Clinicopathological and Genomic Analysis of a Chinese ccRCC Patient with Multiple Morphologies and Rapid Progression : A Case Report


 Background: Renal cell carcinoma (RCC) with sarcomatoid differentiation and multiple metastases is highly aggressive RCC with poor prognosis. However, there are not sufficient report on the genetic alterations and tumor immune microenviroment (TIME) of RCC with complex pathological morphology and aggressive behavior.Case presentation: A rare Chinese RCC case with complex pathological morphology and multiple subcutaneous and soft tissue metastases was reported. The clinical manifestations, histomorphology, immunophenotype and follow-up data were collected and analyzed. We performed target region sequencing and immunohistochemistry staining in different morphological regions of the primary tumor and the peritoneal metastasis. Microscopically, this primary tumor was composed of three different histological variations, including ccRCC like region, eosinophilic papillary structure and sarcomatoid differentiation. The peritoneal metastasis partially showed rhabdoid differentiation. IHC staining didn’t display positivity for characteristic markers. IHC for inflammatory cells showed that CD8+T cells and tumor associated neutrophils (TANs) were significantly increased in the sarcomatous areas and peritoneal metastatic tumor. Genomic analysis indicated that VHL mutations were present in all types of pathological regions and peritoneal metastatic tumor. Therefore, the pathological diagnosis of high-grade ccRCC with sarcomatoid differentiation was established. Additionally, we also found SETD2, TP53 and PDGFRA mutations were observed in sarcomatoid tumor area, whereas BRCA2, ATR, CYLD, YAP1 and COL5A3 mutations were specifically detected in peritoneal metastases. These findings are rather striking because some genes e.g., ATR serine/threonine kinase (ATR) and Hippo signaling (YAP1), PI3K-Akt signaling (PDGFRA) and T cell receptor signaling (COL5A3) were previously reported to be very rare in ccRCC patients.Conclusions: Using next-generation sequencing and TIME analysis, multiple low-frequency mutant genes including PDGFRA, ATR, YAP1 and COL5A3 and increased CD8+ T cells and neutrophils were detected in this rare Chinese ccRCC. These findings potentially provide new evidence and molecular markers for accurately assessing the biological behavior of ccRCC.


Introduction
Renal Cell Carcinoma (RCC) is the most common malignant tumor of kidney with an estimated 403,262 new incidences and 175,098 deaths globally in 2018 and the overall incidence rate has been increasing over the past decade [1,2]. Clear cell renal cell carcinoma (ccRCC) accounts for 75% of kidney cancer diagnoses of RCC [3].ccRCC has long been known to be a highly heterogeneous tumor including morphological heterogeneity and intratumoral genomic heterogeneity. The former is mainly re ected in diverse histological architectural patterns with clear cells or eosinophilic cells [4]. These tumor heterogeneities resulted in variable biological behavior of ccRCC, ranging from low-proliferating localized tumors to highly aggressive metastatic neoplasms. Although the overall survival of ccRCC is good with a 5-year survival rate of 70%, approximately 25% of ccRCC patients show distant metastasis at the rst diagnosis [5]. Thus, accurate assessing the tumor prognosis is important to guide therapeutic intervention and follow-up strategies.
Currently, prognosis evaluation of ccRCC is mainly on the basis of tumor stage and grade. There are also several morphological parameters associated with poor prognosis, such as tumor necrosis, rhabdoid and sarcomatoid differentiation [6]. Studies showed that the 5-year survival rate of the tumor with sarcomatoid differentiation is 15-22% and the median survival for tumors with rhabdoid morphology is 8-31 months [7,8].
As we known, genetic alterations play a critical role in the tumor initiation, progression and evolution. With the rapid development of gene sequencing technology, researchers discovered that VHL, PBRM1, SETD2 and BAP1 are the most commonly mutated genes of ccRCC [9]. Among these genes, BAP1-mutant tumors are typically high-grade and are associated with poor outcome [10]. Mutation in SETD2 is also associated with poor prognosis of ccRCC [11]. Recently, the research from Samra Turajlic et al. identi ed genetic diversity and chromosomal complexity as determinants of patient outcome [12]. Moreover, an integrative genomic study of Chinese ccRCCs revealed that the gene mutation landscape in Chinese cohort is different from TCGA dataset, demonstrating signi cantly higher mutation frequencies in CSMD3 and TMPRSS13 [13]. Therefore, to explore the molecular markers related to ccRCC prognosis in Chinese population is meaningful and important.
In addition, increasing studies indicated that tumor microenvironment (TME) plays an important role in tumor progression, immune escape and drug-resistance [14]. Tumor-associated neutrophils (TANs) and lymphocytes (TILs) are the main components of in ammatory in TME [15]. A study from Song et al. showed that neutrophils are favorably recruited to the RCC cells to promote the RCC migration and invasion [16]. In another study the author developed an architectural pattern-based grading model for ccRCC prognosis evaluation including the intratumoral in ammatory reaction patterns [4].
Herein, we reported a rare Chinese RCC with complicated morphology, extensive metastasis and extremely aggressive behavior. A comprehensive analysis including histopathology, gene alterations and TIME was performed to explore the factors related with aggressive behavior and poor prognosis.

