Description of longitudinal tumor evolution in a case of multiply relapsed clear cell sarcoma of the kidney

Abstract Background Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Case A 2‐year‐old boy was diagnosed with CCSK, which relapsed four times until he yielded to the disease at the age of 7 years. To characterize the longitudinal genetic alterations occurring in the present case, we performed targeted‐capture sequencing by pediatric solid tumors panel (381 genes) for longitudinally sampled tumors, including autopsy samples of metastasis. Internal tandem duplication of BCOR (BCOR‐ITD) was the only truncal mutation, confirming the previously reported role of BCOR‐ITD in CCSK. Conclusion Acquisition of additional mutations along tumor relapses and detection of metastasis‐specific mutations were reminiscent of the tumor progression and therapeutic resistance of this case, leading to clonal selection and a dismal fate.


| INTRODUCTION
Clear cell sarcoma of the kidney (CCSK) makes up 4% of all primary renal malignancies in children. 1 Although its prognosis has significantly improved recently, 1-5 the relapse rate is still high and the prognosis of patients with relapses is extremely poor. 6 Internal tandem duplication of BCOR (BCOR-ITD), 7,8 YWHAE-NUTM2B/E fusion, 9,10 and BCOR-CCNB3 fusion 11 have been reported in CCSK cases; however, the genetic mechanisms associated with tumor recurrence and metastasis are still poorly understood. In this study, we assessed the genetic mechanisms of CCSK recurrence and metastasis by analyzing longitudinally sampled tumors extracted from a single case: specimens at diagnosis, each relapse, and autopsy. Postoperative chemoradiotherapy induced a third remission. At the age of 6 years, he suffered from a third relapse at the para-aortic lymph nodes, the same location as the second relapse. Chemotherapy consisting of ifosfamide, etoposide, vincristine, doxorubicin, and cyclophosphamide resulted in a poor therapeutic response. The patient underwent a subtotal excision of the lesion. Pathological examination of resected specimen detected viable tumor cells among therapy-related necrosis and fibrosis (Supplementary material S1). High dose chemotherapy (busulfan and melpharan) with autologous hematopoietic stem cell transplantation (aHSCT) induced a fourth remission; however, 4 months later, a fourth relapse occurred at the ascending colon, small intestine, duodenum, pancreas, and right hepatic lobe, and he yielded to the advanced disease at the age of 7 years.

| RESULTS
After obtaining informed consent from his parents, DNA was extracted from the peripheral blood and biopsy samples obtained at initial diagnosis, each relapse (second relapse; R2, third relapse; R3, fourth relapse; R4) and autopsy (abdominal lymph node; LN, liver; Liver) except for the sample at first relapse. Direct sequencing identified BCOR-ITD (c.5526_5621dup) at all the analyzed samples ( Figure S2). SNP array analysis did not reveal any copy number alterations (CNAs) at the diagnosis whereas consistent CNAs on chromosomes 1q, 7q, and 15q were detected in R2 through R4 as well as LN and liver ( Figure 2). Each sample possessed several sample specific CNAs. Moreover, we detected a focal loss at chromosome 9q32-33 in the samples of R2 through autopsy, where only the tumor suppressor gene BRINP1 was located ( Figure 3A). We further analyzed other genetic alterations using targeted-capture sequencing (TCS) for 381 genes relating to pediatric solid tumors. 12 Somatic mutations F I G U R E 1 Clinical presentation in this case. Each mutation detected by targeted-capture sequencing was classified based on the detected samples and variant allele frequency, while there were no data available for clone at first relapse. Estimated cell population at each sample is shown were filtered using the peripheral blood sample as germline control.
Candidate somatic mutations located in exonic regions were further filtered by excluding variants: (a) with a VAF < 0.04; (b) listed in SNP databases; and (c) synonymous single-nucleotide variants, according to our previous report. 12 Finally, 13 mutations were detected and classified into 7 clones based on the detected samples and VAF (Table 1 and Figure 3B). Although validation of the candidate mutations was not performed in this study, our sequence depth (mean 600-714) combined with EB call (Empirical Bayesian Mutation calling) analysis 13 in Genomon software (https://github.com/Genomon-Project/GenomonPipeline) was estimated to be sufficient to rule out sequencing artifacts. Only the BCOR-ITD was included in clone 1, whereas three clade mutations (FLT1, JAK2, and MYH7) were additionally detected in relapsed tumor samples (clones 2, 3). The other mutations (PRKRIR, KMT2D, TSHR, CDK6, IL6ST, NF1, KDM6A, OBSCN, and SYNE1) were clone specific (clones 4-7). The number of detected mutations increased during each tumor recurrence. All R3 mutations (clones 1-3) were detected upon metastatic samples from autopsy, suggesting the accumulation of metastasis site-specific mutations during tumor progression. Importantly, the newly acquired mutations at each relapse were not detected even as a minor clone in the previous samples, though variants with a VAF < 0.02 were not detectable due to our mutation calling method (Supplementary Material S2). We analyzed the clonal evolution of the present case based on the VAF of F I G U R E 2 Results of SNP array analysis in each sample. Colorful dots at the top represent signal of each probe (raw data). Red and green lines at the bottom show allele-specific copy number, respectively. Consistent copy number alterations on chromosomes 1q, 7q, and 15q were detected. D, initial diagnosis; LN, lymph node; R2, second relapse; R3, third relapse; R4, fourth relapse each mutation and constructed a node-based phylogenetic tree using a bootstrap resampling technique implemented in ClonEvol. 14,15 Longitudinal sequencing analysis at different time points allowed us to delineate the clonal history of tumor cells, suggesting that each relapse developed based on the preexisting tumors ( Figure 3C).

| DISCUSSION
In this study, we presented a case of CCSK, which underwent four relapses. The first relapse occurred in the lung. This is consistent with the previous reports that the lung was the most common site of relapse in patients treated with DD-4A, 4,5 although the present case relapsed earlier (4 vs 24 months). 1,3 The optimal treatment of relapsed CCSK has not been established as yet. ICE chemotherapy, a salvage regimen for recurrent CCSK, 6,16 resulted in the achievement of the second remission, however, the second relapse developed. Due to the refractory disease, we performed high dose chemotherapy with aHSCT rescue though its effectiveness is unclear. 6

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ETHICAL STATEMENT
The present study was approved by the Ethics Committee of the University of Tokyo. Informed consent for publication was obtained from the patient's parents.