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Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients with Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related

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Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients with Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related ORIGINAL RESEARCH published: 23 June 2021 doi: 10.3389/fonc.2021.646338 Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients With Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related Edited by: Immunosuppressive Marianna Kruithof-de Julio, University of Bern, Switzerland Microenvironment Reviewed by: 1† 1† 1† 2 1 1 Hao Zeng, Shaoxi Niu , Kan Liu , Yong Xu , Cheng Peng , Yao Yu , Qingbo Huang , 1 1 1 1 1 1 Sichuan University, China Shengpan Wu , Bo Cui , Yan Huang , Xin Ma *, Xu Zhang * and Baojun Wang * Ahmet Murat Aydin, Moffitt Cancer Center, United States 1 Department of Urology, The Third Medical Centre, Chinese PLA (People’s Liberation Army) General Hospital, Beijing, China, 2 Department of Urology, The Seventh Medical Center of Chinese PLA General Hospital, Beijing, China *Correspondence: Xin Ma [email protected] Background: Clear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) Xu Zhang [email protected] is associated with a poor clinical outcome. Although several studies have examined the Baojun Wang genomic features of ccRCC, the genetic profile of VTT along with its matched primary [email protected] tumor has not been fully elucidated. †These authors have contributed equally to this work Materials and methods: Samples of VTT tissues and matched primary tumor tissues from ccRCC patients (n = 25), as well as primary tumor tissues from patients without VTT Specialty section: (n = 25) were collected and analyzed using whole-exome sequencing. Four additional This article was submitted to fi Genitourinary Oncology, ccRCC patients who were un t for surgery were treated with an anti-programmed death a section of the journal receptor-1 (PD-1) monoclonal antibody (Toripalimab, 240 mg, Q3W, IV). Frontiers in Oncology Results: By comparing the primary kidney tumors from ccRCC patients with or without Received: 26 December 2020 Accepted: 18 May 2021 VTT, a relatively higher prevalence of BAP1 and KDM5C alterations were found in ccRCC Published: 23 June 2021 patients with VTT, and these alterations were associated with worse overall survival in the Citation: kidney renal clear cell carcinoma (KIRC) database. Based on subclone analysis, VTT was Niu S, Liu K, Xu Y, Peng C, fi Yu Y, Huang Q, Wu S, Cui B, predicted to primarily originate directly from the primary renal mass. A signi cantly higher Huang Y, Ma X, Zhang X and prevalence of CELSR2 and TET2 alterations were identified in the VTTs compared with the Wang B (2021) Genomic Landscape matched primary tumors. An increased prevalence of DNA damage repair genes, of Chinese Clear Cell Renal Cell Carcinoma Patients With Venous especially those involved in homologous recombination repair and non-homologous Tumor Thrombus Identifies end joining, was found in ccRCC patients with VTT. Notably, VTT was characterized by Chromosome 9 and 14 Deletions and Related the increase incidence of copy number loss in the whole exome (p < 0.05), particularly in Immunosuppressive the chromosome 9 and 14 regions. Deletion of chromosome 9 and 14 was associated Microenvironment. with worse survival, unfavorable clinical features, and the presence of an Front. Oncol. 11:646338. doi: 10.3389/fonc.2021.646338 immunosuppressive microenvironment, which was characterized by higher infiltration of Frontiers in Oncology | www.frontiersin.org 1 June 2021 | Volume 11 | Article 646338 Niu et al. Genomic Landscape of ccRCC VTT regulatory T cells, follicular helper T cells, and resting mast cells, but lower counts of resting CD4 memory T cells and CD8 positive T cells. A significantly lower count of CD4+ and CD8+ tumor-infiltrated lymphocytes was identified in the VTT samples comparing with matched primary tumor. Of note, three out of the four ccRCC patients with VTT in our cohort who were treated with the anti-PD-1 therapy exhibited remarkable remission in the renal mass but no notable shrinkage in the VTT mass. Conclusion: Our study revealed the genetic profile of Chinese ccRCC patients with VTT, and identified multiple features associated with known poor outcomes, including gene alterations and copy number loss. The deletions in chromosomes 9 and 14, and the associated immunosuppressive microenvironment may indicate limited sensitivity to anti- PD-1/PD-L1 monotherapy in VTT. Keywords: venous tumor thrombus, ccRCC, genomic feature, copy number variant, immune microenvironment INTRODUCTION different genetic backgrounds and the exposure to aristolochic acid in traditional Chinese medicines. Renal cell carcinoma (RCC) is the second most common In this study, we applied whole-exome sequencing to study genitourinary malignancy in China, with an estimated 66,800 the genomic features of VTT in Chinese patients with clear cell new cases and 23,400 deaths in 2015 (1). Notably, 4–10% of (cc)RCC with the matched primary tumors, and these were also locally advanced RCC patients develop venous tumor thrombus compared with primary