Case Presentation
The patient was a 56-year-old Chinese man accidentally detected a kidney mass by abdominal ultrasound in outer hospital due to repeated hiccups in August, 2019. Then the patient was referred to our hospital for further diagnosis and treatment in September, 2019. No family tumor history was mentioned and no clinical symptoms were presented, such as ank pain, fever and gross hematuria, nevertheless his weight had lost about 5 kilograms since the onset. Physical examination found that multiple subcutaneous soft nodules located in head, face, neck, waist, abdomen, chest, back and limbs with the diameter from 0.8-1.5cm, without redness, swelling, ulceration and purulence. The enhanced computed tomography (CT) of abdomen revealed an uneven-density mass in the upper pole of the right kidney and multiple uneven-density nodules in abdominal cavity, abdominal wall, retroperitoneum, pelvic cavity, bilateral groin, bilateral psoas muscles, iliopsoas and gluteal muscles, front abdominal wall, back and hip subcutaneous and around the anus (Fig. 1A1, A4 and B4). The kidney mass was measured with the size of 46mm×42mm×39mm and shown low-density and no-enhanced necrotic area in portal phase (Fig.   1A2). Another low-density nodule was found in the lower pole of the right kidney with the size of 29.74 × 22.70 × 21.77 mm (Fig. 1A3). Chest enhanced CT showed no metastasis in the lungs, but there were multiple enlarged stuck nodules in the mediastinum, bilateral armpits and anterior chest wall, which was considered as metastases ( Fig. 1B1-B3). A peritoneal metastatic node was removed for frozen-section (yellow triangle) (Fig.1C). Then, a right radical 3D laparoscopic nephrectomy plus peritoneal nodule biopsy was performed. The patient returned to the hospital for review 1 month after surgery and was admitted oral Pazopanib treatment (200mg per tablet, qd) on Oct 13th, 2019. During the treatment of Pazopanib, the patient was getting better physically. Unfortunately, the patient died 1 month later due to excessive medication arbitrarily. The entire course of his clinical treatment is illustrated in Supplementary  Fig. 1.

Macroscopic ndings
Grossly, the tumor protruding from the renal cortex was con ned within the renal parenchyma and measured to be 4.5 × 4.0 × 4.0 cm. The cross-section showed a yellowish and spherical neoplasm in the upper pole of the right kidney. The border with the kidney is sharp. Focal hemorrhage and necrosis were identi ed. In addition, a few dense gray-white nodes were detected in the renal capsule, the perinephric adipose tissue and peritoneum, and their diameter was about 0.3-1.0 cm ( Supplementary Fig. 1).

