A genomic mutation spectrum of collecting duct carcinoma in the Chinese population

Renal collecting duct carcinoma (CDC) is a rare and lethal subtype of renal cell carcinoma (RCC). The genomic profile of the Chinese population with CDC remains unclear. In addition, clinical treatments are contradictory. In this study, we aimed to identify the genomic mutation spectrum of CDC in the Chinese population. Whole-exome sequencing was performed using the Illumina Novaseq™ 6000 platform. MuTect2 detects single-nucleotide variants (SNVs) and small scale insertions/deletions (INDELs). The identified mutations were annotated with ANNOVAR and validated by Sanger sequencing. Control-FREEC was used to detect copy number variation (CNV), and GISTIC was applied to detect frequently mutated altered regions. These data were compared with associated The Cancer Genome Atlas cohorts. Ten normal-matched CDC patients were included. The mean tumour mutation burden was 1.37 Mut/Mb. Six new recurrent somatic mutated genes were identified, including RBM14, MTUS1, GAK, DST, RNF213 and XIRP2 (20% and 2 of 10, respectively), and validated by Sanger sequencing. In terms of common mutated genes, SETD2 was altered in both CDC and other RCC subtypes but not in bladder urothelial carcinoma (BLCA); CDKN2A was a driver gene in both CDC (SNV: 10%, 1 of 10) and BLCA but not in other RCC subtypes. Next, 29 amplifications and 6 deletions of recurrent focal somatic CNVs were identified by GISTIC2.0, which displayed differences from kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP) and BLCA cohorts. Of note, CDKN2A (CNV alteration: 30%, 3 of 10) and CDKN2A-AS1 were the only overlapping genes of these four cohorts. Importantly, the CDKN2A mutation in our cohort differed from previous studies in urinary carcinomas. Moreover, CDKN2A-altered cases had significantly worse overall survival than wild-type cases in both KIRC and KIRP cohorts. In addition, the most frequently altered genomic pathway of our CDC cohort was the CDKN2A-mediated p53/RB1 pathway. Our study offers the first genomic spectrum of the Chinese population with CDC, which differs from that of the Western population. The altered CDKN2A-mediated p53/RB1 pathway might provide new insight into potential therapeutic targets for CDC patients.


Background
Collecting duct carcinoma (CDC) is a rare and lethal subtype of renal cell carcinoma [1] that is still mainly diagnosed based on pathological examination [2,3]. Approximately half of CDC patients are initially diagnosed at an advanced stage with metastatic symptoms of the lymph node, bone, lung, or liver, and most die within 1-3 years [4,5]. Moreover, the clinical treatments are contradictory. The kidney cancer part of the National Comprehensive Cancer Network (NCCN) guidelines recommend platinum-based chemotherapies due to some shared biological features with urothelial carcinoma [6]. However, 23 metastatic CDC patients treated with gemcitabine plus cisplatin or carboplatin showed only an objective response rate (ORR) of 26% and an overall survival (OS) of 10.5 months [7]. In addition, a systemic therapy for CDC, renal cell carcinoma (RCC), has been proposed according to a transcriptome sequencing study [8], yet the outcomes are unsatisfactory. Combined chemotherapies showed an ORR of 30.8% and an OS of 12.5 months [9], which was similar to single chemotherapies [10]. Targeted therapies for metastatic CDC have little clinical benefit [11], and no response to immunotherapy has been observed [4,12].
Therefore, additional comparative studies are urgently needed to better distinguish the dominant molecular signature between CDC and other RCC subtypes or urothelial carcinomas, providing new insight into potential prognostic and therapeutic targets.
Therefore, in this study, we performed deep wholeexome sequencing of 10 paired CDC patient tumour tissues that matched normal kidney tissues to improve our understanding of the genomic profile of Chinese patients with CDC and compared the results with other RCC subtypes and bladder urothelial carcinoma. We found that CDC is not only characterised as a unique type of solid tumour but also shares some specific molecules with other RCC subtypes and urinary tract carcinoma. The CDKN2A alteration-mediated p53/RB1 pathway might provide new insight into potential prognostic and therapeutic targets for these patients.

