PMC

Panels A and C show Kaplan–Meier curves for ERK1/2 expression and PFS or OS where AQUA scores were divided into quartiles. The survival for the highest quartile was shorter than the other three quartiles. Panels B and D show Kaplan–Meier curves for ERK1/2 expression and PFS or OS where scores where dichotomized into “low” (the top three quartiles) and “high” (the fourth quartile). This cut-point corresponds to an AQUA score of 35.485.

AQUA uses S100 to define the tumor mask (bottom-right panel A), 4,6-diamidine-2-phenylindole to define the nuclear compartment (bottom-left panel A) and Cy5 (top-right panel A) for the target (ERK1/2). The cytoplasmic compartment is generated by subtracting the nuclear compartment from the S100 mask (top-left panel A). ERK1/2 expression is then measured within the nuclear and cytoplasmic compartments within the tumor mask, and each spot is assigned a score based on pixel intensity per unit area. ERK1/2 expression in this example corresponds to an AQUA score of 67.38 and it is shown at 10X magnification (top-right panel A) and 60X magnification (panel B).

A) VEGF-R2 expression is higher in patients with CR and PR. B) ERK1/2 expression is higher in patients with SD and PD. C) Chi-square analysis was utilized to compare high/low VEGF-R2/ERK1/2 expression and response to therapy. VEGF-R2 and ERK1/2 scores were arbitrarily dichotomized to high and low categories by the associated median score for each marker (24.426 for ERK1/2 and 32.207 for VEGF-R2). The three groups of patients were defined as follows: one group had low VEGF-R2 and high ERK1/2 expression, the second group had either low VEGF-R2 and low ERK1/2 or high VEGF-R2 and high ERK1/2, and the third group had high VEGF-R2 and low ERK1/2. All of the patients in the high VEGF-R2 and low ERK1/2 group responded to therapy.

Yellow shades represent positive correlations, and turquoise shades represent anti-correlations. The intensity of color in each square represents the strength of the association. Almost all correlations correspond to a p-value of less than 0.0004 (corresponding to the Bonferroni correction for 112 multiple tests at an alpha of 0.05). Note that for ease of reading, scores are represented twice. B and C) High ERK1/2 expression is associated with disease progression and poor prognosis in archival melanoma specimens. B) Analysis of variance comparing the AQUA scores for primary and metastatic specimens. ERK1/2 expression is higher in metastatic specimens than primary specimens. C) Kaplan-Meier survival curves for ERK1/2 AQUA scores divided by the median score for the primary subset of patients. High ERK1/2 expression is associated with poor survival.