tumors from RCC patients without VTT (VTT), and their overall 5-year cancer-specific survival (CSS) is to elucidate the genomic features and their potential clinical only 40–65% (2). The median survival of untreated RCC patients significance in ccRCC with VTT. with VTT is only 5 months, and the 1-year survival rate is less than 30% (3). To date, radical nephrectomy with thrombectomy remains the best therapeutic choice for RCC patients with VTT, MATERIAL AND METHOD and notably, a higher VTT level under the Mayo classification system is associated with worse CSS, increased rates of Sample Source and Ethic Data complications, and increased mortality following surgery (4, 5). We prospectively enrolled 50 ccRCC patients at the Third To improve the safety and therapeutic effects of surgery, Medical Centre of Chinese PLA (People’s Liberation Army) neoadjuvant therapies have been developed to shrink both the General Hospital from 2018 to 2019. Twenty-five were ccRCC primary lesion and the VTT. Although previous studies have with VTT, and twenty-five were ccRCC without VTT. All reported the use of anti-angiogenesis drugs preoperatively, patients had undergone radical nephrectomy with or without including sorafenib and sunitinib, the clinical benefits are thrombectomy and provided written informed consent. This limited (6). Therefore, it is essential to understand the molecular study was approved by the ethics committee of the First mechanisms underlying the formation and development of VTT. Medical Center of PLA General Hospital (S2017-100-01) and A few previous studies have reported the genomic features of conducted under the principles of the Declaration of Helsinki RCC with VTT. As depicted by the TRACERx Renal project, and the Good Clinical Practice guidelines. Patient characteristics most genetic alterations in the VTT tissues were also present in were listed in Table 1. All samples were collected for DNA their matched primary tumor tissue; however, the primary tumor isolation after pathological evaluation, but two VTT samples did tissues also possessed more recently developed driver mutations not yield enough DNA for further testing. Finally, 23 VTT that were absent from the VTT tissue (7). Similarly, another samples (V group), 25 primary tumor samples (VP group), study also suggested that the VTT originated from the primary and 25 renal tumor samples without VTT (NP group) were tumor, as all subclones in the VTT were also shown to match the analyzed by whole-exome sequencing (WES), using the DNA primary renal tumors (8). In the same study, a homologous from peripheral blood as germline control. In addition, another recombination deficiency feature, described as the BRCAness four ccRCC patients with VTT were evaluated as unfitfor mutation signature, was also identified in a subset of RCC tumors surgery and were treated with anti-programmed death with VTT and samples from The Cancer Genome Atlas (TCGA), receptor-1 (PD-1) monoclonal antibody (Toripalimab, 240 mg, indicating a DNA damage repair (DDR) deficiency and potential Q3W, IV). sensitivity to poly(ADP-ribose) polymerase inhibitors or platinum-based therapy in these patients (8). Nevertheless, DNA Isolation most research on the genomic landscape of RCC was DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Inc.) performed in Caucasians, and the results may differ for under the manufacturer’s instructions. The purified gDNA was Chinese RCC patients with VTT, particularly considering the quantified using Qubit 3.0 Fluorometer (Life Technologies, Inc.) Frontiers in Oncology | www.frontiersin.org 2 June 2021 | Volume 11 | Article 646338 Niu et al. Genomic Landscape of ccRCC VTT TABLE 1 | Clinical Characteristics of 50 RCC Patients. Characteristics With VTT n = 25 Non-VTT n = 25 Median age, year (range) 52 (25–84) 55 (34–86) Sex Male 21 20 Female 4 5 ISPU Grade 10 1 1-2 0 1 21212 2-3 6 7 35 3 3-4 2 1 40 0 Histological subtype Clear cell RCC 25 25 IVC wall Yes 19 – invasion No 6 – and StepOnePlus System (Life Technologies, Inc.). For tumor and mutational catalog with known and predefined signatures was non-tumor samples, 100 ng of DNA was sheared with a Covaris computed by non-negative least squares (NNLS). By comparing E210 system (Covaris, Inc.) to generate fragments with a length of the whole genome to WES capture regions, mutational catalog 200 bp. correction was performed to account for the differences in the occurrence of triplet motifs. A set of 30 publicly available WES mutational signatures AC1-AC30 (AC standing for Alexandrov The Accel-NGS 2S HYB DNA LIBRARY KIT (Swift Biosciences, COSMIC) were analyzed. 23096) and HotStart ReadyMix (KAPA, KK2612) for library preparation and amplification were used, respectively. The Homologous Recombination Deficiency amplified libraries were purified by using SPRISELECT The homologous recombination deficiency, also called genomic (Beckman, B23319) and further captured with xGen Exome scar scores was determined by counting the number of loss of Research Panel v2 (IDT), whose target region was 33 Mb. heterozygosity (LOH), large scale transitions (LSTs), and Finally, samples underwent paired-end sequencing on a telomeric allelic imbalances (TAIs).
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