Digital PCR Validates 8q Dosage as Prognostic Tool in Uveal Melanoma

<div><p>Background</p><p>Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in <i>GNAQ</i> and <i>GNA11</i> initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.</p><p>Methods</p><p>A series of 66 UM was analysed with dPCR for three hotspot mutations in <i>GNAQ/GNA11</i> with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.</p><p>Results</p><p>Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, <i>GNAQ/GNA11</i> mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed <i>GNAQ/GNA11</i> mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.</p><p>Conclusion</p><p>Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in <i>GNAQ</i> and <i>GNA11</i> are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.</p></div

GNAQ mutation detection in UM by dPCR.

<p>Two color digital PCR is presented as a 2D plot. On the x-axis, the number of WT amplicons (green) is indicated and on the y-axis the mutant amplicons (blue) are counted. Empty PCRs and negative amplicons (black) end up in the bottom left quadrant. (A) PCRs containing WT and mutant (orange) will end up in the upper right quadrant. Based on Poisson distribution, the number of WT and mutant amplicons can be calculated (bar graph). This shows that 01–074 represent a homogeneous and 04–075 a heterogeneous UM sample. (B) Digital PCR analysis of these samples with the GNAQ Q209P assay did not result in mutant signals but aberrant signals on the x-axis. Manual selection of the aberrant signals in the WT quadrant of the plot learned that the clusters matched the positive clusters in the GNAQ Q209L assay. This indicates that the WT probe hybridized with low efficiency to the GNAQ Q209L allele in UM 01–074 and UM 04–075 in the absence of specific probe.</p

Chromosome 8 heterogeneity in UM 07–050.

<p>One clone presents trisomy 8 (left) while the other clone presents trisomy 8 (triangle) in combination with isochromosome 8q (right). Besides chromosome 8, chromosome 1, 3, 10 and 22 showed clonal abnormalities. Two markers (A) on the bottom indicate the shared origin of these clones.</p

Sequence analysis of the GNAQ Q209P (A>C), GNAQ Q209L (A>T) and GNA11Q209L (A>T) hotspot mutations in UM.

<p>Sequence analysis of the GNAQ Q209P (A>C), GNAQ Q209L (A>T) and GNA11Q209L (A>T) hotspot mutations in UM.</p

Monosomy 3 and chromosome 8 copy number variation: SNP array versus dPCR analysis.

<p>SNP and dPCR thresholds: loss, <1.9: normal, 1.9–2.1: gain, >2.1- <3.2: amplification, >3.1</p><p>* Aberration at chromosome 5, location of TERT, no proper correction possible with TERT dPCR Karyotype chromosome 3: 0; Disomy, 1; MonosomyKaryotype chromosome 8: 0; Disomy, 1; gain, 2; isochromosomen.a.: not analyzed</p><p>Monosomy 3 and chromosome 8 copy number variation: SNP array versus dPCR analysis.</p

Worst survival for patients with amplification of chromosome 8q.

<p>The presence of monosomy 3 (n = 43) in UM is highly prognostic for death due to metastases compared to the presence of disomy 3 (n = 23) (p<0.0001). (A) Significant differences in survival between classes of tumours with normal (n = 14), gain (n = 24), or amplification (n = 28) of chromosome 8q. A significant difference in survival was observed between the three categories (p<0.0001), and between the different individual classes: normal vs. amplification (p<0.0001), and gain vs. amplification (p = 0.00125). Between normal and gain of 8q a trend towards significance was observed (p = 0.07). (B) On the background of monosomy 3, 8q amplification (n = 24) increased the risk significantly (p = 0.011) compared to monosomy 3 with 8q gain (n = 16). Three UM presented monosomy 3 without aberration on 8q: none died due to UM metastasis. (C)</p

GNAQ A209H mutation in UM 06–046.

<p>(A) GNAQ mutation in 06–046 involves the c.627 A>C that encodes for the Q209H substitution. (B) For comparison, the WT sequence analysis of 05–034 is provided. Digital PCR with a newly designed GNAQ Q209H probe validates the mutation in 06–046.</p

Patient characteristics.

<p>Frequencies of relevant parameters of 133 UM</p><p>Patient characteristics.</p

Relationship of location and eye color.

<p>Light eye color in UM patients reflects the population distribution though choroidal UM may present the lower estimate of light eye color while ciliochoroidal and iridociliary UM present the upper limit of the population frequency.</p><p>Relationship of location and eye color.</p

Correlation between dPCR and SNP with regard to chromosome 3 and 8q copy numbers.

<p>Copy numbers of chromosome 3 and 8q in UM calculated with SNP and dPCR display a strong correlation (r = 0.921 and r = 0.922, p<0.0001), indicating that these methods cross validate each other.</p