**2. Ovarian cancer: A model for greater insight into genetic instability**

This review concerns high-grade serous ovarian cancer (HGSC) characterized by a higher genomic instability (see below) and very frequent *TP53* gene mutations. Germline mutations in breast cancer susceptibility 1 (BRCA 1) or BRCA 2 may lead to hereditary HGSC, whereas 90% of HGSC are sporadic (no BRCA mutations but frequent BRCA inactivation or modulation with promoter hypermethylation, loss of heterozygosity in over 50% of cases) [15].

Four hundred and eighty-nine HGSC were recently studied by the Cancer Genome Atlas Research Network [16]. *TP53* mutations were present in 96% of cases followed by mutations in *NB1, BRCA1, BCA2, RB1,* and *CDK12* genes. Homologous recombination (and the DNA damage signaling pathway) is defective in about half of the cancers analyzed [16]. Taken together, all these findings can explain why ovarian cancer may be a relevant model to explore genetic instability.

Recent histopathologic and molecular studies enabled to describe a model of tumorogenesis comprising two pathways [15]:


True occult intraepithelial cancerous lesions of the tube (serous tubal intraepithelial carcino‐ ma (STIC)) have been reported unusually often in the distal portion of tubes obtained after prophylactic adnexectomy for BRCA mutation, prompting systematic and meticulous examination of these tubes [18–23]. They show epithelial stratification, nuclear atypicalities with an increase in the nucleocytoplasmic ratio, loss of nuclear polarity, nuclear pleiomorphism, and loss of ciliated cells. Immunohistochemical analysis may reveal intense and diffuse expres‐ sion of *p53* (the p53 signature) and a high proliferative index (Ki67> 40%). There could be a high level of genetic instability, as demonstrated by the high positive level of γH2AX [15, 16, 18–23]. In other words, high-grade ovarian serous carcinoma could originate in the fallopian tube, and all these findings encouraged us to study STIC lesions and genomic instability.

As it has been demonstrated that telomere shortening occurs early during carcinogenesis (being found in 89% of preinvasive lesions of the bladder, cervix, colon, and also the esopha‐ gus) [5], we investigated genetic instability in precancerous ovarian lesions, which has been

**2. Ovarian cancer: A model for greater insight into genetic instability**

with promoter hypermethylation, loss of heterozygosity in over 50% of cases) [15].

This review concerns high-grade serous ovarian cancer (HGSC) characterized by a higher genomic instability (see below) and very frequent *TP53* gene mutations. Germline mutations in breast cancer susceptibility 1 (BRCA 1) or BRCA 2 may lead to hereditary HGSC, whereas 90% of HGSC are sporadic (no BRCA mutations but frequent BRCA inactivation or modulation

Four hundred and eighty-nine HGSC were recently studied by the Cancer Genome Atlas Research Network [16]. *TP53* mutations were present in 96% of cases followed by mutations in *NB1, BRCA1, BCA2, RB1,* and *CDK12* genes. Homologous recombination (and the DNA damage signaling pathway) is defective in about half of the cancers analyzed [16]. Taken together, all these findings can explain why ovarian cancer may be a relevant model to explore

Recent histopathologic and molecular studies enabled to describe a model of tumorogenesis

**•** Type I tumors: These are low-grade tumors for which the pathogenesis would consist of a sequence of cystadenomas/adenofibromas, then borderline tumors and finally progression towards cancer. A lesional continuum has indeed been found, that is, molecular mutations that are common to benign, borderline, and invasive lesions (BRAF and KRAS mutations in over 60% of cases, less frequently mutated PTEN (20%–46%) and β-catenin (≈30%). Note also the recent discovery of ARID1A gene mutations in endometriosis and cancers associ‐

**•** Type II tumors: These are high-grade tumors from the outset. At the molecular level, *TP53* gene mutations are found in 50%–80% of cases. These tumors show high genomic instability and are essentially represented by HGSC. This histologic subtype is the most frequent and consequently has been the subject of many studies, and a tubal carcinogenic sequence has

True occult intraepithelial cancerous lesions of the tube (serous tubal intraepithelial carcino‐ ma (STIC)) have been reported unusually often in the distal portion of tubes obtained after prophylactic adnexectomy for BRCA mutation, prompting systematic and meticulous examination of these tubes [18–23]. They show epithelial stratification, nuclear atypicalities with an increase in the nucleocytoplasmic ratio, loss of nuclear polarity, nuclear pleiomorphism, and loss of ciliated cells. Immunohistochemical analysis may reveal intense and diffuse expres‐ sion of *p53* (the p53 signature) and a high proliferative index (Ki67> 40%). There could be a high level of genetic instability, as demonstrated by the high positive level of γH2AX [15, 16, 18–23].

ated with endometriosis (clear cell and endometrioid cancers) [17].

described very recently.