Study design and samples information
Patients were collected retrospectively from the First Affiliated Hospital (Changhai Hospital), Naval Military Medical University. Paired tumour and normal formaldehyde-fixed paraffin-embedded (FFPE) samples were obtained prior to any treatment. Pathological diagnoses were reconfirmed by two experienced uropathologists. Patients with urothelial carcinoma involving the upper tract, papillary RCC, clear cell carcinoma RCC, chromophobe RCC, unclassified RCCs and other malignant tumours were excluded.
Ethics approval was obtained from the institutional review board of Changhai Hospital (CHEC2021-064). The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983, and the Good Clinical Practice guidelines. All research participants or their legal representatives signed informed consent forms for participation in the research.

DNA extraction and quantification
Genomic DNA was extracted using QIAamp DNA FFPE Tissue Kit (QIAGEN). DNA quality and yield were measured and assessed using a Qubit fluorometer and Qubit dsDNA HS Assay Kit (Thermo Fisher) following the manufacturer's protocol.

WES library generation and sequencing
Before library generation, genomic DNA was fragmented by sonication to a median size of 350 bp. Then, the KAPA hyperprep kit (Roche) was used for library preparation, and xGen ® Hybridization and Wash Kit (IDT) was used Keywords: Collecting duct carcinoma, Somatic mutations, Copy number variants, CDKN2A for exome capture before sequencing. Next, genomic DNA fragments were end-repaired, ligated with Illumina sequencing adapters, and amplified. Finally, DNA libraries were subjected to WES using the Illumina Novaseq ™ 6000 platform (2 × 150-bp paired-end reads).

Sequencing data analysis
Paired-end reads were quality checked by FastQC (v0.11.9) and processed to high quality using Trimmomatic [17]

Somatic mutations calling
Somatic mutations, including single-nucleotide variants (SNVs) and small-scale insertions/deletions (INDELs), were detected using the Mutect2 pipeline in Genome Analysis Toolkit (GATK, v4.1.9.0). ANNOVAR [18] was applied to annotate filtered variant call format files using multiple annotation databases. Briefly, mutations in segmental duplications (genomicSuperDups) or repetitive elements (RepeatMasker) were removed. Non-synonymous exonic mutations with minor allele frequency > 5% in the 1000 Genome Project, Exome Aggregation Consortium database with allele frequencies in East Asia (EAS), dbSNP 138, or exome sequencing project (ESP) were removed; all COSMIC variants were retained. Mutations with a variant allele frequency (VAF) greater than 0.03 after filtering were reviewed manually using integrated genomics viewer (IGV). Finally, mutations within the blacklist [19] were also filtered and removed. Next and importantly, recurrent mutated genes were experimentally validated by Sanger sequencing.
Further sequencing analyses, including the significantly mutated genes (SMGs), mutation signature pattern and tumour mutation burden, were also performed. Briefly, the prepared mutation annotation format file was analysed to determine SMGs using MutSigCV (v2.0), with a cut-off value of p < 0.05. The deconstructSigs R package [20] was adopted to calculate the ratios of 30 types of defined COSMIC mutation signatures [21] in each sample. Tumour mutation burden was defined as in our previous study [22], with 34.2 Mb of exonic region.

Comparison of mutation landscapes and pathways across CDC and associated TCGA cohorts
The integrated SNV and CNV sequencing data of associated The Cancer Genome Atlas (TCGA) cohorts, including kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and bladder urothelial carcinoma (BLCA), were downloaded from cBioPortal (https:// www. cbiop ortal. org). Next, RCircos [25] and Venn diagrams were applied to visualise and compare the different distributions of the above CNVbased genes in our CDC, KIRC, KIRP and BLCA cohorts.
In addition, the mutation frequency of all key genes in 10 common and 3 specific cancer-related pathways related to SNV and CNV were used to compare the mutation landscape and pathway enrichment across these four study cohorts and to determine the putative critical pathway in our CDC cohort. The components of all the altered genes in each pathway were also calculated.

CDKN2A mutation spectrum in CDC and associated TCGA cohorts
Available SNV, CNV and overall survival data of CDKN2A for KIRC, KIRP, BLCA and breast cancer (BRCA) patients were downloaded from cBioPortal. The percentages of patients with SNVs, CNV alterations and wild-type CKDN2A were summarised, and the mutation spectrum of CDKN2A in these cohorts was analysed.

