New SOMAmer-based assay to discover biomarkers relevant to malignant melanoma
Authors: William Robinson, Malti P. Nikrad, Steven Robinson, Stephen Williams, Rachel Ostroff
Journal: Clinical Cancer Research (2010)

Abstract Malignant melanoma is a serious form of skin cancer with approximately 138,000 new cases predicted worldwide in 2010 and an estimated 48,000 deaths. The number is expected to double in less than a decade. There is an unmet need for new molecular technologies that provide for cost-effective, reliable and accurate detection of malignant melanoma at tumor burden levels below those currently observable with standard imaging or histopathological techniques. It would be desirable if a new test could predict a) sentinel lymph node (SLN) positivity in order to avoid SLN biopsy, an invasive and expensive procedure, b) recurrence to help monitor disease and make treatment decisions and c) survival time in metastatic melanoma. Secreted proteins and those released during apoptosis from tumor cells and surrounding tissues undoubtedly contain important biologic information that would theoretically enable early diagnosis and prognostic and therapeutic decisions in oncology. However, there is great difficulty in finding and quantifying such signals for large numbers of low abundance proteins. SomaLogic therefore created a highly multiplexed proteomic assay which is continuously expanding in breadth. It currently measures 825 proteins simultaneously from ~15ul blood, with throughput of 300 samples /day. The average dynamic range of each protein in the assay is &amp;gt;3 logs — with nearly seven logs of dynamic range achieved through multiple dilutions — and the median lower limit of quantification is below 1 pM. The median coefficient of variation for each protein is &amp;lt;5%. This assay performance arises from the selection of high affinity aptamers (SOMAmers) that bind selectively to their target proteins with slow off-rates. Retrospective analysis of serum, collected from patients before or after wide local excision / sentinel lymph node removal was done using this aptamer proteomics platform. In patients with metastatic stage IV disease at the time of diagnosis follow-up data was collected for several years to monitor time to survival. A naïve Bayesian statistical program was used for analysis of each comparison, negative to positive SLN cases, recurrence and non recurred cases and in case of metastatic melanoma alive vs. dead of disease within a year of blood draw. Of the 39 patients who had SLN biopsy, 25 were negative (stage II) and 14 were positive (stage III) by pathological analysis. 13 proteins were significantly differentially expressed (p&amp;lt;0.01). A three protein marker panel generated a Receiver Operating Characteristic (ROC) curve area under the curve (AUC) of 0.9 and could identify SLN positive patients with a specificity and sensitivity of 0.92 and 0.93 respectively. Comparison of patients alive with stage IV disease (N=22) verses those dead of disease in 1 year after blood draw (N=15) to yielded 37 proteins significantly different (p=&amp;lt;0.01). A signature of 2 proteins had an area under the ROC curve of 0.89 with a sensitivity and specificity of 0.87 and 0.91 respectively. Thus in a preliminary study we have discovered new proteins associated with a regional verses local disease at the time of diagnosis in patients with melanoma lesions &amp;gt;1mm. In metastatic melanoma, we were able to build classifier to predict survival time within a year. These results will be further validated on a larger sample set.