Ovarian carcinoma patient derived xenografts reproduce their tumor of origin and preserve an oligoclonal structure

Abstract

Advanced Epithelial Ovarian Cancer (EOC) patients frequently relapse by 24 months and develop resistant disease. Research on EOC therapies relies on cancer cell lines established decades ago making Patient Derived Xenografts (PDX) attractive models, because they are faithful representations of the original tumor. We established 35 ovarian cancer PDXs resulting from the original graft of 77 EOC samples onto immuno-compromised mice. PDXs covered the diversity of EOC histotypes and graft take was correlated with early patient death. Fourteen PDXs were characterized at the genetic and histological levels. PDXs reproduced phenotypic features of the ovarian tumors of origin and conserved the principal characteristics of the original copy number change (CNC) profiles over several passages. However, CNC fluctuations in specific subregions comparing the original tumor and the PDXs indicated the oligoclonal nature of the original tumors. Detailed analysis by CGH, FISH and exome sequencing of one case, for which several tumor nodules were sampled and grafted, revealed that PDXs globally maintained an oligoclonal structure. No overgrowth of a particular subclone present in the original tumor was observed in the PDXs. This suggested that xenotransplantation of ovarian tumors and growth as PDX preserved at least in part the clonal diversity of the original tumor. We believe our data reinforce the potential of PDX as exquisite tools in pre-clinical assays.

Keywords: CNC; PDX; mutations; oligoclonality; ovarian cancer.

Figure 1. PDX faithfully replicate the morphology of the EOC of origin

Most representative examples are shown here. O-3312 is a Grade 3 (Silverberg classification) Serous Carcinoma characterized by papillae proliferation and extended necrotic areas; O-2878 is a Grade 2 Serous Carcinoma with papillae architecture and psammoma; O-3006 is an undifferentiated Grade 3 Serous Carcinoma with solid architecture and large necrotic areas; O-1912 is a Clear Cell Carcinoma; O-1217 Grade 2 is a Carcino-Sarcoma (Mixed Mixed Müuuml;llerian tumors) showing inclusions of sarcomatous tissue within an undifferentiated epithelial carcinoma; O-2781 is a mucinous carcinoma with mucus material accumulating inside and outside tumor cells. Note that the characteristics observed in the original tumors were strikingly reproduced in the PDXs that were generated.

Figure 2. EOC of origin and corresponding PDXs show similar CNC profiles

CNC profiles of patient tumors and corresponding PDXs were analyzed by unsupervised hierarchical clustering showing systematic co-clustering. When several PDXs generated from the same original tumor could be analyzed they regrouped as tight clusters. Samples are in columns, chromosomal localization in rows. Chromosomes are indicated as alternating green and white vertical bars and identified by their number. Each family of EOC and derived PDXs are highlighted in colored boxes below the dendogram (no code associated to the colors used). Colored horizontal lines indicate the histological type of the EOC grafted; black: serous ovarian carcinoma, blue: clear cell carcinoma, red: carcino-sarcoma, green; mucinous carcinoma.

Figure 3. Fraction of the genome involved in CNCs differing in the PDX as compared with cognate patient EOC

The X axis represents CNC originally present in the patient tumor and absent in the PDX (Tumor of origin specific), the Y axis CNCs occurring de novo in the PDX while not scored in the tumor of origin (PDX specific). Vertical and horizontal blue lines indicate the median levels of tumor specific (X) or PDX specific (Y) CNCs. Each color ball represents the comparison of the tumor of origin and one PDX. Tumors occurring only once in the graph are those where only one PDX was analyzed. Tumor IDs are indicated and color highlights indicate the fraction of tumor cells for each tumor of origin; green 70 to 90%, purple 50 to 70%, yellow < 50%.

Figure 4. PDX established from tumor O-2878 and subsequent metastatic recurrences O-0005D and O-0005S show fluctuating CNC patterns at 8p12 and 20q13 indicating oligoclonality

A. Schematic representation of the clinical history of patient 2878 and graft trees of the PDXs established from the 3 original tumors. Each represents a PDX. Boxes in blue filling correspond to cases that were analyzed by array-CGH, color lining identifies PDXs presented in B. B. zoomed representations of array-CGH profiles at chromosomes 8p12 (left box) and 20q13 (right box) showing variation in copy number levels as well as in the size of the region of gain. Variations observed by array-CGH were confirmed by interphase FISH on frozen tumor sections using probes to FGFR1 (8p12) and an anonymous probe to 20q13.

Figure 5. mutational profiles of O-2878, distant nodules O-0005D and O-0005S and the PDX that were derived from them

A. mutations detected were indicated in shades of red ranging from dark red (100% of the calls) to light red (10% of the calls). White boxes indicate samples in which the mutation was not detected. B. Phylogenetic tree of the 3 tumor samples and their PDX offsprings. The 3 patient samples show greater proximity to each other than they do to their corresponding PDXs.