The purpose of this work was to initiate development of a multiplex, plasma-based protein biomarker panel to differentiate between patients with resected melanoma and melanoma patients with unresected disease. To achieve our goal, we screened a number of abundantly expressed candidate genes identified in early passage melanoma cell strains which encoded for secreted proteins, plasma membrane proteins, and extra-cellular matrix proteins known to be associated with cancer, as these were felt to be more likely to found in the plasma of patients with unresected disease. Seven proteins were found to have higher levels in unresected stage IV patients compared with age and gender matched patients with resected, early stage disease. Among the stage I/II patients, levels of none of these markers was associated with stage or Breslow depth, indicating that they might require a larger burden of unresected disease than primary melanomas to result in elevation in the plasma at the time of diagnosis, and thus might have value for monitoring patients. In combination, these proteins were superior in discriminating between the two groups of patients (resected stage I/II and unresected stage IV) than each protein alone, and the proteins were clearly superior to serum LDH in differentiating between these patient populations. A more compact model containing a subset of these variables (ICAM-1, GDF-15 and OPN) resulted in an improved misclassification rate. Our findings were validated in a separate test set.
The biological basis for finding these specific proteins in the blood of our metastatic melanoma patients varied from marker to marker. CEACAM (CEA-related Cell Adhesion Molecule) expression has been shown to markedly enhance melanoma cell invasion and migration, and the molecule is expressed in melanoma tumor-stroma interface of invading melanoma masses 15
. It is therefore likely that CEACAM would be secreted into the blood system in melanoma patients. ICAM-1 (intercellular adhesion molecule) is expressed on the surface of melanoma cells, and interacts with polymorphonuclear leukocytes, which facilitate melanoma cell extravasation through the endothelium and into the circulation 16
. ICAM-1 is therefore likely to be associated with hematogenous melanoma dissemination. In mouse models, osteopontin (OPN) levels are increased during the progression from primary to metastatic melanoma 17
. In patient tissue samples, OPN is associated with melanoma progression 18-19
. Serum levels of MIA (melanoma-inhibiting activity) have been shown by others to be associated with melanoma progression, as reviewed 20
. GDF-15 (growth differentiation factor 15, also known as Macrophase Inhibitory Cytokine-1) has been shown to be up-regulated in advanced melanoma tumors in a number of reports 21
. Given that this is a secreted protein, its utility as a plasma marker for metastatic melanoma has been suggested by Boyle et al 22
. TIMP-1 (tissue inhibitor of metalloproteinase-1) has been shown in a small series to be elevated in patients with stage IV melanoma when compared with healthy controls and patients with thin primary melanomas 23
. S100B is expressed on over 90% of melanoma cells and has been shown to be elevated in blood of patients with metastatic melanoma 13
While a number of published studies have assessed our blood-based biomarkers for surveillance of melanoma recurrence, individual markers are associated with high misclassification rates. S100B has perhaps been the most widely studied and has been validated as a single biomarker in samples from a large multi-center randomized clinical trial 13
. Rangel et al. showed that elevated OPN levels in tumors are associated with metastatic relapse, but plasma OPN levels in metastatic and primary melanoma have not been studied 18
. Elevated levels of MIA were found in peripheral blood mononuclear cells in 26.8% of samples from stage I/II patients and 86.% of patients with clinically evident, untreated stage IV disease 27
. The stage IV cohort in this study, however, only included 13 patients with clinically evident disease. Both OPN and MIA have been shown to be associated with metastases in uveal melanoma 28
. Yamada et al showed increasing elevation in levels of ICAM-1 in two patients whose melanoma metastasized 29
. Levels of TIMP-1 were elevated in plasma of a small sample or 19 stage IV patients compared to stage I-III patients 23
. We did not find similar studies on the other biomarkers in our panel. In our study, these markers in combination had a stronger association with metastatic disease than any single marker. The CART analysis resulted in a more compact model with improved sensitivity and specificity.
To the best of our knowledge, this is the first published study that assesses these markers in combination. As is the case with other clinically used biomarker studies, multiplex analysis of our biomarker panel improved the error rate for predicting metastatic disease. The area under the receiver operating curve for our biomarker panel was 0.84 in both the training and test set, and 0.898 in the reduced variable model, which compares well to that of other clinically used assays, such as the Oncotype Dx model, which is used for predicting relapse in breast cancer 24
The sensitivity of our combined marker set of seven variables was 74% and 87% using the reduced variable model. Ideally, none of the stage I/II patients would have elevated marker levels. Given that we are proposing a screening test that can be used to select out individuals who need surveillance imaging, however, such a test, once validated, could still eliminate unnecessary scans in asymptomatic patients with normal blood work and normal marker levels. Our test, however, compares favorably to use of serum LDH in melanoma, and is in the ballpark of other clinically used assays for cancer surveillance. For example, in a large study of patients with resected early stage colon cancer under surveillance in the United Kingdom, CEA was associated with a sensitivity of 64% in identifying metastatic disease 25
. In patients with resected ovarian cancer, serum CA-125 was 23.3% sensitive in identifying disease recurrence 26
. Ongoing and future studies will focus on including other secreted proteins to increase the sensitivity of our assay while preserving or improving specificity.
Plasma markers associated with metastatic disease can be useful in identifying patients who might benefit from early intervention. There are a number of lines of evidence to suggest that early detection of metastases and resection of oligometastases can result in improved survival 30-32
. Most of these series assess the benefit of metastatectomy compared to historical controls, although one phase III trial showed benefit to aggressive surgical resection 32
. Other studies suggest that survival rates for patients with stage IV disease that has been resected (called stage IV NED for stage IV with No Evidence of Disease) have improved over the past years, suggesting that active radiographic surveillance and immediate intervention might improve survival in patients whose disease metastasizes 33
. In patients with unresectable metastases, the likelihood of response to systemic therapy is sometimes inversely related to disease burden, although it is unclear whether this is directly related to overall survival 34
. Nonetheless, early detection of metastases can result in treatment of patients with a better performance status and less symptoms, and as newer, more effective therapies for metastatic disease become available, tools for early detection of metastases and subsequent early intervention are likely to be highly useful. This is supported by a recent study demonstrating improved overall survival in metastatic melanoma in the United States compared to Australia; the difference in overall survival was attributable to more active surveillance in the United States and early detection of metastases 35
In summary, we have identified a group of markers that are elevated in unresected metastatic melanoma compared to individuals with resected stage I/II disease. Particularly when used in combination, these markers can be used to monitor patients for disease recurrence and might be useful for complimenting surveillance imaging for resected stage I-III patients, or increasing the interval between imaging studies, especially in high risk stage IIC and III patients. Prospective validation of these findings in an independent cohort of stage I-III patients with resected melanoma at high risk for disease recurrence is warranted.