Genome Analysis of Osteosarcoma Progression Samples Identifies FGFR1 Overexpression as a Potential Treatment Target and CHM as a Candidate Tumor Suppressor Gene

Abstract

Osteosarcoma (OS) is the most common primary malignant tumor of bone, showing complex chromosomal rearrangements but with few known consistent changes. Deeper biological understanding is crucial to find new therapies to improve patient survival. We have sequenced the whole exome of two primary tumors (before and after chemotherapy), one metastatic tumor and a matched normal sample from two OS patients, to identify mutations involved in cancer biology. The metastatic samples were also RNA sequenced. By RNA sequencing we identified dysregulated expression levels of drug resistance- and apoptosis-related genes. Two fusion transcripts were identified in one patient (OS111); the first resulted in p53 inactivation by fusing the first exon of TP53 to the fifth exon of FAM45A. The second fusion joined the two first exons of FGFR1 to the second exon of ZNF343. Furthermore, FGFR1 was amplified and highly expressed, representing a potential treatment target in this patient. Whole exome sequencing revealed large intertumor heterogeneity, with surprisingly few shared mutations. Careful evaluation and validation of the data sets revealed a number of artefacts, but one recurrent mutation was validated, a nonsense mutation in CHM (patient OS106), which also was the mutation with the highest expression frequency (53%). The second patient (OS111) had wild-type CHM, but a downregulated expression level. In a panel of 71 clinical samples, we confirmed significant low expression of CHM compared to the controls (p = 0.003). Furthermore, by analyzing public datasets, we identified a significant association between low expression and poor survival in two other cancer types. Together, these results suggest CHM as a candidate tumor suppressor gene that warrants further investigation.

Fig 1. Schematic timeline of the disease and treatment courses of the two patients for (A): OS106 and (B): OS111.

Time is shown in months. P-1: primary tumor treatment naïve, P-2: primary tumor treatment experienced. N: normal, M: metastasis, MAP: methotrexate, doxorubicin and cisplatin, IE: ifosfamide and etoposide, NED: No evidence of disease.

Fig 2. Transcriptome analysis of OS106 and OS111.

(A) The relative expression level of known drug resistance-related genes and apoptosis-related genes. The values are shown as log2 of the expression level ratio, compared to the average of the four normal osteoblasts. Asterisk (*) indicates low number of reads in the osteoblasts. (B) The expression of all exons in TP53 and FAM45A, respectively, shown as total number of reads in patient OS111. Red squares indicate the exons that are fused to form the resulting fusion transcript. ex: exon; no.: number (C) The relative expression level of TP53 and FAM45A. The values are shown as log2 of the expression level ratio, compared to the average of the osteoblasts. The expression of all transcripts are included, both wild-type and aberrant. (D) The expression of all exons in FGFR1 and ZNF343, respectively, shown as total number of reads in patient OS111. Red squares indicate the exons that are fused to form the resulting fusion transcript. (E) The relative expression level of FGFR1 and ZNF343. The values are shown as log2 of the expression level ratio, compared to the average of the osteoblasts. The expression of all transcripts are included, both wild-type and aberrant.

Fig 3. WES analysis of OS106 and OS111.

(A) The total number of somatic mutations identified in each sample for both patients. Black represents nonsynonymous mutations in the coding sequence and/or mutations in the splice sites, whereas grey represents all other mutations. The respective numbers of mutations are indicated in the bars. aa: amino acids, P-1: primary tumor treatment naïve, P-2: primary tumor after treatment, M: metastasis. (B) The mutation spectrum showing the proportions of transitions and transversions in percent (adding up to 100%). (C) Venn diagram showing the distribution of mutations (only nonsynonymous mutations in the coding sequence and mutations in the splice sites) between the matched tumor samples in each patient. The total number of mutations in each tumor is shown in parenthesis.

Fig 4. CHM analysis.

(A) The identified nonsense mutation p.G646* in CHM results in the loss of the last eight amino acids. (B) The relative expression level of CHM in OS106 and OS111 compared to the average of the four osteoblasts. The nonsense mutation was only identified in OS106 at a frequency of 53% revealed by the RNA-seq (illustrated with grey bars). Mut: nonsense mutation, wt: wild-type. C) Expression level of CHM in osteosarcoma cell lines (n = 19), osteosarcoma clinical samples (n = 71) and normal control samples (n = 6). The difference was statistical significant with p-values of 0.004 and 0.003, for cell lines and clinical samples, respectively (Mann-Whitney U test) (D) Survival analysis of CHM expression in 71 clinical OS samples. The samples are sorted according to low (below median) or high (above the median) expression level. The difference was not significant with a p-value of 0.980 (Log Rank/Mantel-Cox test). (E) Survival plot obtained from DRUGSURV showing expression of CHM in patients with large diffuse B-cell lymphoma. Low expression was significantly associated with poor survival, p = 1.68e-05 (Chi-square 18.5 on 1 degree of freedom). (F) Survival plot obtained from DRUGSURV showing expression of CHM in patients with breast cancer. Low expression was significantly associated with poor survival, p = 8.49e-03 (Chi-square 6.9 on 1 degree of freedom).