Low expression of pro-apoptotic proteins Bax, Bak and Smac indicates prolonged progression-free survival in chemotherapy-treated metastatic melanoma

Abstract

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

Fig. 1. Low expression of pro-apoptotic proteins Bax, Bak and Smac correlates with increased progression-free survival (PFS) in chemotherapy-treated metastatic melanoma.

a Tissue cores stained by IHC for Bax, Bak and Smac. Representative original (left panels) and mark-up images (right panels) of cores with low, medium and high expression of the three proteins are shown. The mark-up images were quantified to compute automated H scores. Table insert shows cohort information. b Distribution of H scores across the analysed melanoma tissue cores. Only stained cores that passed the quality control were retained for subsequent analyses (Bax n = 100 cores from 52 patients, Bak n = 100 cores from 51 patients, Smac n = 104 cores from 53 patients). c Survival analysis based on H scores for Bax, Bak and Smac. Median H scores were used as cutoff to separate the patients with high (red line) and low (blue line) expression of each protein. Log-rank test was used to compare the Kaplan–Meier curves for progression-free survival from the date of sample procurement.

Fig. 2. Manual scoring confirms association of low Bak, Bax and Smac protein expression with improved PFS.

a Correlation between automated and manual H scores. Manual scores were obtained from two independent pathologists blinded to patient follow-up. Correlation was analysed using Spearman’s rank correlation coefficient. b Median H scores were used as cutoff to separate the patients with high (red line) and low (blue line) expression of each protein. Log-rank test was used to compare the Kaplan–Meier curves for progression-free survival from the date of sample procurement.

Fig. 3. Combined low expression of Bax, Bak and Smac is a combinatorial marker candidate for improved progression-free survival.

a Expression profiles based on the H scores of Bax, Bak and Smac (blue: automated H score below median, red: above median) in n = 50 patients. b Survival analysis for the cohort based on the expression profiles shown in a. Log-rank test for trend was used to compare three Kaplan–Meier curves representing patients with low Bax, Bak and Smac H scores (blue) vs. mixed and high expression (black and red, respectively).

Fig. 4. TCGA-SKCM-based analysis validates the prognostic Bax, Bak, Smac signature.

Independent validation of the prognostic signature at transcriptome level in the SKCM-TCGA metastatic sub-cohort. a Survival analysis in the SKCM-TCGA sub-cohort (n = 79 patients diagnosed with stage III or IV metastatic melanoma before 2010). An optimised chi-square-based cutoff was determined to divide patients with high (red) and low (blue) normalised BAX, BAK1 and DIABLO (Smac) expression (log₂(FPKM-UQ + 1)). Kaplan–Meier curves (follow-up from sample procurement) were compared by log-rank test. b mRNA amounts for BAX, BAK1 and DIABLO (Smac) (blue: mRNA level below cutoff, red: mRNA level above cutoff). c Survival analysis in the metastatic TCGA-SKCM sub-cohort based on the expression profiles in Fig. 4b. Log-rank test for trend was used to compare three Kaplan–Meier curves representing patients with combined low BAX, BAK1 and DIABLO (Smac) expression (blue), combined high expression (red) or with mixed expression (black).

Fig. 5. Pattern recognition allows predicting patient prognosis.

a A principal component analysis was performed on the H scores of nine apoptotic proteins. To provide a visualisation of the spatial clustering, patients samples were positioned in a 3D scatter plot defined by the first three principal components and colour-coded according to their PFS time (red < 12 months, n = 33; blue > 12 months, n = 17). Linear discriminant analysis (LDA) correctly segmented 72% of the patients. Leave One Out Cross Validation (LOOCV) combined with LDA predicted the correct class for 74% of the patients. b 3D scatter plot showing the spatial clustering of patients with short and long PFS based on the H scores for Bax, Bak and Smac. LDA correctly segmented 80% of the patients and LOOCV-LDA achieved 78% prediction accuracy. c Comparison of the performance of the two classifiers shown in a and b. The receiver operating characteristic curves (ROCs) and respective areas under the curve (AUC) are shown.