Patient clinical information
Ten CDC patients with matched tumour and normal renal tissues were included, and their clinical information is summarised in Fig. 1 and Additional file 1: Table S1. Of these patients, eight were male and two female. The median age was 57.3 years old (range: 33-67). The ratio of left to right tumour side was 1: 1.5. Two patients were diagnosed with a high clinical T stage (T3 and T4), though most of the others were at T2. More than half of the patients had local lymph node infiltration, and two patients had distant metastases.
Next, compared with previous studies with CDC and associated TCGA cohorts, including KIRC, KIRP and BLCA, TP53 mutation was detected in all six studies. examined, and those of 1, 3, 5, 8 and 9 were the most common (Additional file 6: Figure S3).

CDKN2A alteration is important in CDC and associated TCGA cohorts
Since CDKN2A is a key mutated gene according to the somatic mutation and copy number alteration landscapes, we further explored CDKN2A alteration in CDC and associated TCGA cohorts, including BLCA, KIRC, KIRP and BRCA.
There was a higher percentage of CNV alterations than SNV alterations of CDKN2A in each cohort. The percentages of somatic mutated SNVs in CDC, BLCA, KIRC, KIRP and BRCA were 10%, 5.5%, 1.1%, 0.7% and 0.2%, respectively (Fig. 3A). Surprisingly, the CDKN2A mutation in our study, which encodes a protein change of p. R24Gfs*16 on exon 1, did not overlap with the previously reported CDKN2A mutation spectrum in urinary carcinomas and BRCA (Fig. 3B).
Moreover, patients with CDKN2A alteration displayed a significantly worse overall survival than patients with the wild-type gene in both the KIRC and KIRP cohorts. Despite no significant differences between CDKN2Aaltered and wild-type cases in the BLCA and BRCA cohorts, these patients exhibited the same tendency in the early follow-up period (Fig. 3C). Overall, clinical data on CDKN2A in CDC are lacking due to its rarity.
The CDKN2A-mediated p53/RB1 pathway is mostly altered in the CDC population Genomic alterations are known to target common cancer pathways, even though not all component genes are altered at an equal frequency [27]. Next, we compared the mutational landscape among the CDC, KIRC, KIRP and BLCA cohorts in a pathway-centric manner. We focused on 10 important common pathways and three cancerspecific pathways based on both the SNV and CNV data, which suggested that the overall pathway-level mutation burden was different among the four cohorts (Fig. 4A, B; Additional file 10 and 11: Table S6 and S7).
Notably, the most frequently altered cancer-specific pathway in both the CDC and BLCA cohorts was the p53/RB1 pathway (Fig. 4C). However, individual altered genes varied significantly. In our CDC cohort, CDKN2A was most frequently altered, whereas TP53 was the major mutated gene altered in the BLCA cohort. In addition, genes in the p53/ RB1 pathway in the BLCA cohort were altered to different degrees. The KIRC cohort of TCGA typically showed specific VHL pathway alteration (Additional file 12: Fig.  S5), which offers clinical drug targets, such as sunitinib and pazopanib. None of the key genes in the VHL pathway were altered in our CDC cohort, which might help to explain the limited efficacy of target therapies in CDC patients. In addition, MET was most frequently mutated in the specific MAPK pathway in the KIRP cohort but not in our CDC cohort (Additional file 13: Fig. S6).
Taken together, the CDKN2A alteration-mediated p53/ RB1 pathway is most common in the CDC population, which might offer new insight into the clinical treatment of CDC patients.

