EXCEPCIÓN AL DECRETO DE PRUEBA DE OFICIO EN SEDE DE TUTELA

Correlation in methylation levels between the endometrial and ovarian part of the carcinoma was confirmed by their overall 24-gene methylation measurements. Twelve carcinoma pairs showed strong to very strong correlation between methylated genes in tumor pairs (r²=0.69- 97), and one carcinoma pair showed moderate correlation (Patient ID 9, r²=0.45). Therefore, we concluded that methylation in the ovarian location of widespread serous carcinomas was highly correlated to its endometrial counterpart.

The average methylation of the 24-gene set was a bit higher in the ovarian location of the widespread serous carcinoma (0.28 vs 0.21), as was the CMI (for ovarian 7.15, for endometrial 5.45 (p=0.047)). This was also reflected in the number of genes methylated above the threshold (for ovarian 9.8, for endometrial 4.9).

Discussion

Methylation levels for CDH13 are significantly different between OSC and UPSC cases and the optimal level of CDH13 to discriminate OSC from UPSC was ≥0.32. Other analyzed promoter regions of commonly methylated tumor suppressor genes did not have discriminative capability in assayed samples and showed highly identical methylation levels in the OSC and UPSC cases.

The tumor suppressor gene, CDH13, or cadherin 13 (T-cadherin) is suggested to be involved in various cancers, including ovarian and endometrial cancer25_{. In comparison to normal}

endometrial or ovarian tissue CDH13 methylation is significantly increased in endometrial and ovarian carcinomas10,26_{. The CDH13 protein is a cell surface transmembrane protein, and has}

a role in cell adhesion. Silencing of T-cadherin expression by aberrant promoter methylation in cancers is associated with metastatic potential, and may play a role in tumor cell invasion

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and progression of cancers25,27_.

Methylation levels for CDH13, as found in the current study, were comparable with levels reported by others, using a similar MS-MLPA probe mix9,10,17_{. Seeber et al.}17_{, found frequent}

hypermethylation of CDH13 in UPSC cases with mean methylation levels of 0.44, which is comparable to the 0.40 as found in the current study. Bol et al.9_{, used the MS-MLPA probe}

mix to investigate methylation profiles within a cohort of ovarian carcinoma, consisting of OSC in 88%. They reported a mean methylation level for CDH13 of 0.22, which is comparable to the 0.28 we reported for solitary OSC9_{. Studies using other methods to analyze CDH13}

methylation in OSC reported hypermethylation in 9-24%12,13_.

We compared results for the 24 tumor suppressor genes of UPSC and OSC in the study cohort with results obtained from the publicly available ‘The Cancer Genome Atlas Research Network (TGCA)’ data3,6_{. Data included results from 17 cases diagnosed with UPSC and a}

large number of cases with OSC. A total of 65 OSC cases were selected from this database, based on the following criteria: bilateral disease, high FIGO stage, serous histology type and Caucasian. Average methylation levels were similar to results from the current study (0.19 vs 0.21 for serous ovarian and endometrial cancer, respectively). Their CMI was slightly lower with a mean level of 4.89 for UPSC and 4.38 for OSC, compared to 5.44 and 5.07 in the current study.

Interestingly, the mean methylation levels from the UPSC and OSC cases of the TGCA database differed significantly in more than half of analyzed tumor suppressor genes between both groups, including methylation levels for CDH13. However, compared to the current study different probes and methods for DNA methylation measurements were used. Moreover, the UPSC and OSC data had to be retrieved from two digital TGCA databases and comparing their results might not be correct because of putative differences in analysis, other than DNA methylation platforms. Finally, the number of UPSC cases analyzed is relatively small.

In this study, we also tested the value of CDH13 methylation to support in classifying widespread serous carcinoma, involving the endometrium and ovaries as primary endometrial or ovarian. Primary carcinoma location determined by histological criteria was confirmed by CDH13 methylation levels when applying the cut-off level of ≥0.32. All widespread serous carcinomas histologically suggestive for primary UPSC, showed a CDH13 methylation level of >0.32. In those cases with inconclusive primary location, 2 out of 6 cases showed a CDH13 methylation below this cut-off level suggestive for a primary ovarian origin, and four were suggestive for a primary endometrial origin.

