Intratumor Heterogeneity in Uveal Melanoma BAP-1 Expression

Abstract

Malignant tumors are rarely homogenous on the morphological, genome, transcriptome or proteome level. In this study, we investigate the intratumor heterogeneity of BAP-1 expression in uveal melanoma with digital image analysis of 40 tumors. The proportion of BAP-1 positive cells was measured in full tumor sections, hot spots, cold spots and in scleral margins. The mean difference between hot spots and cold spots was 41 percentage points (pp, SD 29). Tumors with gene expression class 1 (associated with low metastatic risk) and 2 (high metastatic risk) had similar intratumor heterogeneity. Similarly, the level of intratumor heterogeneity was comparable in tumors from patients that later developed metastases as in patients that did not. BAP-1 measured in any tumor region added significant prognostic information to both American Joint Committee on Cancer (AJCC) tumor size category (p ≤ 0.001) and gene expression class (p ≤ 0.04). We conclude that there is substantial intratumor heterogeneity in uveal melanoma BAP-1 expression. However, it is of limited prognostic importance. Regardless of region, analysis of BAP-1 expression adds significant prognostic information beyond tumor size and gene expression class.

Keywords: BAP-1; survival; tumor heterogeneity; uveal melanoma.

Figure 1

Example of intratumor heterogeneity in BRCA1 associated protein 1 (BAP-1) expression. In a uveal melanoma growing close to the optic nerve head (arrowhead), both BAP-1 positive and negative cells can be identified. Positive nuclei are stained lilac with a red chromogen over blue hematoxylin. Negative cells are stained with blue hematoxylin only. Areas with relatively low (left circle), intermediate (center circle) and high (right circle) proportions of BAP-1 positive cells can be found. Areas with artefacts, uneven staining, necrosis, bleeding, abundant pigmentation or poor focus were excluded from analysis (red-striped areas). Tissue for gene expression classification has been removed from the center of this tumor (asterisk). This case was designated class 1A, implying a low risk for metastasis. Magnification ×20, circles ×400.

Figure 2

BAP-1 expression heat map by digital image analysis. (A) A choroidal melanoma growing on top of the optic nerve head is outlined. (B) A region of interest is drawn around the edges of the tumor. In this region, digital image analysis identified BAP-1 positive and negative cells. (C) Tumor regions with relatively high (red), intermediate (green) and low (blue) proportions of BAP-1 positive cells can be visualized. The hot spot was defined as the circular region with a diameter of 0.5 mm (corresponding to an area of 0.2 mm² per tumor, or one high-power field in a light microscope at 400×) with the highest proportion of BAP-1 positive tumor cells in any part of the tumor. The cold spot was defined as the circular region with a diameter of 0.5 mm with the lowest proportion of BAP-1 positive tumor cells in any part of the tumor. Scleral margin was defined as the circular region with a diameter of 0.5 mm with the lowest proportion of BAP-1 positive tumor cells within 1 mm from the tumor base towards the sclera. Scale bars: (A), 5 mm; (B,C): 1 mm.

Figure 3

Distribution of the proportion of BAP-1 positive tumor cells in full tumor sections, hot spots, cold spots and scleral margins. (A) Cumulative frequency polygon. (B) Box plots. The mean proportion of BAP-1 positive cells was significantly different in full sections, hot spots, cold spots and scleral margins (one-way ANOVA p < 0.0001). Error bars represent 95% confidence interval.

Figure 4

Receiver operating curves. (A) For metastases, AUCs of 0.86 to 0.89 were achieved, depending on tumor region (p < 0.0001), with sensitivities in the range of 75 to 88% and specificities in the range of 75 to 92%. (B) For gene expression class 2, AUCs of 0.85 to 0.86 were achieved (p = 0.001 to 0.002), with sensitivities in the range of 82 to 100% and specificities in the range of 73 to 83%. See also Table 2. Abbreviations: AUC, area under the curve.

Figure 5

Kaplan–Meier curves. Cumulative metastasis-free survival for patients with high versus low BAP-1 expression, measured in (A) full tumor sections, (B) hot spots, (C) cold spots, and (D) scleral margins.