In this retrospective study of 364 melanoma patients, we identified an independent seven-marker signature of prognosis. Notably, the predictive power of the signature was carefully validated and confirmed on a secondary independent external test cohort (n
225). With a total of 27,055 specimens of primary MMs analyzed by IHC, this TMA study is unmatched in the literature. An individual patient’s risk score can easily be calculated given the immunoreactivity scores for the seven markers and the estimated coefficients (). shows stained TMA specimens illustrating the Seven-Marker Signature for a patient with a high-risk and another patient with a low-risk melanoma.
Immunohistochemically stained TMA specimens illustrating the Seven-Marker Signature for a patient with a high-risk and another patient with a low-risk melanoma.
One of the main statistical problems in large scale IHC studies are missing values in the design matrix due to missing or corrupt spots on the TMA. The more markers are investigated the higher the chance that at least one value is missing per patient. Frequently this problem is tackled by either sacrificing a larger number of patient records or by employing volatile multiple imputation techniques. In this study 9.3% of values were missing, reducing the set of patients with all IHC measurements from 364 to 170. Algorithms like random survival forests 
and ensemble learning with gradient boosting 
are capable of dealing with missing values, but lead to models that are not intuitively interpretable and difficult to implement in clinical practice. To overcome these problems we employed a learning procedure which is invariant to missing values and results in an easily interpretable and practically applicable linear model.
In early disease stages, application of an IHC based test to examine a MM patient’s tumor tissue at a molecular level suggests itself. Even though melanoma was among the first cancers recognized as a target for practical application of microarray analysis starting in 1996 
, the transition of gene expression results to diagnostic applications with clinical impact has not been shown yet in MM. Microarray studies produced a plethora of data and have provided useful insights into the molecular biology of melanoma (reviewed in 
). However, analysis of different histological subtypes of melanoma, expression analysis of mainly melanoma metastases, and lack of homogeneity of patient cohorts hampered the interpretation of these data 
. Furthermore, routine supply of fresh frozen MM tissue for microarray based assays seems virtually impossible. Also, use of RT-PCR based tests are complicated by the need for pure populations of neoplastic melanoma cells, and has not yet resulted in a breakthrough in melanoma diagnosis or management of melanoma patients. Our defined seven-marker signature was of independent prognostic relevance in two independent patient cohorts. For the practitioner, the assessment of this set of seven IHC markers promises to be a helpful tool to answer the crucial question “whom to treat, and how to treat”, especially in the adjuvant setting after surgical excision of early-stage and localized primary MM (Stage I to IIa). With MTAP, COX-2 and Bcl-X, three markers of the seven-marker signature offer direct therapeutic implications, since the corresponding drugs have already been approved by the FDA.
Currently, in the adjuvant treatment of MM, interferon alpha is the only clinically accepted therapeutic agent providing a significant (recurrence-free) survival benefit for a small but distinct percentage of patients 
. On account of the serious side effects and the high costs of the therapy, only those patients with a realistic chance to benefit from interferon should receive this treatment. We have recently shown that there is a clear association between MTAP expression in the primary melanoma and melanoma progression and, even more importantly, response to interferon treatment 
. This gives rise to the hypothesis that interferon response may be correlated with the expression of interferon response genes such as MTAP.
Cyclooxygenase 2 (COX-2) may represent another promising therapeutic target. Cyclooxygenases catalyze the first rate-limiting step in the conversion of arachidonic acid to prostaglandins. COX-2 is expressed in various tumor types and levels of COX-2 expression have been shown to correlate with invasiveness and prognosis in some tumor entities, including epithelial and melanocytic skin cancer 
. So far the benefit of COX-2-inhibitors has not been studied in the adjuvant treatment of early-stage melanomas to prevent metastasis. In the second-line treatment of advanced metastatic melanoma disease, however, a survival benefit was shown for targeted combined therapy using COX-2 inhibitors and PPARG-agonists for anti-inflammatory treatment together with low-dose metronomic chemotherapy 
. Considering this observation and the fact that melanoma patients with COX-2-positive primary tumors bear a significantly higher risk of tumor recurrence 
, introduction of COX-2 inhibitors for primary adjuvant treatment of these patients seems obvious.
