Protein signatures for survival and recurrence in metastatic melanoma

Abstract

Patients with melanoma metastatic to regional lymph nodes exhibit a range in tumor progression, survival, and treatment. Current approaches to stratify patients with this stage of disease predominantly involve clinical and histological methods. Molecular classification thus far has focused almost exclusively on genetic mutations. In this study, proteomic data from 69 melanoma lymph node metastases and 17 disease free lymph nodes acquired by histology-directed MALDI imaging mass spectrometry were used to classify tumor from control lymph node and to molecularly sub-classify patients with stage III disease. From these data, 12 survival associated protein signals and 3 recurrence associated signals in the acquired mass spectra were combined to generate a multiplex molecular signature to group patients into either poor or favorable groups for recurrence and survival. Proteins represented in the signature include cytochrome c, s100 A6, histone H4, and cleaved forms of thymosin β-4, thymosin β-10, and ubiquitin. In total over 40 protein signals from the tissue were identified.

Fig. 1

A) Histology-directed MALDI IMS. Cellular regions are selected by a pathologist to ensure melanoma foci are targeted. B) The MALDI spectra from these regions were averaged and compared by SAM (control LN versus tumor-positive LN) and by CPH to determine proteins associated with survival and recurrence.

Fig. 2

H & E stain of stage III melanoma infiltrating lymph node. Brown pigmentation and enlarged nuclei characterize melanoma cells, while lymphocytes appear with much smaller nuclei and with little cytoplasm. Dashed circles indicate the area of a typical 200 μm diameter dried MALDI sample spot.

Fig. 3

Comparison of protein signals between control lymph nodes and metastatic melanoma. A) Comparison of averaged spectra of control lymph nodes (black) and melanoma lymph node metastases (red). B) SAM analysis revealing select differentially expressed proteins between LN and melanoma. *Indicates truncated molecular form with 2 amino acids absent from the carboxy-terminus.

Fig. 4

A) Model algorithms to classify control lymph node and melanoma lymph node metastases by their proteomic signature. B) 3D PCA plot of control lymph node (green) and melanoma lymph node metastases (red).

Fig. 5

Training and test patient groups were used to generate and validate survival and recurrence compound predictor (CP) protein signatures. The CP was generated from the sum of the significant proteins in the training set, weighted by the proteins’ predictive ability, for both survival and recurrence. A) top: the patients in the training set were ranked and divided by their CP score into 2 groups: “poor” (med. survival ≤12 months) and “favorable” (med. survival >12 months). A) bottom: CP range for “poor” and “favorable” survival was evaluated in the test set, showing comparable median 12 month survival in the “poor” group. B) top: recurrence CP scores divided the training set into “favorable” (med. rec. >12 months) and into two poor groups: “poor” (med. rec.=8 months) and “poorer” (med. rec.=5 months). B) bottom: recurrence CP score evaluated in the test set, which successfully showed comparable “poor” (med. rec.=9 months) and “poorer” (med. rec.=4 months) groups, in line with the predicted score. *The “favorable” group in the recurrence test set was omitted for clarity, see text.

Fig. 6

Graphical representation of proteins related to survival and recurrence. Patients with a high compound predictor (unfavorable outcome, red diamond) were plotted with patients having a low compound predictor (favorable outcome, blue triangle) for both survival and recurrence. A) top: plot of the weighted intensity of two proteins associated with poor survival, m/z 4049 and m/z 4737 (TYB-10, modified). A generally better prognosis is observed as both proteins decrease, as shown in the survival plot (bottom). B) top: plot of the weighted intensity of two proteins associated with favorable recurrence outcome, m/z 16,791 (calmodulin) and m/z 12,275 (CyC), with better prognosis as the intensity of both proteins increase, displaying a longer time to recurrence (bottom).

Fig. 7

Top–down identification of m/z 4748 (mono-isotopic m/z of 4745) by MALDI TOF–TOF. A) MALDI spectrum of tissue homogenate, with in-source LIFT cell isolation of m/z 4748. B) MALDI MS/MS spectrum of m/z 4748. The protein was identified as thymosin β-4 with the 2 C-terminus amino acids missing. (The final M.W. is given by: 5053–M (131)–ES (277)+acetylation (42)=4748 Da).