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, 7 (6), e38222

A Seven-Marker Signature and Clinical Outcome in Malignant Melanoma: A Large-Scale Tissue-Microarray Study With Two Independent Patient Cohorts

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A Seven-Marker Signature and Clinical Outcome in Malignant Melanoma: A Large-Scale Tissue-Microarray Study With Two Independent Patient Cohorts

Stefanie Meyer et al. PLoS One.

Abstract

Background: Current staging methods such as tumor thickness, ulceration and invasion of the sentinel node are known to be prognostic parameters in patients with malignant melanoma (MM). However, predictive molecular marker profiles for risk stratification and therapy optimization are not yet available for routine clinical assessment.

Methods and findings: Using tissue microarrays, we retrospectively analyzed samples from 364 patients with primary MM. We investigated a panel of 70 immunohistochemical (IHC) antibodies for cell cycle, apoptosis, DNA mismatch repair, differentiation, proliferation, cell adhesion, signaling and metabolism. A marker selection procedure based on univariate Cox regression and multiple testing correction was employed to correlate the IHC expression data with the clinical follow-up (overall and recurrence-free survival). The model was thoroughly evaluated with two different cross validation experiments, a permutation test and a multivariate Cox regression analysis. In addition, the predictive power of the identified marker signature was validated on a second independent external test cohort (n=225). A signature of seven biomarkers (Bax, Bcl-X, PTEN, COX-2, loss of β-Catenin, loss of MTAP, and presence of CD20 positive B-lymphocytes) was found to be an independent negative predictor for overall and recurrence-free survival in patients with MM. The seven-marker signature could also predict a high risk of disease recurrence in patients with localized primary MM stage pT1-2 (tumor thickness ≤2.00 mm). In particular, three of these markers (MTAP, COX-2, Bcl-X) were shown to offer direct therapeutic implications.

Conclusions: The seven-marker signature might serve as a prognostic tool enabling physicians to selectively triage, at the time of diagnosis, 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.

