Tracking sub-clonal TP53 mutated tumor cells in human metastatic renal cell carcinoma

Abstract

Renal Cell Carcinomas (RCCs) are heterogeneous tumors with late acquisition of TP53 abnormalities during their evolution. They harbor TP53 abnormalities in their metastases. We aimed to study TP53 gene alterations in tissue samples from primary and metastatic RCCs in 36 patients followed up over a median of 4.2 years, and in xenografted issued from primary RCCs. In 36 primary RCCs systematically xenografted in mice, and in biopsies of metastases performed whenever possible during patient follow-up, we studied p53-expressing tumor cells and TP53 gene abnormalities.We identified TP53 gene alterations in primary tumors, metastases and xenografts. Quantification of tumors cells with TP53 gene alterations showed a significant increase in the metastases compared to the primary RCCs, and, strikingly, the xenografts were similar to the metastases and not to the primary RCCs from which they were derived.Using laser-microdissection of p53-expressing tumor cells, we identified TP53-mutated tumor cells in the xenografts derived from the primary RCC, and in a lung metastasis later developed in one patient. The mutation enabled us to track back their origin to a minority sub-clone in the primary heterogeneous RCC. Combining in situ and molecular analyses, we demonstrated a clonal expansion in a living patient with metastatic RCC.

Keywords: TP53 mutation; human RCC; metastases; sub-clonal tumor cells; tumor heterogeneity.

Figure 1. p53-expressing tumor cells and TP53 copy number abnormalities in primary RCCs and corresponding metastases

A. In the 36 primary RCCs, the percentage of tumors expressing p53 (threshold >1% p53-positive cells) significantly increases from the primary RCCs (PRCC) with no metastasis to the PRCCs with metastases during evolution or at diagnosis. B. p53-expressing cells are found around vessels (V) in primary RCC, and are scattered in the corresponding lung metastasis. Indirect immunoperoxydase, bar =50μm. In 8 patients with biopsy samples of the primary RCCs (PRCC) and their corresponding metastases (CM), the percentage of p53-expressing cells significantly increases in the metastases. C. TP53 gene copy number abnormalities are assessed by FISH in the same 8 patients, with TP53 gene labeled in red and chromosome 17 centromere in green. Nuclei are colored in blue by DAPI. The upper panel shows one trisomic nucleus (three red spots) and two monosomic nuclei (one red spot in each) for TP53 gene. The lower panel shows one trisomic cell and one disomic cell. Counts of TP53 gene copy numbers show a significant decrease in the percentage of disomic cells in the metastases, while the percentage of monosomic cells significantly increases. *p < 0.05, **p < 0.01.

Figure 2. p53-expressing tumor cells and TP53 copy number abnormalities in primary RCCs, corresponding metastases and xenografts derived from the primary RCC

A. In the 4 patients with biopsy samples of the primary RCCs (PRCC), of their corresponding metastases (CM), and of the xenografts (Xe), the percentage of p53-expressing cells significantly increases in the metastases and in the xenografts for Patient 35 (in purple) and for Patient 36 (in red), without any difference between the metastasis and the xenograft of a same patient. B. In the same 2 patients, the xenograft is reflecting the metastasis, and both are different from the primary RCC, with increased numbers of TP53 monosomy and trisomy in the corresponding metastasis and in the xenograft.ns = not significant, *p < 0.05.

Figure 3. TP53 signaling pathway in the lung metastasis and the two xenografts compared to the primary RCC of patient 36

In tumor samples from patient 36, the TP53 cell signaling pathway is significantly altered in the metastasis and in the xenografts compared to the primary tumor, with 22 genes differentially expressed. The fold changes are indicated with a color gradient from blue (primary tumor) to pink (metastasis or xenografts). The fold changes are indicated with a color gradient from blue (metastasis or xenografts, CM or Xe) to pink (primary tumor, PRCC). When a gene is significantly overexpressed in the xenografts and in the lung metastasis, the box is colored blue (for example ATR). When significantly overexpressed in the primary tumor, the box is colored pink (for example BAX).

Figure 4. Tracking TP53 mutated tumor cells in p53 expressing cells by laser-microdissection

A. In the RCC lung metastasis sample, p53-expressing cells are laser-microdissected for further molecular analyses. B. TP53 status is assessed by PCR-HRM screening on exons 5 to 8 in p53-expressing cells microdissected from the primary RCC (PRCC), the two xenografts derived from it (Xe1, Xe2), and the lung metastasis (CM). A shift on exon 6 is identified in the four samples when compared to wild-type profile (WT). C. Sequencing of exon 6 of TP53 identifies an identical missense mutation c.605G>A p.R202H in laser-microdissected p53-expressing cells from the primary RCC, in the two tumor xenografts derived from it, and in the lung metastasis.