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Supervised clustering of primary HGSOC (n=421) from the TCGA data using 59/126 immune related genes

Purpose: Most high-grade serous ovarian cancer (HGSOC) patients develop recurrent disease after first-line treatment, frequently with fatal outcome. This work aims at studying the molecular biology of both primary and recurrent HGSOC.Experimental Design: Gene expression profiles of matched primary and recurrent fresh-frozen tumor tissues from 66 HGSOC patients were obtained by RNA sequencing. Clustering analyses and pairwise comparison of the profiles between matched samples and subsequent functional alignment were used for the identification of molecular characteristics of HGSOC.Results: Both primary and recurrent HGSOC samples presented predominant gene expression differences in their microenvironment, determined by a panel of genes covering all major pathways of immune activation together with a number of genes involved in the remodeling of extracellular matrix and adipose tissues. Stratifying tumor tissues into immune active and silent groups, we further discovered that although some recurrent tumors shared the same immune status as their primary counterparts, others switched the immune status, either from silent to active or active to silent. Interestingly, genes belonging to the B7-CD28 immune checkpoint family, known for their major role as negative regulators of the immune response, were overexpressed in the immune active tumors. Searching for potential tumor antigens, CEACAM21, a member of the carcinoembryonic antigen family, was found to be significantly overexpressed in immune active tissues in comparison with the silent ones.Conclusions: The results illustrate the complexity of the tumor microenvironment in HGSOC and reveal the molecular relationship between primary and recurrent tumors, which have multiple therapeutic implications. Clin Cancer Res; 23(24); 7621–32. ©2017 AACR.

Functional assignment of the 142 genes with the highest expression difference in the two clusters revealed by unsupervised clustering of recurrent samples

Supplementary figure legends and the title and notes of supplementary tables

Routine molecular diagnostic tests have yet to be introduced to guide personalized cervical cancer treatment in contrast to other solid cancers. To assess cancer functional events (CFEs), the RAIDs consortium (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) accrued consecutive tumour tissues, whole blood and sera from 419 cervical cancer patients in 18 centers from 7 European Union countries, between 2013-2016 (BioRAIDs: NCT02428842). Whole exome sequencing (WES) is presently available for the first 98 patients, for 20 CC cell lines (list available in supplementary data) and Reverse Phase Protein Array (RPPA) data for 154 patients with a common core set of 91 patients. By comparing the proportion of mutated tumours for each gene and after correction for multiple testing, we detected no significant difference in the frequency of significantly mutated genes (SMG) between the TCGA and RAIDs datasets. However, alterations in epigenetic regulator genes, which are of potential high clinical interest, are not discussed in the TCGA paper. Mutations, frameshift deletions, insertions or stop codons in MLL2 and MLL3 genes, are present in 17% and 19 % respectively (total: 30%) of the first 98 RAIDs tumours, as well as in all 20 cervical cancer cell lines analyzed. Similar to the TCGA results, frequent mutations in genes coding for acetyltransferases such as EP300/CREBBP (9%) were detected in RAIDs tumours and cell lines (15%/25%). With the exception of SHKBP1, all other novel and previously confirmed genes by TCGA were equally detected in the RAIDs dataset, such as PIK3CA (33%), FBXW7 (14%), NFE2L2 (7%), KRAS (4%), ERBB2 (4%), PTEN (5%), CASP8 (3%). Similarly to TCGA, RAIDs RPPA data defined 3 stable clusters. TCGA and RAIDs cluster 1 regrouped proteins frequently associated with EMT (epithelial mesenchymal transition). Significant features of RAIDs cluster 1 were high phospho-YAP (p= 3.11e-06) and phospho-Met (p=4.74e-03), high Met (p=2.42e-11), high Notch (p=4.14e-17), low E-cadherin (p=2.41e-06) as well as significant levels of the phosphorylated forms of EGFR, HER2-3 and AKT and high PD-L1 and B7-H4 expression. While TCGA cluster 2 had been associated with “Hormone” signaling, RAIDs cluster 2 was associated with “DNA damage” signaling. Antibody selection was not identical between TCGA and RAIDs and further comparisons are warranted. In RAIDs, pre-treatment activation of DNA repair proteins (Rad50, Phospho-Chk2, Phospho-DNA-PK, Phospho-FANCD2, Phospho-53BP1) together with low levels of Ki67 (proliferation) appeared predictive for a complete response to standard radio-chemotherapy. This needs validation on an independent population. RAIDs and TCGA cluster 3 were associated with p38 MAPK and PI3K signaling. The RAIDs consortium's future objectives are to better define actionable CFE in relation to outcome and patient's quality of life, allowing the focus on relevant CFEs for patients at high risk. Citation Format: Maud Kamal, Els Berns, Windy Luscap Rondof, Leanne De koning, Gemma Kenter, Attila Kerezst, Marina Popovic, Choumouss Kamoun, Balazs Balint, Suzy Scholl, RAIDs Consortium. RAIDs: Future prospects for innovative targeted treatments in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3686.