Complete response to pembrolizumab in a patient with recurrent and metastatic urothelial bladder carcinoma reflecting coexisting sarcomatoid subtype and glandular differentiation: a case report

In advanced urothelial carcinoma (UC), approximately 20% of patients respond to pembrolizumab, an anti-programmed cell death-1 (PD-1) antibody. Herein, we reported a single case of UC showing coexistence of sarcomatoid subtype and glandular differentiation. Notably, only glandular differentiation was recurrent, probably progressive, and metastatic, which showed complete response to pembrolizumab. An 80-year-old woman presented with hematuria and dysuria, and an intra-vesical tumor was detected on ultrasound. Transurethral resections (TUR) were performed three times. In the first TUR, a sub-pedunculated tumor and a flat lesion were closely but independently located. Pathologically, the sub-pedunculated tumor was an invasive UC, sarcomatoid subtype. Meanwhile, the flat lesion was invasive UC with glandular differentiation. Despite the second and the additional TUR, the tumor was growing and a lymph node metastasis was detected. The third TUR specimen showed UC with glandular differentiation, and a positive PD-L1 expression as well as high density CD8-positive lymphocytic cells infiltration were observed. Pembrolizumab was administered for four courses after terminating the chemotherapy. The CT scan revealed shrinkage of both primary tumor and metastases. Cystectomy and lymph nodes dissection were performed, and no residual carcinoma was detected. The therapeutic effect was regarded as pathological complete response. Pembrolizumab could be effective for special subtype or divergent differentiation of UC, particularly in an event of an ‘immune hot’ tumor.


Introduction
Immune checkpoint inhibitors (ICIs) are widely administrated to patients with advanced-staged urothelial carcinoma (UC). In KEYNOTE-045 trial, approximately 20% of patients respond to pembrolizumab, anti-PD-1 antibody, which is one of the ICIs [1]. Positive programmed deathligand 1 (PD-L1) expression [2,3], high density of CD8positive lymphocytes [4] are considered as potential prognostic marker for ICIs in UC. Also, Bellmunt et al. reported that subtype UCs had higher overall survival (OS) after receiving pembrolizumab than patients with conventional UCs [3]. In this study, we report a single case of urothelial carcinoma with coexisting sarcomatoid subtype and glandular differentiation.

Case report
An 80-year-old woman presented with hematuria and dysuria at a clinic. On ultrasound, an intra-vesical tumor was detected and she was subsequently referred to our hospital.
She had a medical history of gastrointestinal stromal tumor (GIST) of the small intestine and had taken partial resection ten years ago. She had been taking imatinib for GIST since her diagnosis. She had a healthy daily lifestyle, and is a non-smoker.
In the cystoscope, a sub-pedunculated, non-papillary tumor was detected in the left lateral wall and the first TUR was performed. We diagnosed the tumor as UC, sarcomatoid subtype. Moreover, UC with glandular differentiation, which was close to the sub-pedunculated tumor but independently located, was also detected through the biopsy specimen obtained from the flat lesion. Two months following the first TUR, a new recurrent tumor was detected and a second TUR was performed. A year after the first TUR, she undertook additional TUR due to the recurrence of the tumor in the left lateral wall. After 19 months following the first TUR, a computed tomography (CT) scan revealed swelling of the primary tumor and a new left obturator lymph node metastasize. She consented to undergo cystectomy, and was administered with gemcitabine and cisplatin for one course as neoadjuvant chemotherapy. However, severe neutropenia was observed and the chemotherapy was discontinued. After 20 months following the first TUR, the CT scan revealed swelling of the primary tumor (Fig. 1a) and metastasis in the left obturator lymph node. In addition, a new metastasis was detected in the right obturator lymph node (Fig. 1c). Thus, pembrolizumab was administered for four courses every three weeks. After 22 months following the first TUR, the CT scan revealed shrinkage of all the primary tumor and lymph node metastases (Fig. 1b, d). After 24 months following the first TUR, cystectomy was performed and no residual carcinoma in either primary or metastatic sites was detected. At present, neither recurrence nor metastasis of the disease were observed. Table 1 showed the illustrative summary of the clinical course.

Pathological findings
The Pathological and genetic findings of each specimen is shown in Table 2.

First TUR
A sub-pedunculated tumor located in the left lateral wall was found during cystoscopy. In addition, a red, flat lesion was observed near the tumor (Fig. 2). However, these subpedunculated tumor and flat lesion were histologically different (Fig. 3). The sub-pedunculated tumor was composed of spindle cells, which had severe nuclear atypia and plump eosinophilic cytoplasm; moreover, conventional UC component was not detected.
In immunohistochemistry, the tumor cells were diffusely positive for p63 and CK7. cKIT and DOG1 were also negative, and thus metastatic GIST was excluded. We diagnosed it as invasive UC, sarcomoatoid subtype without the heterologous element, pT1. Meanwhile, the flat lesion was composed of malignant urothelial cells that formed glandular architecture, and a sarcomatoid component was not detected. We diagnosed it as invasive UC with glandular differentiation, pT1. Glandular cystitis was observed in the background.
Several molecular analyses which reflect genetic variants typically seen in invasive UC, such as CDKN2A/p16 homologous deletion in fluorescence in situ hybridization (FISH), p53 and RB1 expression in immunohistochemistry, TERT promoter and FGFR3 hot spot mutations by sanger sequencing, were performed to verify whether these sub-pedunculated tumor and flat lesion are of the same origin. There were no differences in the genetic variants between these tumors; absence of p16 deletion, mutant-type-pattern p53 expression, wild-type-pattern RB1 expression, and absence of FGFR3 hotspot mutations (Table 2). Notably, C228T, one of TERT promotor hotspot mutations, was detected in both tumors. The protocols for immunohistochemistry, FISH and genetic mutation analyses were described in the Supplementary Data.

