Estimation of the timing of BAP1 mutation in uveal melanoma progression

Abstract

Uveal melanoma is the most common primary intraocular malignancy. A vast majority of metastasizing tumors have mutations in the BAP1 gene. Here, we investigate the spatiotemporal timing of these mutations. The size of 177 uveal melanomas and 8.3 million individual tumor cells was measured. BAP1 sequencing results and BAP1 IHC were available and for 76 (43%) and 101 (57%) of these, respectively. Tumors with a BAP1 mutation had significantly larger volume (2109 vs. 1552 mm³, p = 0.025). Similarly, tumor cells with loss of BAP1 protein expression had significantly larger volume (2657 vs. 1593 μm³, p = 0.027). Using observations of the time elapsed between mitoses, the BAP1 mutation was calculated to occur when the primary tumor had a size of a few malignant cells to 6 mm³, 0.5 to 4.6 years after tumor initiation and at least 9 years before diagnosis. We conclude that BAP1 mutations occur early in the growth of uveal melanoma, well before the average tumor is diagnosed. Its timing coincides with the seeding of micrometastases.

Figure 1

Results of whole exome sequencing and digital image analysis of BAP1 immunohistochemistry. (A) Distribution of BAP1 mutations and copy number variations (CNV) across the 76 included patients from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/projects/TCGA-UVM). Twenty-two patients had a BAP1 mutation, 1 patient had a BAP1 CNV gain, and 1 patient had a BAP1 CNV loss. (B) Distribution of the most frequently mutated genes as proportion of the cohort. (C) BAP1 immunohistochemistry. In this example, most tumor nuclei have retained their BAP1 expression (lilac) whereas some have lost their expression (blue). (D) Excerpt from digital image analysis of the same tumor section shown in B. Tumor cells with retained and lost BAP1 expression are indicated in red and blue, respectively. (E) Tumors with a BAP1 mutation had significantly larger mean volume than those with wild-type BAP1 (2109 vs. 1552 mm³, p = 0.025). (F) tumor cells with loss of BAP1 expression had significantly larger mean volume (2657 μm³, SD 1283) than those with retained expression (1593 μm³, SD 602, p = 0.027). Freq, Frequency. CNV, Copy number variation. * < 0.05.

Figure 2

Cumulative metastasis-free survival proportions. (A) Survival after diagnosis for patients with a BAP1 mutation or low immunohistochemical BAP1 expression (red) versus no BAP1 mutation or high immunohistochemical BAP1 expression (yellow, Log-Rank p < 0.0001). (B) Survival after tumor initiation, which was estimated to occur 22.3 years before diagnosis (SD 1.4). (C) Survival after BAP1 mutation, which was estimated to occur 20.6 years before diagnosis (SD 2.5). Green areas represent 95% confidence intervals.

Figure 3

A model for the growth and dissemination of uveal melanoma. (A) In a tumor’s infancy, mutations in G-protein subunits including GNA11 or GNAQ are accumulated. (B) In about half of uveal melanomas, subsequent mutations in BAP1 occur after 0.5 to 5 years, at a tumor volume of < 10 mm³. At this point, micrometastases are seeded to the liver. (C) As the primary tumor keeps growing, the BAP1 mutated clone has a survival advantage over BAP1 wild type tumor cells, gradually replacing the latter. The average primary tumor is diagnosed at a volume of 350 mm³, 9.5 to 38 years after tumor initiation. Micrometastases may remain dormant and undetectable for years. (D) At a later stage, the primary tumor is composed of BAP1 mutants only, with few exceptions. Eventually, liver micrometastases leave dormancy and start growing to radiologically detectable macrometastases. The entire process from primary tumor initiation to death from metastatic disease takes one to five decades. The minimum estimate is 9.5 years between tumor initiation and diagnosis, and 0 years from diagnosis to metastatic death for an overall course of 9.5 years. The maximum estimate is 38 years between tumor initiation and diagnosis, and 15 years from diagnosis to metastatic death for an overall course of 53 years. Later metastases are rare^,. Note: The presence of micrometastases does not necessarily imply a worse prognosis. Subsequent events may be necessary for micrometastases to leave dormancy and start proliferating. Importantly, more than half of patients do not develop macrometastases at all^,,,,.