Molecular profiling of patient-matched brain and extracranial melanoma metastases implicates the PI3K pathway as a therapeutic target

Abstract

Purpose: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches.

Experimental design: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors.

Results: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases.

Conclusions: These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors.

Fig. 1

Copy number variation (CNV) profiling of brain metastases and extracranial metastases. (A) CNV histograms of 10 brain metastases (BM) and the matched 10 extracranial metastases (EM). Frequencies of CN gains (blue, pointing up) and CN losses (red, pointing down) were plotted to their genomic locations. (B) Results of unsupervised hierarchical clustering analysis of CNVs in the matched 10 BM and 10 EM. Genome-wide CN gains (light blue=one copy gain, dark blue=high copy gain) and losses (light red=one copy loss, dark red=homozygous copy loss) in each sample are plotted next to the dendrogram. BM are in orange and EM are in black; matched samples are connected by arced lines. Solid lines: matched samples clustering together; dotted lines: matched samples clustering apart. (C) CNV frequencies of 13 genes in matched 10 BM (orange) and 10 EM (black). Genes are sorted by the mean CNV frequency in BM and EM, with high on top and low on bottom.

Fig. 2

Gene expression profiling of brain and extracranial metastases. (A) Hierarchical clustering of log2 expression values of 8050 probes in 27 brain metastases (BM, orange) and 25 extracranial metastases (EM, black). Probes that showed less than 1.5-fold change from the median in more than 80% of the samples were excluded. Matched tumors are connected with arcs. (B) Differences in expression of 13 genes between paired BM and EM. Fold changes of gene expression from EM to BM were converted into log2 ratios. Log2 ratios in six pairs of samples were plotted as dots for each gene, means and standard deviations were plotted as horizontal bars. (C) Expression of SGK3, SGSM2 and ELOVL2 in matched BM and EM of six patients. Each column represents the expression value of one sample. (D) Expression of SGK3, SGSM2 and ELOVL2 in unmatched BM (N=21) and EM (N=19). Each dot represents the expression value of one sample.

Fig. 3

Protein expression profiling of brain and extracranial metastases. (A) Unsupervised hierarchical clustering of RPPA data from 152 proteins in nine brain metastases (BM, orange) and 20 extracranial metastases (EM, black). Matched tumors are connected by arced lines. The cyan box highlights five phospho proteins of the PI3K/AKT pathway that clustered together. (B) Log2 expression levels of AKT_pS473 in seven pairs of matched metastases. Each column represents log2 expression value in one tumor. (C) Log2 expression levels of PTEN in seven pairs of matched tumors. (D) Unsupervised hierarchical clustering of 29 unmatched tumors using expression data of five phospho proteins highlighted in (A), which are activation markers of the PI3K/AKT pathway. The magenta box highlights a cluster of tumors with high PI3K/AKT pathway activity.

Fig. 4

Immunohistochemistry (IHC) of PI3K/AKT pathway proteins in matched melanoma metastases. (A) PTEN IHC results in 20 pairs of brain metastases (BM) and matched extracranial metastases (EM) were categorized and the incidences displayed. (B) Log2 ratio of GSK3α/β_pS21/S9 percentage-adjusted intensity between BM and EM in each of the 26 patients. Patients were sorted from high to low percentage-adjusted intensity. (C) Images of GSK3α/β_pS21/S9 IHC in the BM and EM of patient 57. BM_57 was photographed at a lower magnification than EM_57 to show the negative stroma.