Discussion
In this study, we identified eight recurrently somatically mutated genes: RBM14, MTUS1, GAK, DST, ASPM, CDC27, RNF213 and XIRP2. Except for ASPM and CDC27, six of these genes were validated by Sanger sequencing. In terms of the biological functions of these six genes [28][29][30][31][32][33][34][35], surprisingly, only RBM14 and MTUS1 have been reported to be associated with urothelial carcinomas. RBM14 encodes a ribonucleoprotein that functions as a general nuclear coactivator and an RNA splicing modulator in a PARP-dependent DSB repair process [29]. MTUS1, a tumour-suppressor gene encoding angiotensin-II type 2 receptor-interacting proteins, is downregulated in clear cell renal cell carcinoma [31]. However, whether these mutations alter the clinical treatment of CDC patients remains unclear. Hence, these six genes might be new targets for CDC molecular therapy. For the remaining mutated genes shown in Fig. 1, TP53 is a known tumour-suppressor gene. SETD2 was altered in both CDC and RCC subtypes but not in BLCA. Additionally, CDKN2A was a driver gene in both the CDC and BLCA but not in other RCC subtypes. In one patient in our study, arotinib target therapy achieved good efficacy after gemcitabine plus cisplatin chemotherapy. Taken together, these results suggest that CDC might present some shared therapeutic target molecules with other RCC subtypes and BLCA, which offers new insight into the systemic treatment of CDC patients.
Next, when we compared copy number variants in our CDC cohort with other subtypes of renal cell carcinomas and bladder urothelial carcinoma and found a unique mutation spectrum. In total, 29 amplified and 6 deleted recurrent focal CNVs were identified. In terms of driver genes in Cancer Gene Census, oncogenes SKI, MUC4, TLX3, NPM1, KAT7, H3F3B, GNA11, MAP2K2, FSTL3, SH3GL1 and U2AF1 and tumour-suppressor genes RAD51B, DICER1, BCL11B, CCNB1IP1 and BAZ1A were detected. However, none of these genes are reported in association with renal cell carcinoma or urothelial carcinoma.
Notably, CDKN2A, a typical tumour-suppressor gene, was the shared gene in these four cohorts. Afterwards, we investigated the mutation spectrum of CDKN2A. CDKN2A encodes p16 and p14 to inactivate p53 [36]. Recently, a study reported 12.5% heterozygous losses and half homozygous losses in 16 patients with CDC [15]. Similar results were identified in 12% of 17 CDC patients, including one homozygous deletion and one truncation [13]. We also explored the role of CDKN2A in different subtypes of RCC, BLCA and BRCA. We detected a frameshift-deleted CDKN2A mutation in our CDC cohort, which suggested a novel mutated site different from any of the previously reported CDKN2A mutated sites in other urinary carcinomas and BRCA [37][38][39][40]. Furthermore, the most frequently cancer-specific altered pathway of this Chinese CDC cohort was the p53/RB1 pathway, which was consistent with our finding of CDKN2A. In addition, CDKN2Aaltered patients displayed a significantly worse OS than patients without alterations in both the KIRC and KIRP cohorts, as validated by Girgis et al. [41], who found that KIRC with biallelic inactivation of CDKN2A indicates a poor prognosis. Importantly, CDKN2A upregulates expression of cyclin-dependent kinase 4 (CDK4)/CDK6 [15], the corresponding selective inhibitors of which, such as palbociclib, ribociclib, and abemaciclib, interfere with cell cycle progression, induce cell senescence, and promote cancer cell disruption through a cytotoxic CD8+ T cell-mediated effect [22]. No CDKN2A alteration was found to correlate with clinical outcome in patients with platinum-refractory metastatic urothelial carcinoma, and palbociclib did not show meaningful clinical efficacy [42]. Moreover, CDKN2A loss is not associated with further benefit from palbociclib in combination with letrozole in the Palbociclib Ongoing Trials in the Management of Breast Cancer (PALOMA)-1 trial for patients with advanced ER+/HER2− breast cancer [43]. Nevertheless, a preclinical study of RCC cell lines reported that decreased CDKN2A is associated with sensitivity to CDK4/6 inhibitors [44], with an effective response in KIRC with wild-type VHL and CDKN2A mutations due to palbociclib [45]. Pal et al. [46] also observed a meaningful response to palbociclib in a patient with metastatic CDC harbouring a CDKN2A homozygous deletion, which suggests a direction for further study of CDC patients. Taken together, our findings indicate that this CDKN2A alteration-mediated pathway represents a rational and novel therapeutic strategy target for CDC, even for all urothelial carcinomas, which needs further validation in patients.
There are some limitations to our study. First, this was a single-centre, retrospective study with a small sample size due to the rare incidence of the cancer. Second, it was a study of single omics, which might also lack sufficient validation information. Third, there was a lack of collecting duct carcinoma cell lines and animal models to complete validations in vivo and in vitro. Hence, in the future, we will conduct a prospective, randomised and multicentre clinical trial to obtain multi-omics data to validate our findings.