Histopathological and immunohistochemical (IHC) ndings
Microscopic evaluation of the renal tumor showed the tumor was multi-nodular and separated by brous tissue. The tumor histology was very complicated with three different histological morphologies, including conventional ccRCC like region, eosinophilic papillary structure and sarcomatoid (spindle) differentiation ( Fig. 2A). In ccRCC like region, the pattern of growth was predominantly solid, with formation of nest and acinar patterns separated by the stroma that was characteristically endowed with a prominent network of small, thin-walled blood vessels (Fig. 2Aa1). The area of eosinophilic papillary structure was composed of papillae formed by delicate brovascular cores that contain foamy macrophages. Most of these tumor cells showed abundant eosinophilic cytoplasm with occasional small nuclei (Fig. 2Ab1). The sarcomatoid area was lack of distinct borders with the area of clear cells which transition zone between them could be seen. In this region, some little abscesses with lots of neutrophils and tumor necrosis were observed (Fig. 2Ac1). In addition, histopathological examination of the peritoneum node revealed epithelioid sheets of cells exhibiting great pleomorphism and frequent mitoses (Fig. 2B). These tumor cells with eccentric nuclei and abundant eosinophilic cytoplasm resembled rhabdomyoblasts, which was designated as rhabdoid differentiation (Fig. 2B1).
To identify the speci c subtype of this tumor, we completed serious of IHC staining in the primary kidney tumors and peritoneal metastatic node. These immunostainings showed that CK8/18 and EMA were diffuse or patchy strong positivity in the classical ccRCC like area and eosinophilic papillary area ( Gene mutation analysis with target capture sequencing To con rm the accurate subtype of tumor and further reveal the underlying molecular mechanism related heterogeneous morphology and extremely aggressive biological behavior, genomic pro ling of the four distinct morphology tissue slides from resected tumor specimens was achieved by using target capture sequencing with a designed panel of 620 key cancer-related genes (GloriousMed Clinical Laboratory Co., Shanghai, China). The distribution of somatic mutations (SNV, InDel) among four different morphological tissues from this patient are summarized in a Venn diagram (Fig. 4A).
The most common VHL mutations in ccRCC were detected in all of them, including primary and peritoneal metastatic foci (Fig. 4B). Therefore, we identi ed this patient as a high-grade clear cell renal cell carcinoma (ccRCC) with with sarcomatoid differentiation and systemic multiple metastases (pT3aN1M1). Further analysis indicated tumor of eosinophilic papillary area carries the fewest number and types of mutated genes among all tumor tissues, only VHL and BAP1mutation, suggesting that these two genes might be early events in ccRCC tumorigenesis. From the perspective of genomic changes, eosinophilic morphology tumor seems to occur in the earliest stage of the disease due to the minimum number of driver gene alterations and mutation types. Notably, histone modi cation gene SETD2 mutations were detected in all of tumor regions except for eosinophilic papillary area, suggesting SETD2 could play an important role in ccRCC progression.
Remarkably, the genetic alterations in sarcomatoid regions are similar to those in clear cell carcinoma areas, both sharing some identical gene mutations such as VHL(p. R64fs), SETD2(p.N1628T), SMAD4(p.Q534fs*3), and PTPRT(p.A696V). BAP1 variant P190R and ATM variant E1199 deletion were observed only in conventional clear cell carcinomas, whereas there were exclusive TP53 variant A347G and PDGFRA variant S890 frameshift deletion in sarcomatoid-like tumor, hinting that TP53 and PDGFRA may play a signi cant role in the sarcomatoid transformation from ccRCC morphology. BRCA2 and ATR involved in DNA damage response pathway, NF-kappa B signaling gene CYLD and Hippo signaling gene YAP1, protein digestion and absorption gene COL5A3 were speci cally detected in peritoneal metastatic node but not in other primary areas (Fig. 4B).
Besides, the tumor mutation burden (TMB) value of the four tumor areas present some certain regularities. The higher the grade, the higher the TMB value, as expected, the worse the prognosis, especially with sarcomatoid lesions. The TMB of peritoneal metastatic tumor was higher than the primary tumor (Fig. 4C). The TMB evidence also supports that tumor of eosinophilic papillary area and ccRCC area occurred at the early stage, then the tumor developed into the sarcomatoid morphology, and lastly metastasized to multiple organs including the peritoneum.
The last main thing is that this patient harbored several somatic mutations of certain genes including histone methyltransferase SETD2 (SET domain containing 2), ATR serine/threonine kinase (ATR), ATM serine/threonine kinase (ATM) and TP53 associated with DNA damage response signaling and Hippo signaling (YAP1), PI3K-Akt signaling (PDGFRA) and T cell receptor signaling (COL5A3) (Fig. 4B), some of which contribute to the development of the RCC in light of previous data [9,17,18].