Conflict of interest statement

Competing Interests: SM, TJF, AKB, and PJW are inventors on pending patent B70261. FH, AP, VR, J. Brandner, IM, NA, J. Buhmann, KI, HM, ML, and TV have declared that no competing interests exist. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Hazard Ratios of the Nine-Marker Signature learned by the FDR selection procedure.
Markers with a hazard ratio smaller than 1.00 represent “protective markers” (MTAP, β-Catenin). Those with hazard ratios larger than 1.00 represent “risk markers” (Bax, Bcl-X, infiltration with CD20 positive B-lymphocytes, CD49d, COX-2, MLH1 and PTEN).
Figure 2
Figure 2. The Seven-Marker Signature and Survival of 362 Patients with Primary MM.
Panels A and B show Kaplan-Meier estimates of overall and recurrence-free survival for high risk patients (red) and low risk patients (green) from the primary cohort according to the final seven-marker signature (Panels A, B). Equality in survival expectance of the subgroups is assessed by the log-rank test. The difference between high risk patients and low risk patients is highly significant (p<0.001) for the seven-marker signature. Panel C shows the IHC expression profiles of 362 tumor specimens from the primary cohort ordered by their predicted risk score. Each column represents an individual patient consisting of the expression values of the seven-marker signature (5 risk markers and 2 protective markers). The magnitude of the corresponding risk score is plotted below for 181 low risk patients (green) and 181 high risk patients (red). IHC expression values were scaled between 0 (light blue) and 1 (dark blue) for plotting only. White cells represent missing values (n.a.).
Figure 3
Figure 3. Dot blots of risk scores for different Clark levels in the training (Panel A) and testing (Panel B) cohort.
Horizontal lines represent median risk scores for each subgroup.
Figure 4
Figure 4. Dot blot of risk scores for the various histological subtypes of melanoma as classified by the ICD-10 (International Statistical Classification of Diseases and Related Health Problems, 10th revision).
SSM, superficial spreading melanoma; LMM, lentigo maligna melanoma; NM, nodular melanoma; NOS, not otherwise specificed; ALM, acral lentiginous melanoma. Horizontal lines represent median risk scores for each subgroup.The aim of this study was to provide a maximum of prognostic and therapeutically relevant information by a minimum of markers combined in a clear signature. For the sake of clinical feasibility and cost saving, an IHC marker set suitable for routine clinical assessment should be based on a limited number of antibodies. Accordingly, the nine-marker signature was reduced by the risk marker with the lowest Cox regression coefficient β, i.e. MLH1 (β = 0.254).
Figure 5
Figure 5. Statistical Analyses. Panel A, B. The Seven-Marker Signature and Survival of Patients with a Tumor Thickness ≤2.0 mm (TMA 1).
Kaplan-Meier estimates show a significantly lower overall (p<0.01, Panel A) and recurrence-free survival (p<0.01, Panel B) for patients with a comparatively low tumor thickness ≤2.0 mm but high-risk score. Panel C, D. Leave-One-Out Cross Validation. To investigate the generalization error of the models produced by the FDR signature learning procedure a leave-one-out cross validation experiment was conducted on the primary cohort of 362 MM patients. The resulting risk score could significantly (p<0.001) differentiate between patients with higher or lower overall survival expectance. The two patient groups also significantly (p<0.01) differ in recurrence-free survival. Panel E, F. 10-Fold Cross Validation. In addition, the FDR marker selection procedure was tested by a 10-fold cross validation experiment on the 362 patients of the primary cohort (TMA 1) resulting in still significant estimates for overall survival (p<0.001; Panel E) and recurrence-free survival (p<0.05; Panel F). Panel G. Permutation Test. In addition to the cross validation experiments a permutation test was conducted to assess if the signature learning procedure is over fitting the data set. The resulting signature, which was learned on permuted overall survival data, was not able (p = 1) to discriminate between patients with differing survival expectance. This result indicates that the proposed learning procedure does not over fit the data. Panel H. Coefficients and Confidence Intervals of the Seven-Marker Signature. The coefficients from the univariate Cox proportional hazard models are used in a weighted linear combination to predict the risk score for each patient. Markers with negative coefficients represent protective markers (MTAP, β-Catenin); those with positive coefficients risk markers (Bax, Bcl-X, PTEN, COX-2, and presence of CD20 positive lymphocytes,).
Figure 6
Figure 6. Validation of the Seven-Marker Signature and the FDR Marker Selection Procedure.
Kaplan-Meier estimates of overall (Panel A) and recurrence-free survival (Panel B) for the independent external test cohort of 225 patients (TMA 2) confirm the predictive prognostic power of the signature (p<0.001).
Figure 7
Figure 7. Immunohistochemically stained TMA specimens illustrating the Seven-Marker Signature for a patient with a high-risk and another patient with a low-risk melanoma.
The low-risk melanoma (Column C) showed a strong cytoplasmic staining for β-Catenin and MTAP, respectively. Immunoreactivity of these two protective markers was not found in the high-risk melanoma (Column D). In contrast, the high-risk melanoma demonstrated a moderate to strong cytoplasmic staining for Bax, Bcl-X, PTEN, COX-2, and infiltration with CD20 positive B-lymphocytes.
Figure 8
Figure 8. High resolution images of case no. 137 on the tissue microarray.
Serial sections of the tissue microarray (TMA 1) was immunohistochemically stained with CD20 (Panel A, B) and HMB45 (Panel C, D) to show CD20 positive B-lymphocytes within and adjacent to melanoma cells (case no. 137).
Figure 9
Figure 9. The Six-Marker Signature (without CD20) and Survival of Patients with Malignant Melanoma.
Kaplan-Meier estimates show a significantly lower overall (p<0.00001, Panel A) and recurrence-free survival (p<0.01, Panel B) for melanoma patients with high-risk score.

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