The CDH13 methylation levels were not significantly different between locations within carcinoma pairs, and one level was thought representative for the entire carcinoma. However,

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locations, only the endometrial part of the carcinoma showed a methylation level suggestive for a primary ovarian location (Patient ID 12). In the latter, a double primary carcinoma could be one explanation for this wider range in CDH13 methylation levels.

Most widespread serous carcinomas included in this study were classified as primary endometrial in origin (UPSC). Remarkably, the 24-genes methylation profile of the ovarian location of the widespread serous carcinoma shows significantly higher methylation levels compared to their endometrial counterpart, or compared to the solitary OSC and UPSC cases. Although, the correlation for methylation in the endometrial and ovarian location within cases was high, higher methylation levels in the ovarian counterpart could represent an effect needed for spread of disease or metastasis of the primary carcinoma.

Further, in all analyzed groups of cases relatively high, as well as relatively low methylation levels were found. These DNA methylation clusters have been reported before in OSC and were attributed to significant differences in age at diagnosis and overall survival3_{. Our current}

results do not show a correlation between the cumulative methylation index and age at diagnosis, but sample sizes are relatively small, and data on 5-year overall survival were not available yet in most cases.

Another limitation of the current study is the restricted number of genes analyzed. Ideally methylation profiling of both primary carcinoma would be performed by analyzing promoter regions of a much larger set of genes to identify a panel of discriminative markers. We were unaware of studies specifically looking at methylation differences between serous carcinomas, and since it has been speculated that methylation might even be an earlier event than mutation, we chose to look at promoter regions of TSGs and oncogenes commonly altered in carcinomas.

Besides methylation levels for CDH13, levels for the other frequently silenced promoter regions in cancers were highly identical for OSC and UPSC in the current study. Total genome- wide studies already provided evidence for highly similar genetic profiles of OSC and UPSC, and the current study suggests that these serous carcinomas also have highly comparable methylation profile. Kolbe et al., compared gynecologic carcinomas of endometrioid histology type and also found highly comparable methylation levels in solitary endometrial and ovarian endometrioid carcinoma18_{. A common methylation phenotype for endometrioid ovarian and}

endometrial carcinomas is in line with the hypothesis that all endometrioid carcinomas might originate following a similar pathway involving the malignant development of endometriosis, originally arising from endometrial cells. However, OSC and UPSC are thought to develop following different pathways of carcinogenesis28_.

Comparable histological and (epi-)genetic tumor characteristics for UPSC and OSC support that UPSC is more and more treated as OSC in the last decade. Despite of highly similar profiles, immunohistochemical staining for Wilms tumor 1 has been suggested to show different expression in serous endometrial and serous ovarian carcinomas, and for example

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the frequency of PPP2R1A mutations has been reported as a potential molecular distinction between UPSC and OSC5,29-31_{. Possibly, CDH13 methylation levels might represent another}

discriminative feature between both serous carcinoma. Although, efforts still need to be made to elucidate the biology of these serous subtypes and to personalize treatment management further.

In conclusion, OSC and UPSC are not only genetically highly similar, but also their methylation profiles are comparable. However, CDH13 methylation might have discriminative capability between OSC and UPSC and might have value in determining the primary carcinoma location in cases with widespread serous carcinoma.

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OSC (N=15) UPSC (N=15) Paired OSC (N=13) Paired UPSC (N=13)

