, is a major tumor suppressor in melanoma, and PTEN gene alterations are commonly found in CMM. However, we found no difference in PTEN protein expression by IHC between CDKN2A carriers and sporadic cases (P=0.08, Table 1 and Figure 1d), indicating that deficiencies of the PI3K pathway occur in both sporadic and familial melanoma.
In conclusion, this is the only study, to our knowledge, investigating genome-wide molecular changes in primary melanomas from CDKN2A mutation carriers. Although larger numbers of familial tumors could potentially identify additional subtle molecular alterations between familial and sporadic cases, our results strongly suggest that melanomas from CDKN2A mutation carriers are not associated with a distinct gene expression signature and that overall they have similar molecular characteristics to sporadic melanomas.
Acknowledgments
We thank Kristina Lövgren and Björn Nodin for assistance with TMA construction and immunohistochemical staining. This project received support from the European Commission under the 6th Framework Programme, Contract: LSHC-CT-2006-018702 (GenoMEL), the Swedish Cancer Society, the Swedish Research Council, the Nordic Cancer Union, the Berta Kamprad Foundation, the Gunnar Nilsson Cancer foundation, the Gustav Vth Jubilee foundation, BioCARE, the National Health and Medical Research Council of Australia, and governmental funding for medical health research (ALF).
Diffuse large B-cell lymphomas with CDKN2A deletion have a distinct gene expression signature and a poor prognosis under R-CHOP treatment: a GELA study.
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