Second and additional TUR
The recurrent tumors were located near the left lateral wall in the first TUR scar. Histologically, these tumors were composed of malignant urothelial cells, which formed glandular architecture and similar morphology to the biopsy specimen in the flat lesion at the first TUR. However, sarcomatoid component was not observed in the second and additional TUR specimens. We diagnosed the tumor as invasive UC with glandular differentiation, pT1.
In immunohistochemistry for the additional TUR specimen, high density of CD8-positive lymphocytic infiltration was observed. The expression of PD-L1 was also examined using clone SP263 (Roche diagnostics, Tuscon, AZ). The expression of PD-L1 was positive according to the SP263 assay scoring algorithm [5]. Figure 4 showed microphotographs of the recurrent tumor at the additional TUR.

Cystectomy and lymph nodes dissection
Macroscopically, ulcer scar was only detected in the urinary bladder. A subtle swelling was observed in one of the left obturator lymph nodes, which is 10 mm in diameter. Histologically, no residual carcinoma was detected in either the bladder or left obturator lymph node. Instead, they were  replaced with fibrosis. The pathological therapeutic effect was regarded as complete response.

Discussion
We reported a single case of invasive UC with coexistence of sarcomatoid subtype and glandular differentiation in the urinary bladder. Sarcomatoid subtype comprises 0.6% of bladder cancer and is characterized by more aggressive behavior than conventional UC [6]. Most sarcomatoid subtype UCs are composed of spindle cells not otherwise specified [7], such as in this case. According to a histological review, sarcomatoid subtype UC exhibits variable degree of several kinds of carcinoma such as urothelial, and glandular or small cell [8]. However, in this case, sarcomatoid subtype UC and UC with glandular differentiation were not 'combined' but 'coexisted' as indicated by the formation of independent lesions as shown in the macroscopy. In addition, sarcomatoid subtype UC was cured with the first TUR, and only UC To presume molecular subtype of these coexisting tumors, we performed immunohistochemical analysis. Both tumors showed focally positive for CK5/6 and negative for CK20, high density of CD8-positive lymphocytic infiltration, and positive expression of PD-L1. Thus, we regarded them as basal subtype based on previous report [9]. In addition, to check the genetic differences between these sub-pedunculated tumor and flat lesion, we examined the presence of mutant-type pattern expressions of p53 and Rb, hotspot mutations in TERT promoter and FGFR3, and homozygous deletion in CDKN2A/p16, which are typically seen in UC. There were no genetic variations observed between these tumors.
Identifying special subtype of UC or UC with divergent differentiation is important in the pathological diagnosis for an effective response to ICI therapies. Recently, Kobayashi et al. analyzed data from 755 patients with advanced UC who received pembrolizumab, and reported that patients with sarcomatoid subtype UC (19 cases) had better objective response rate, disease control rate, and OS compared with conventional UC [10]. Meanwhile, glandular differentiation (21 cases) was not associated with a response. However, each subtype had smaller cases compared with conventional UC. Thus, additional cases are needed to validate the association between therapeutic effect and each subtype or divergent differentiation.
In this case, the recurrent UC with glandular differentiation at the additional TUR (before pembrolizumab administration) showed positive expression of PD-L1 and high density of CD8-positive lymphocytes. Reis et. al reported that when the cutoff was set to 5% tumor reactivity, more than half of the cases with glandular differentiation are PD-L1 positive in UC [11]. Positive expression of PD-L1 [2,3] and high density of CD8positive lymphocytes [4] seemed to be prognostic in the context of second-line therapy in UC. Indeed, 'immune hot' tumor such as this case are features associated with increased response to anti-PD-1 or anti-PD-L1 monotherapy and are good candidates for combination immunotherapy [12].
After receiving pembrolizumab for four courses, our patient exhibited no residual carcinoma. Accordingly, cystectomy might not be needed. Meanwhile, patients with advanced bladder carcinoma usually undergo cystectomy after neoadjuvant chemotherapy, even if they achieve a clinical complete response. As a result, physicians sometimes encounter pathologically complete response cases, such as this patient. However, we cannot definitively confirm whether a patient with a clinical complete response has residual carcinoma without surgery. Nevertheless, with continued accumulation of data on neoadjuvant immune checkpoint inhibitor therapy, we may be able to precisely predict pathologically complete responses and avoid unnecessary surgery in the near future.
In conclusion, we reported a single case of invasive UC showing coexistence of the sarcomatoid subtype and glandular differentiation. While sarcomatoid subtype UC was cured at the initial TUR, UC with glandular differentiation was residual, recurrent, and most likely progressive and metastatic. Moreover, the tumor showed complete response to pembrolizumab. ICIs could be effective for special subtype UC or UC with divergent differentiation, especially in case of positive expression of PD-L1 and high density of CD8-positive lymphocytic cells infiltration.