Discussion
This is a really complex, di cult and rare RCC case even though it was identi ed high-grade advanced ccRCC with sarcomatoid differentiation by gene sequencing nally. But making an accurate pathological diagnosis for this case is very di cult in the beginning because it presented complicated pathological morphology as mentioned before and lacked the expression of characteristic immune markers. For example, a classical ccRCC marker CD10 was negative in all of tumor areas. Meanwhile, we excluded chromophobe renal cell carcinoma (ChRCC), MiT family translocation carcinoma, renal medullary carcinoma and ALK rearrangement-associated RCC because of the lack of CK7, CD117, MelanA, HMB45, TFE3 and ALK expression and retaining SMARCB1 (INI-1) expression. Additionally, we also excluded clear cell papillary RCC due to the staining pattern of CA-without the basolateral positivity. As mentioned above, this tumor was highly invasive and metastasis had occurred in the multiple parts of the body including subcutaneous, soft tissue, lymph nodes and so on at the beginning of diagnosis. Sarcomatoid differentiation in the primary tumor was undoubtedly one of the important reasons of its high aggressiveness. Published data showed that this feature is found in 5-8% of ccRCC, 8-9% of ChRCC, and 2-3% of PRCC [19][20][21][22]. Additionally, in the context of the current 2016 WHO classi cation system, tumors in the unclassi ed RCC (uRCC) category can be those with a combination of features of more than one recognized subtype, with mucin production or with unrecognized epithelial cell subtypes, low-or high-grade unclassi ed oncocytic neoplasms, and other unclassi able renal tumors [23]. Sarcomatoid differentiation is also included in the morphology spectrum of uRCC. Some research data from a few relatively large series of uRCC suggest a comparably poor or worse outcome than high-stage ccRCC [24]. Hence, it is critical to determine the tumor subtype and explore the underlying molecular mechanism of highly aggressive biological behavior via target gene sequencing in four different tumor areas including metastatic foci In this study, we detected VHL mutations in all of tumor areas including the primary tumor with three different morphologies and metastasis. It con rmed that this tumor was ccRCC and its different components are originated from the same progenitor cell.
Because of huge tumor heterogeneity, the mutation pro les between in situ foci and metastases are varied, especially for driver mutations. BRCA2 and ATR involved in DNA damage response pathway, NFkappa B signaling gene CYLD and Hippo-YAP signaling gene YAP1, protein digestion and absorption gene COL5A3 was speci cally detected in peritoneal metastatic node but not in other primary areas, indicating that these gene mutations might play an important role in promoting tumor metastasis. Importantly, the alterations (especially deleterious gene mutations) of DDR genes exhibited potential value of predicting response to immune checkpoint inhibitors for metastatic RCC patients [25]. Given the key role of these signaling pathways in the feature of DNA repair, cell proliferation and metastasis, these mutations might contribute to this patient's rapid progression and high aggressiveness.
It was reported that patients with sarcomatoid carcinoma have a poorer prognosis than classical ccRCC [8,26], therefore uncovering the genomic characterization of sarcomatoid transformation in ccRCC is vital. The most altered pathways of sarcomatoid RCC (sRCC) involved VHL (72%), chromatin remodeling genes (72%), MTOR pathway (50%), DNA repair (30%), and the Hippo-YAP pathway (20%) [27]. The research from Wang Z and colleagues indicated more mutations in TP53, PTEN and RELN occurred in sRCC compared with ccRCC [28]. In addition, tyrosine kinase PDGFR-α (PDGFRA) gain of function mutations play a crucial role in RCC pathogenesis. Higher expression of tyrosine kinase receptor PDGFRα was observed in the sRCC part (81.2%) in comparison with the epithelial part (43.7%) and more common mutation of PDGFRA in a subset sRCC cases where sarcomatoid differentiation showed overexpression of this proteins [29]. In this study, SETD2 variant N1628T, TP53 variant A347G and PDGFRA variant S890 frameshift deletion were detected in sarcomatoid-like tumor, which may be the intrinsic cause of sarcoma tumor progression and suggested that patient can bene t from the imatinib drug. This example among others provides a model of how oncogenic activation of tyrosine kinase maintains the highly aggressive clinicopathological features and poor prognosis.
The tumor microenvironment consisting of several cellular and extracellular matrix components is closely related to the outcome of tumor progression by promoting tumor growth, tumor invasion as well as metastatic growth [30]. In immune cell TME compartments of RCC, studies indicated that subdivided macrophages and T cells are the main immune cells [31,32]. In July 2018 Wang et al. described a new RCC subtype, namely the in amed pan-RCC subtype (IS) and revealed that IS RCCs are enriched for Tregs, NK cells, neutrophils, macrophages, B-cells, TH1 cells, and CD8 + T cells and are importantly associated with poor prognosis and poor survival [33]. Some papers also reported that the immune microenvironment of RCC is unique compared with other solid tumors [2]. Resected RCC tumors are usually wide-spread in ltrated by CD8 + T cells and an increased level of in ltrating CD8 + T cells is associated with worse outcome in RCC [34], while in the other solid tumors in ltrating CD8 + T cells are associated with a better prognosis [35]. In our study, CD8 + T cells and tumor associated neutrophils (TANs) signi cantly increased in the more aggressive tumor components (sarcomatous area and peritoneal metastasis tumor) compared with ccRCC. It explained the highly aggressive biological behavior from the modi cation of tumor microenvironment (TME) and suggested that the patient may bene t from immunotherapy due to extensive in ltrated of CD8 + T cells and neutrophils. Further data are needed to determine whether CD8 + T cell density might be a biomarker for immunotherapy. This study has several limitations, including its small sample size, lack of transcriptome analysis due to insu cient quality of RNA and non-coding gene analysis. Despite the limitations, based on molecular features and TIME analysis, we have identi ed a rare Chinese case of ccRCC with sarcomatoid differentiation, extensive tumor metastasis and poor prognosis. Furthermore, multiple low-frequency mutant genes including PDGFRA, ATR, YAP-1 and COL5A3, activated tumor in ltrating CD8 + cells and increased neutrophils were detected in this study, which provided genetic explanation of the highly heterogeneous nature of ccRCC and poor prognosis. These ndings provide new evidence and potential for accurately assessing the biological behavior of ccRCC.