One additional marker, Bcl-X, has been targeted in preclinical tests and several targeting agents are in the clinical testing phase by now 
: Bcl-X is related to the anti-apoptotic Bcl-2 protein family. Overexpression of these anti-apoptotic proteins protects cancer cells against death signals of apoptosis. Interestingly, tumors expressing high levels of Bcl-2 or Bcl-X are often found to be resistant to chemotherapeutic agents or radiation therapy 
. In recent years, non-peptidic cell permeable “small molecule inhibitors” (SMIs) against antiapoptotic proteins like Bcl-2 or Bcl-X have been identified. SMIs inhibit distinct protein-protein interactions by blocking specific binding sites of the target molecule, thus supporting the apoptotic machinery 
. Inhibition of Bcl-X may exert a synergistic effect with conventional treatments like chemo- or radiation therapy. Regarding MM therapy, this effect would be a decisive therapeutic success.
Presence of CD20-positive B-lymphocytes within or adjacent to MM tissue ( ) was among the top seven biomarkers. However, the role of B-lymphocyte infiltration in MM is unknown and needs further investigation. The CD20-antigen is known to be an effective therapeutic target in the treatment of patients with CD20-positive B-Cell-Non-Hodgkin-Lymphomas. The monoclonal chimeric antibody Rituximab is indicated for alternative immunotherapy 
. In MM, several subpopulations - some with stem cell-like characteristics - have been described including one with expression of CD20 (reviewed in 
). In our study, only very few cases showed infiltrating CD20 positive B-lymphocytes (), comprising a very narrow dynamic range of this marker. However, even a six-marker signature without CD20 was significantly associated with overall and recurrence-free survival ().
High resolution images of case no. 137 on the tissue microarray.
The Six-Marker Signature (without CD20) and Survival of Patients with Malignant Melanoma.
The tumor-suppressor gene phosphatase and tensin homolog (PTEN) is one of the most commonly inactivated genes in human cancer and has been identified as lost or mutated in melanoma 
. An established consequence of PTEN inactivation is the constitutive aberrant activation of the phosphatidylinositol-3-kinase (PI3K)-signaling pathway that drives uncontrolled cell growth, proliferation, and survival 
. In general, prediction of PI3K-signaling pathway activation based on PTEN IHC expression status is unfeasible since inactivation of PTEN is achieved by either gene mutation or deletion 
. Figure S5
shows that some melanoma cell lines show PTEN expression and concomitant activation of the PI3K/AKT cascade. Alterations of the tumor suppressor PTEN have already been linked with disease outcome in patients with MM: Mikhail et al. have shown that loss of nuclear PTEN expression was associated with aggressive tumor behavior 
. In contrast, we could show that strong cytoplasmic PTEN expression was found only in high risk patients.
Most oncological findings regarding the Wnt/β-Catenin signaling are derived from the analysis of colon, breast and kidney carcinoma 
where activation of the pathway has been directly implicated in disease pathogenesis. The majority of colorectal carcinomas carry inactivating mutations in the Adenomatous polyposis coli (APC) tumor suppressor which lead to stabilization of β-Catenin, mimicking Wnt stimulation. Additionally, mutations in the β-Catenin gene CTNNB1
were found in colon cancer leading to the constitutive activation of β-Catenin/LEF/TCF-dependent canonical signaling 
. However, such mutations are rarely found during melanoma development. In contrast to findings in colon carcinoma and in line with a study by Kuphal et al. 
, we show that β-Catenin protein was basically cytoplasmic in melanomas in vivo.
Regarding our risk score, loss of cytoplamic β-Catenin expression was associated with worse outcome of melanoma patients. Kuphal and co-workers also demonstrated that the transcriptional activity of β-Catenin regulating expression of β-Catenin target genes was not observed in several melanoma cell lines, suggesting a cell type specific regulation of β-Catenin function 
According to the data presented here, the seven-marker signature might serve as a prognostic tool enabling physicians to selectively triage, at the time of diagnosis and initial surgery, the subset of high recurrence risk stage I–II patients for adjuvant therapy. Selective treatment of those patients that are more likely to develop distant metastatic disease could potentially lower the burden of untreatable metastatic melanoma and revolutionize the therapeutic management of MM. Prospective clinical trials are necessary to validate the prognostic and therapeutic value of this seven-marker signature and its benefit for routine clinical assessment of MM. The utility of the algorithm in a practice setting, where full heterogeneous tissue sections are used, is currently being analyzed in a prospective clinical trial at the Department of Dermatology, University of Regensburg, Germany.