Combination of Nivolumab Plus Ipilimumab for Mucinous Tubular and Spindle Cell Carcinoma of the Kidney with Bone Metastases: The First Case Report

Background: Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma (RCC) and generally considered a low-grade renal epithelial neoplasm composed of tubules, spindle cells, and extracellular mucin. MTSCC with distant metastases has been reported, with some cases showing a poor prognosis. While only a few case reports regarding the treatment of metastatic MTSCC have been reported, targeted agents and monotherapy of immune checkpoint inhibitors have shown some ecacy. To our knowledge, this is the rst reported case of metastatic MTSCC of the kidney treated with combination therapy of nivolumab plus ipilimumab. Case presentation: A 26-year-old man consulted our hospital after a 72-mm tumor was detected at the upper pole of the left kidney with multiple osteolytic bone metastases by computed tomography (CT). A CT-guided biopsy of the renal tumor and bone metastases resulted in a diagnosis of MTSCC of the kidney with bone metastases (cT2aN0M1) and an ‘intermediate risk’ according to the International Metastatic Renal Cell Carcinoma Database Consortium criteria. He therefore received combination therapy of nivolumab plus ipilimumab therapy. After 4 cycles of combination nivolumab plus ipilimumab and 9 cycles of nivolumab monotherapy, he underwent cytoreductive nephrectomy because the tumor had shrunk, and sclerotic changes in the bone metastases were noted. In the excised specimen, the programmed cell death ligand 1 expression was higher in the spindle components than in the tubular components, but CD4- and CD8-positve T cells showed greater inltration in the tubular components than in the spindle components. Furthermore, CD-4- and CD-8-positive T cells in both components of the resected specimen showed greater inltration than they had at the pretreatment biopsy of the renal tumor and bone metastases. Combination immunotherapy of nivolumab and ipilimumab effective

nivolumab plus ipilimumab therapy. After 4 cycles of combination nivolumab plus ipilimumab and 9 cycles of nivolumab monotherapy, he underwent cytoreductive nephrectomy because the tumor had shrunk, and sclerotic changes in the bone metastases were noted. In the excised specimen, the programmed cell death ligand 1 expression was higher in the spindle components than in the tubular components, but CD4-and CD8-positve T cells showed greater in ltration in the tubular components than in the spindle components. Furthermore, CD-4-and CD-8-positive T cells in both components of the resected specimen showed greater in ltration than they had at the pretreatment biopsy of the renal tumor and bone metastases.
Conclusions: Combination immunotherapy of nivolumab and ipilimumab may be an effective treatment option for metastatic MTSCC of the kidney.

Background
Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma (RCC) rst de ned in the 2004 edition of the World Health Organization Classi cation (1) and generally considered a low-grade renal epithelial neoplasm composed of tubules, spindle cells, and extracellular mucin. Fewer than 100 total cases have been reported in the literature (2). However, with the increasing recognition of MTSCC of the kidney, more cases of MTSCC with distant metastases are being reported, some of which have a poor prognosis. Only case reports have been reported regarding the treatment of metastatic MTSCC, and targeted agents and monotherapy with immune checkpoint inhibitors have shown some e cacy (3-6).
To our knowledge, this is the rst case of metastatic MTSCC of the kidney treated with an immune checkpoint inhibitor via combination therapy of nivolumab plus ipilimumab.

Case Presentation
Clinical history This patient was a 26-year-old man with a 1-month history of intermittent back pain. He went to a hospital because he had asymptomatic gross hematuria. He consulted our hospital because a left renal tumor had been detected by echography and computed tomography (CT). CT revealed a 72-mm tumor at the upper pole of the left kidney, well-circumscribed, that was slightly enhanced heterogeneously in the corticomedullary phase and excretory phase but showed no strong enhancement in the corticomedullary, as is usually observed in clear cell RCC. In addition, multiple osteolytic changes were con rmed in bone (Fig. 1). Combination therapy (once every 3 weeks, intravenously) of nivolumab (240 mg/body) and ipilimumab (1 mg/kg) was administered as the rst-line therapy. At the same time, 120 mg of denosumab was subcutaneously injected every 4 weeks, and palliative radiotherapy was also performed for bone metastases to control the bone pain in the rst cervical vertebra and second lumbar vertebra. After 4 cycles of the combination of nivolumab plus ipilimumab, CT revealed that the non-enhanced area was increased in the left renal tumor, and sclerotic changes had appeared in the bone metastases. Nivolumab monotherapy (once every 2 weeks, 240 mg/body) was subsequently continued. He developed immunerelated adverse events (irAEs) after four courses of nivolumab (diarrhea, Grade 3). CT showed no thickening of the intestinal tract. However, colonoscopy showed a slightly edematous mucosa and poor vascular visibility, and a colon biopsy showed colonic mucosa with mild chronic in ammatory in ltration. The diarrhea improved immediately after prednisolone was started at 1 mg/kg/day to control the AEs. Subsequently, nivolumab was restarted, and CT to evaluate the therapeutic effect after eight courses revealed that the left renal tumor had shrunk slightly, and the non-enhanced area was even further increased, as were the osteosclerosis changes in the bone metastases (Fig. 4). Therefore, the patient underwent cytoreductive nephrectomy (laparoscopic radical nephrectomy) after nine courses.
He resumed nivolumab treatment 2 weeks later after surgery and has been receiving nivolumab (480 mg/body) and denosumab every 4 weeks. At present, the observation period is quite short at 12 months, but no progressive disease or irAEs has been observed.