Genes Mean SD Mean SD Mean SD Mean SD

APC 0.28 0.15 0.31 0.21 0.44 0.12 0.36 0.23 ATM 0.17 0.13 0.19 0.16 0.27 0.07 0.19 0.14 BRCA1 0.17 0.16 0.16 0.14 0.22 0.07 0.16 0.11 BRCA2 0.16 0.12 0.18 0.15 0.25 0.07 0.17 0.10 CASP8 0.23 0.21 0.22 0.16 0.26 0.07 0.20 0.12 CD44 0.27 0.16 0.25 0.13 0.32 0.07 0.26 0.09 CDH13 0.28 0.09 0.40 0.10 0.44 0.17 0.39 0.13 CDKN2A 0.14 0.07 0.15 0.08 0.18 0.05 0.14 0.06 CDKN1B 0.18 0.11 0.22 0.14 0.29 0.09 0.21 0.14 CDKN2B 0.33 0.21 0.31 0.21 0.43 0.08 0.34 0.16 CHFR 0.12 0.12 0.15 0.14 0.20 0.07 0.13 0.12 DAPK1 0.12 0.11 0.13 0.11 0.17 0.05 0.12 0.08 ESR1 0.26 0.16 0.25 0.17 0.35 0.08 0.27 0.10 FHIT 0.07 0.07 0.09 0.09 0.09 0.04 0.06 0.05 GSTP1 0.20 0.12 0.21 0.13 0.28 0.08 0.23 0.10 HIC1 0.19 0.15 0.20 0.18 0.29 0.07 0.25 0.17 IGSF4 0.15 0.13 0.17 0.15 0.22 0.07 0.17 0.09 MLH1 0.17 0.14 0.17 0.14 0.24 0.06 0.17 0.13 MLH1b 0.09 0.08 0.10 0.11 0.14 0.06 0.09 0.08 PTEN 0.25 0.15 0.26 0.17 0.32 0.07 0.25 0.12 RARB 0.15 0.11 0.18 0.13 0.23 0.06 0.17 0.11 RASSF1a 0.29 0.24 0.29 0.19 0.38 0.23 0.28 0.15 RASSF1b 0.37 0.30 0.36 0.18 0.44 0.23 0.35 0.23 TIMP3 0.16 0.14 0.20 0.17 0.30 0.16 0.21 0.13 TP73 0.17 0.08 0.23 0.13 0.29 0.10 0.22 0.09 VHL 0.07 0.06 0.08 0.07 0.10 0.04 0.07 0.06 CMI 5.07 5.44 7.15 5.45

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OSC UPSC Paired OSC Paired UPSC

Genes N=15 % N=15 % p value N=13 % N=13 % p value

APC 4 27% 6 40% 0.700 11 85% 4 31% 0.015 ATM 3 20% 5 33% 0.682 8 62% 2 15% 0.041 BRCA1 3 20% 4 27% 1.000 1 8% 2 15% 1.000 BRCA2 3 20% 4 27% 1.000 5 39% 2 15% 0.378 CASP8 4 27% 5 33% 1.000 4 31% 2 15% 0.645 CD44 3 20% 4 27% 1.000 5 39% 2 15% 0.378 CDH13 4 27% 13 87% 0.003 12 92% 10 77% 0.593 CDKN2A 3 20% 6 40% 0.427 8 62% 2 15% 0.041 CDKN1B 2 13% 3 20% 1.000 3 23% 1 8% 0.593 CDKN2B 5 33% 5 33% 1.000 6 46% 2 15% 0.202 CHFR 3 20% 5 33% 0.682 5 39% 2 15% 0.378 DAPK1 2 13% 4 27% 0.651 2 15% 1 8% 0.480 ESR1 3 20% 4 27% 1.000 5 39% 2 15% 0.378 FHIT 2 13% 2 27% 1.000 0 0% 0 0% 1.000 GSTP1 2 20% 4 27% 0.651 3 23% 1 8% 0.593 HIC1 3 20% 4 27% 1.000 5 39% 3 23% 0.673 IGSF4 3 20% 5 33% 0.682 4 31% 1 8% 0.322 MLH1 4 27% 4 27% 1.000 6 39% 2 15% 0.202 MLH1b 2 13% 4 27% 0.651 1 8% 1 8% 1.000 PTEN 3 20% 5 33% 0.682 5 39% 2 15% 0.378 RARB 3 20% 6 40% 0.427 6 46% 2 15% 0.202 RASSF1a 6 40% 8 53% 0.464 7 54% 8 62% 0.691 RASSF1b 4 27% 8 53% 0.264 6 39% 3 23% 0.411 TIMP3 4 27% 5 33% 1.000 10 67% 3 23% 0.017 TP73 1 7% 6 40% 0.080 4 31% 1 8% 0.322 VHL 1 7% 4 27% 0.330 1 8% 1 8% 1.000 Total; mean 80; 5.3 20% 133; 8.9 34% 0.296 133; 10.2 38% 62; 4.8 19% 0.110

Supplementary Table 2: Frequency of methylation above the threshold compared between serous carcinomas located

Chapter 8