Genome-wide high-resolution aCGH analysis of gestational choriocarcinomas

Abstract

Eleven samples of DNA from choriocarcinomas were studied by high resolution CGH-array 244 K. They were studied after histopathological confirmation of the diagnosis, of the androgenic etiology and after a microsatellite marker analysis confirming the absence of contamination of tumor DNA from maternal DNA. Three cell lines, BeWo, JAR, JEG were also studied by this high resolution pangenomic technique. According to aCGH analysis, the de novo choriocarcinomas exhibited simple chromosomal rearrangements or normal profiles. The cell lines showed various and complex chromosomal aberrations. 23 Minimal Critical Regions were defined that allowed us to list the genes that were potentially implicated. Among them, unusually high numbers of microRNA clusters and imprinted genes were observed.

Figure 1. Genotype profile obtained with an automated sequencing apparatus of choriocarcinoma M176 with msat 9A from the PMP22 locus on 17p.

. A Patient/mother genotype with a 154 bp and a 180 bp 9 A alleles ; B father genotype with a 159 bp and a 169 bp 9 A alleles; C Tumor M176 : only a single 159 bp paternal allele is detected. Its absence in the mother allowed us to conclude that it is an andromonospermic tumor; sz = size in base pairs; ar = area; ht = height.

Figure 2. aCGH choriocarcinomas M26, M176, M232.

Respectively, loss of 18q, gain of 14q and loss of X. The vertical lines along the chromosomes indicate losses on the left, gains on the right side of chromosomes.

Figure 3. FISH on choriocarcinomas.

A = M176 Xq10 r & Yq10 g : left nucleus with 3Xq10, right ones with 5 spots. B = M232 Xq10 r & Yq10 g : only one Xq10 spot for each cell. C = M176 14q32 g & 16q23 r : 2 to 3 green spots (14q) and 1 to 2 red ones(16q). D = JAR 16q22 CBFB r+g : 3 to 4 spots for 16q. E = JEG 16q22 CBFB r+g : 4 spots for 16q. F = BEWO 16q22 CBFB r+g: 4 spots for 16q and a cell with eigth spots; these two types of cells are not distinct by aCGH. G = JEG BCL2 r+g : only one spot of 18q. H = BeWo BCL2 r+g : three spots of BCL2 per cell, that are considered as a small loss compared to JEG. I = BeWo Xq10 r & Yq10 g : two Xq10 and one Yq10 spots. J & K = JAR 12p12 ETV6 r+g : ETV6 is amplified ×8 in the right nucleus of picture J and, on picture K, this amplification is located on a marker chromosome while 3 copies are sitting on probably normal 12p. L = JAR 12q10 r & 12q15 MDM2 g. An amplification of MDM2 is observed (green) from 6 copy on a der(12) with one (or 2) chromosome 12 centromere(s); 3 copies of MDM2 seem present on apparently normal chromosomes 12.

Figure 4. aCGH choriocarcinoma cell lines BeWo, JEG, JAR.

Recurrent gain of 5q, 12p, 14q, 20q and loss of 8, 18q, Xp. The CNAs following optimal normalization for the X (see results) are indicated in blue along the X chromosomes in the three cell lines.

Figure 5. Representative images of IHC.

MMP-2: (A) Normal placenta 12 weeks = no labeling. (B) CK M176 = strong cytoplasm and membrane labeling of the of CTB cells and MEC. SERPINB2: C) Placenta 9 weeks = immunostaining of cytoplasm STB cells and microvillosities. D) CK M176 = no labeling.