Abstract 4833: Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration
Authors: Hernandez Leanora S. , Bradley Amanda , Gaiser Timo , Andersson Sonia , Gertz E. Michael , Chowdhury Salim A. , Schwartz Russel , Schäffer Alejandro , Heselmeyer-Haddad Kerstin , Ried Thomas 
Published: August 2015

Abstract
In human cervical carcinogenesis, the gain of the long arm of chromosome 3 is the decisive genetic event that determines progression of dysplasia to invasive disease. Other recurrent genomic imbalances, such as gains on 1q, 5p, 8q, and 20q, and losses on 2p, 3p, and 11q are also observed. To better understand the patterns of clonal evolution during disease progression we developed a multiplex FISH assay with three multicolor probe panels for sequential hybridizations of cervical intraepithelial neoplasia (CIN3) and invasive cervical carcinoma (ICC), including synchronous CIN3/ICC lesions. Our FISH assay targets oncogenes COX2 (1q), TERC (3q), TERT (5p), MYC (8q), ZNF217 (20q), and tumor suppressor genes ING5 (2p), FHIT (3p), CHEK1 (11q), and centromere 7 as a ploidy control.
Our results confirmed the dominant role of the gain of 3q: nineteen out of twenty lesions showed a major clonal population with TERC amplification. Nine out of the 20 lesions showed a concomitant loss of FHIT (3p) suggesting an isochromosome 3q formation, which makes the loss of FHIT the second most frequent clonal event in this group of samples. Other imbalances were present, but, as expected, less common. The copy number increases of 3q can occur either in diploid cells or after tetraploidization of the genome. Interestingly, in our samples only three CIN3 lesions showed major clones with signal patterns consistent with near-tetraploidy, while all other lesions, including all ICC lesions and the two synchronous CIN3/ICC lesions showed major signal patterns consistent with near-diploid cell populations. Although tetraploidization can occur already in premalignant lesions, and there is independent evidence that such chromosome doublings increase the risk of chromosome segregation errors, and therefore promote the subsequent acquisition of genomic imbalances, our data indicate that a majority of cervical precancerous and cancerous lesions acquire aberrations in a diploid genome. Independent of whether the 3q gain and other aberrations are acquired in a diploid or tetraploid background, it selects cells with a proliferative advantage, which is supported by our observations that (i) cells with identical patterns of 3q gain are adjacent to each other, hence indicating a clonal proliferation, and (ii) that the only commonality between the major clones in the synchronous CIN3 and ICC lesions was a TERC gain. We further examined overall cell-to-cell heterogeneity in CIN3 versus ICC samples. While overall levels of clonal heterogeneity were indistinguishable between CIN3 and ICC, phylogenetic models of single tumors suggested greater average numbers of mutations per cell in ICC versus CIN3. These findings improve our understanding of clonal development during cervical carcinogenesis and with that our understanding of genome dynamics at early stages of carcinogenesis.
Citation Format: Leanora S. Hernandez, Amanda Bradley, Timo Gaiser, Sonia Andersson, E. Michael Gertz, Salim A. Chowdhury, Russel Schwartz, Alejandro Schäffer, Kerstin Heselmeyer-Haddad, Thomas Ried. Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2015-4833