Pathological ndings
Grossly, the tumor at the left kidney was mostly necrotic with hemorrhaging and white-toned solid parts (Fig. 5). Histologically, the sections showed biphasic proliferation of a papillary or tubular pattern with atypical epithelial cells and clear or eosinophilic cytoplasm as well as diffuse proliferation of atypical spindle-shaped cells, accompanied by in ammatory cell in ltration and necrosis. Nucleoli were prominent and easily visualized at low-power magni cation, showing an International Society of Urological Pathology grade was 3. Mucin was observed in the interstitium of the tumor on alcian blue staining (Fig.  6). An immunohistochemical analysis revealed that the tumor cells of the tubular/spindle components were positive for AE1/AE3(+/+), EMA(+/+), vimentin(+/+), PAX8(+/+), AMACR(+/+) in the resected specimen after nivolumab plus ipilimumab.
In addition, the biopsy of the pretreatment renal tumor and bone metastasis and the resected specimen after nivolumab plus ipilimumab was evaluated by immunostaining of programmed cell death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), CD4, and CD8. The PDL-1 expression was higher in the spindle components than in the tubular components, but CD4-and CD8-positve T cells showed greater in ltration in the tubular components than in the spindle components (Fig. 7, 8). Furthermore, CD-4-and CD-8-positive T cells in both components of the resected specimen showed greater in ltration than they had at the pretreatment biopsy of the renal tumor and bone metastases (Fig.  2, 3). There was no marked difference in the CTLA-4 expression between the spindle and tubular components in the resected specimen (Fig. 7, 8).

Genomic ndings
The nonsense variant S562* in the FBXW7 gene was reported. The tumor mutation burden was 2.52 mutations per megabase, and microsatellite instability was absent.

Discussion
MTSCC was originally reported as a rare histological type of RCC with low malignant potential (7,8). Patients with MTSCC of the kidney treated with surgical resection tend to have generally favorable outcomes. However, fatal cases of MTSCC with distant metastases have been reported (9)(10)(11)(12)(13)(14). No therapeutic strategy has yet been established for metastatic MTSCC, and the e cacy of targeted agents and monotherapy of immune checkpoint inhibitors has been reported only in a few case reports (3-6).
Ipilimumab is a humanized monoclonal IgG1 anti-cytotoxic T-lymphocyte antigen-4 antibody that, in combination with nivolumab, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, was recently approved by the Food and Drug Administration as a rst-line treatment of IMDC intermediate-and poor-risk metastatic clear cell RCC after the Checkmate 214 trial showed a signi cantly improved progression-free survival and overall survival (OS) with combination therapy (15). However, non-clear cell RCC histologies were excluded from the trial, so the activity of nivolumab plus ipilimumab for non-clear cell RCC remains unknown.
According to the National Comprehensive Cancer Network guidelines, sunitinib is a preferred systemic regimen for non-clear cell histology (16). A meta-analysis of three studies concluded that sunitinib results in favorable outcomes compared with everolimus for rst-line therapy (17). However, combination therapy of nivolumab plus ipilimumab was chosen in the present case because there was a possibility that combination immunotherapy might be more effective for bone metastases and show a more durable response than targeted agents like sunitinib.
A retrospective analysis of the combination of nivolumab plus ipilimumab for non-clear cell RCC has already been reported in a small sample, but that study did not include MSTCC of the kidney (18). To our knowledge, the present case was the rst to be treated with combination therapy of nivolumab plus ipilimumab for MTSCC of the kidney with bone metastases. In addition, the present patient underwent nephrectomy, and the immunological therapeutic effect in the resected specimen was also evaluated.
Impressively, two different components-tubular and spindle-were con rmed in the same preparation of the excised specimen and showed a differing expression on immunohistochemical staining (Fig. 6-8).
Staining showed that the PD-L1 expression was higher than in the spindle components in the tubular components. The expression of the immune checkpoint PD-L1 is reportedly increased at the surface of sarcomatoid RCC cells compared to non-sarcomatoid RCC cells, regardless of the parent histology or nonsarcomatoid RCC tumor grade (19,20).
Regarding sarcomatoid RCC, while a consensus conference discussed the de nition of sarcomatoid RCC, no consensus was ultimately obtained (21). The largest percentage (41%) of participants felt that a tumor was sarcomatoid if it consisted of atypical spindle cells and resembled any form of sarcoma.
However, another group (22%) considered a tumor sarcomatoid if it had a spindle cell pattern. In the present case, the tumor did not show any sarcomatoid changes but did have spindle components with grade 3 nuclear grading. Therefore, the spindle components of the present case may have similar characteristics to sarcomatoid RCC. CD4 and CD8 staining revealed a difference between the tubular and spindle components, and both CD4-and CD8-positve T cells showed greater in ltration in the tubular components than in the spindle components in the resected specimen (Fig. 7, 8).
Transforming growth factor receptor-β (TGF-β) signaling in the tumor microenvironment has been associated with a poor prognosis (22), and transcriptional analyses have suggested that different pathways are enriched in sarcomatoid RCC, particularly TGF-β signaling (23). As a pleiotropic cytokine, TGF-β maintains immune homeostasis through regulation of essentially every cell type of the innate and adaptive immune system. Speci cally, TGF-β suppresses the proliferation, differentiation, and effector functions of multiple immune cell types, especially T lymphocytes, and induces the generation of immunosuppressive cells or phenotypes (24,25). TGF-β may thus be a factor that caused the weak in ltration of CD4-and CD8-positve T cells in spindle components compared to tubular components.

Conclusion
We reported the rst case of MTSCC of the kidney with bone metastases treated with the combination of nivolumab plus ipilimumab therapy. MTSCC of the kidney is a rare subtype of RCC, and no standard therapeutic care has been established for metastatic MTSCC of the kidney. However, the present case suggested that combination immunotherapy might be an effective treatment option for metastatic MTSCC of the kidney. More studies with a larger number of patients with this rare disease are needed.