Conclusions
Using next-generation sequencing and TIME analysis, multiple low-frequency mutant genes including PDGFRA, ATR, YAP1 and COL5A3, activated in ltrating CD8 + T cells and increased neutrophils were detected in a rare Chinese ccRCC with sarcomatoid differentiation and extremely aggressive biological behavior. These ndings potentially provide new evidence and molecular markers for accurately assessing the biological behavior of ccRCC.

Funding
The authors have no funding to disclose Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Ethics approval and consent to participate The present study was approved by the ethics committee of Guangzhou First People's Hospital and adhered to the tenets of the Declaration of Helsinki.

Consent for publication
Patient signed an informed consent for publication of the case report. The entire course of his clinical treatment is illustrated and contrast-enhanced computed tomography in portal phase of the thoraco-abdomen. (A1-A4) Abdomen enhanced CT showed an uneven-density mass (black arrow) in the upper pole of the right kidney and multiple uneven-density nodules abdominal cavity, abdominal wall, retroperitoneum, pelvic cavity, bilateral groin, bilateral psoas muscles, iliopsoas and gluteal muscles, front abdominal wall, back and hip subcutaneous (red arrow) and around the anus. (A3) Another low-density nodule was found in the lower pole of the right kidney (blue arrow). (B1-B3) Chest enhanced CT showed multiple enlarged stuck nodules formed a huge irregular mass (yellow arrow) in the mediastinum and a metastatic nodule was in the anterior chest wall (red triangle). (C) A peritoneal metastatic node was removed for frozen-section (yellow triangle).

Figure 1
The entire course of his clinical treatment is illustrated and contrast-enhanced computed tomography in portal phase of the thoraco-abdomen. (A1-A4) Abdomen enhanced CT showed an uneven-density mass (black arrow) in the upper pole of the right kidney and multiple uneven-density nodules abdominal cavity, abdominal wall, retroperitoneum, pelvic cavity, bilateral groin, bilateral psoas muscles, iliopsoas and gluteal muscles, front abdominal wall, back and hip subcutaneous (red arrow) and around the anus. (A3) Another low-density nodule was found in the lower pole of the right kidney (blue arrow). (B1-B3) Chest enhanced CT showed multiple enlarged stuck nodules formed a huge irregular mass (yellow arrow) in the mediastinum and a metastatic nodule was in the anterior chest wall (red triangle). (C) A peritoneal metastatic node was removed for frozen-section (yellow triangle).

Figure 1
The entire course of his clinical treatment is illustrated and contrast-enhanced computed tomography in portal phase of the thoraco-abdomen. (A1-A4) Abdomen enhanced CT showed an uneven-density mass (black arrow) in the upper pole of the right kidney and multiple uneven-density nodules abdominal cavity, abdominal wall, retroperitoneum, pelvic cavity, bilateral groin, bilateral psoas muscles, iliopsoas and gluteal muscles, front abdominal wall, back and hip subcutaneous (red arrow) and around the anus. (A3) Another low-density nodule was found in the lower pole of the right kidney (blue arrow). (B1-B3) Chest enhanced CT showed multiple enlarged stuck nodules formed a huge irregular mass (yellow arrow) in the mediastinum and a metastatic nodule was in the anterior chest wall (red triangle). (C) A peritoneal metastatic node was removed for frozen-section (yellow triangle).      bar=100mm; (B1-B5) H&E and immunohistochemistry staining for LCA, CD4, CD8, MPO in the peritoneal node, respectively. HE Scale bar=50mm; IHC Scale bar=100mm (C-F) Statistical results of TILs, CD8+ T cells, CD4+ T cells and neutrophils distributed in these three areas. Ordinary one-way ANOVA was taken. * P 0.05.