38 Telomere - A Complex End of a Chromosome

genetic instability.

comprising two pathways [15]:

been identified [15].

We started by assessing the telomere length in 12 STICs, 36 non-cancerous controls, and 43 high-grade serous cancers [24, 25]. Laser microdissection of paraffin-embedded samples was used in all cases. A DNA extraction was followed by purification step. Telomere length was measured by real-time quantitative polymerase chain reaction (PCR) according to Cawthon's method [26].

STICs had the shortest telomeres (p=0.0008). Telomeres of invasive cancer were shorter than those in benign controls but longer than telomeres found in STICs. A significant correlation was also found between overexpression of p53 and H2AX proteins and shortened telomeres in STICs (p<10−7) [24]. Kuhn *et al* [27] used fluorescence in situ hybridization technique to analyze telomere length of a series of 22 STICs in comparison with non-cancerous controls and high-grade cancers: 82% of STICs had the shortest telomeres, followed by the cancers. The controls had the longest telomeres. These findings suggest that STIC lesions are the most unstable genetically and are likely to represent one of the first steps in tubo-ovarian carcino‐ genesis.

We also studied these STICs by array comparative genomic hybridization (aCGH) using oligonucleotide microarrays (Agilent 180 K) [24].

The size of rearrangements in STICs was high, with an average of 2363.37 Kb (488–8161).

We found common gains at chromosomes 19q (6/12, 50%), 16p (5/12, 41.6%), 12q (5/12, 41.6%), 10q (5/12, 41.6%), 11p (4/12, 33.3%), 4p (3/12, 25%), and 8q (3/12, 25%), and common losses at chromosomes 3q (6/12, 50%), 2q (5/12, 41.6%), 11q (4/12, 33.3%), 6p (4/12, 33.3%), 22q (4/12, 33.3%), 18q (3/12, 25%), 19p (3/12, 25%), 20p (3/12, 25%), and 2 p (2/12, 16.6%) [24]. These early rearrangements could be key steps in these first phases of carcinogenesis, and the correspond‐ ing candidate genes could be involved directly in the tumor triggering process, meaning that they could represent diagnostic and/or treatment targets. More studies are required.

Finally, in another study, we investigated the level of DDR activation in STICs by immuno‐ histochemistry (pATM, pChk2, γH2AX, 53BP1, and TRF2) [28]. We constructed a tissue microarray, including 21 benign fallopian tubes, 21 STICs, and 30 HGSCs. We demonstrated that the expression of all DDR proteins increased from benign fallopian tubes to STICs. Analysis of staining variance within cases showed that 53BP1, γH2AX, pATM, pCHK2, and TRF2 expressions were significantly higher in STICs than in HGSC [28].

Taken together, all our results have shown evidence of genomic instability in the precancerous lesions known as STICs. Of note among the frequent chromosomal breakpoints in STICs, more than half occurred at terminal bands, which is characteristic for a telomere crisis with the occurrence of telomere fusions, leading to chromosomal aberrations.

It could also be interesting to study human telomerase reverse transcriptase (hTERT) expres‐ sion, which is a determinant for telomerase activity, and also the telomere architecture proteins (shelterin complex proteins TRF1, TRF2, and POT1) in STICs and HGSC [29–33]. TRF1 and TRF2 proteins are involved in negative regulation of telomere lengthening and interact with telomerase [29, 30]. POT1 appears to play a dual antagonist role, depending on cell conditions, acting as positive or negative regulator of telomere length depending on telomerase [6, 34]. In our study [28], the level of expression of TRF2 was increased in STICs in comparison with HGSC (p=0.012). It has been shown that TRF2 may shorten telomeres without telomerase inactivation [12]. TRF2 is also phosphorylated by ATM to enable DNA damage repair in response to DNA damage [12].

Telomerase would probably be activated after telomere shortening at the invasive stage and would thus counteract further telomere shortening: stabilization of telomere length at this stage would moreover represent an advantage in terms of tumor proliferation and escaping apoptosis [3].

Wang *et al* [35] investigated the relationship between telomere length and telomerase activity in 15 ovarian cancers. The authors found telomeric dysfunction in 9/15 (60%) and telomerase activation in 11/15 (73.3%) ovarian cancers. However, they did not study precancerous lesions.

Other authors demonstrated that telomerase activity was higher in ovarian carcinoma than in borderline tumors (considered as premalignant tumors) and normal ovary [36–38]. This was confirmed in cell cultures [39]. However, telomerase activity in STICs has not yet been studied.
