Irradiation plus myeloid-derived suppressor cell-targeted therapy for overcoming treatment resistance in immunologically cold urothelial carcinoma.

BACKGROUND: Radiotherapy (RT) has recently been highlighted as a partner of immune checkpoint inhibitors. The advantages of RT include activation of lymphocytes while it potentially recruits immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). This study aimed to investigate the mechanism of overcoming treatment resistance in immunologically cold tumours by combining RT and MDSC-targeted therapy. METHODS: The abscopal effects of irradiation were evaluated using MB49 and cisplatin-resistant MB49R mouse bladder cancer cells, with a focus on the frequency of immune cells and programmed cell death-ligand 1 (PD-L1) expression in a xenograft model. RESULTS: MB49R was immunologically cold compared to parental MB49 as indicated by the fewer CD8+ T cells and lower PD-L1 expression. Polymorphonuclear MDSCs increased in both MB49 and MB49R abscopal tumours, whereas the infiltration of CD8+ T cells increased only in MB49 but not in MB49R tumours. Interestingly, PD-L1 expression was not elevated in abscopal tumours. Finally, blocking MDSC in combination with RT remarkably reduced the growth of both MB49 and MB49R abscopal tumours regardless of the changes in the frequency of infiltrating CD8+ T cells. CONCLUSIONS: The combination of RT and MDSC-targeted therapy could overcome treatment resistance in immunologically cold tumours.

[A Case of Sigmoid Colon Cancer with Simultaneous Solitary Adrenal Metastasis Refractory to Preoperative Diagnosis].

A 72-year-old man was referred to our urology department due to a giant adrenal tumor detected by computed tomography( CT). Endocrine screening showed that cortisol, renin, aldosterone, adrenaline, and noradrenaline levels were all normal, and there was no evidence of adrenal hyperfunction. The adrenal tumor was so large that we suspected malignancy. Contrast-enhanced CT of the abdomen was performed for qualitative diagnostic purposes, and showed wall thickening of the sigmoid colon extending for approximately 6 cm. Lower gastrointestinal endoscopy was performed and revealed a full circumferential type 2 tumor in the sigmoid colon. Biopsy results showed intermediate differentiated ductal adenocarcinoma. Tumor markers were as follows: CEA 23.1 ng/mL, CA19-9 962 U/mL. The adrenal tumor was suspected of being malignant due to its size, but imaging examinations did not lead to a diagnosis of primary or metastatic disease. There were no tumors other than those in the sigmoid colon and adrenal glands. Since complete resection was deemed possible, sigmoid colon resection and combined left adrenalectomy were performed for both a diagnosis and treatment. A histopathological examination revealed that the histology of the adrenal tumor resembled that of colorectal cancer, leading to a diagnosis of left adrenal metastasis from sigmoid colon cancer.

Successful case of olaparib treatment for castration-resistant prostate cancer with multiple DNA repair gene mutations: Use of comprehensive genome profiling for treatment-refractory cases.

Herein, we report a case of a 59-year-old man with advanced castration-resistant prostate cancer with rectal invasion. Multimodal treatment, including drug therapy, surgery, and radiation therapy was sequentially performed; however, lymph node metastases repeatedly occurred. Tumor genomic profiling using FoundationOne CDx identified pathogenic alterations in three DNA repair genes, including BRCA2 frameshift mutation. Olaparib, a poly-ADP ribose polymerase inhibitor, showed marked response. Castration-resistant prostate cancer with multiple DNA repair genes was successfully treated with olaparib; comprehensive genome profiling can lead to its optimal clinical management.

[A Case of Metastatic Urothelial Carcinoma with Pseudoprogression of Liver Metastasis during the Treatment with Pembrolizumab].

A 75-year-old woman with a complaint of gross hematuria was referred to our hospital. The patient was diagnosed as having bladder cancer (cT3bN1M0) and received two cycles of chemotherapy with gemcitabine and cisplatin. Radical cystectomy with pelvic lymph node dissection and bilateral ureterostomy was performed after achieving partial response in a lymph node metastasis following chemotherapy. Based on the pathological diagnosis of high-grade (G3) urothelial carcinoma (ypT3aN2), two more cycles of adjuvant chemotherapy with gemcitabine and cisplatin were administered. Four months after completing adjuvant chemotherapy, pulmonary and hepatic metastases appeared, and treatment with pembrolizumab was initiated. The size of the lung metastasis decreased, while that of the liver metastasis increased 2 months after administering pembrolizumab. However, considering treatment beyond progression using checkpoint inhibitors, pembrolizumab was continued, resulting in marked tumor shrinkage of the liver metastasis. After that, pembrolizumab treatment was temporarily discontinued, and radiation therapy was administered for a new lymph node metastasis at the tracheal bifurcation. Eventually, the lymph node metastasis shrank, and the treatment with pembrolizumab was recommenced for 1 year and the metastases remained shrumken.

A retrospective study on optimal number of cycles of the first-line platinum-based chemotherapy for metastatic urothelial carcinoma.

BACKGROUND: Recently, switch maintenance with avelumab has been approved for the treatment of advanced or metastatic urothelial carcinoma (UC), with no progression after four to six cycles of first-line platinum-based chemotherapy. However, the optimal number of cycles of platinum-based chemotherapy has not been determined. OBJECTIVE: To analyze the clinical characteristics of patients with advanced UC who were treated with platinum-based chemotherapy and investigate the association between the number of cycles of the treatment and the patients' overall survival. METHODS: A total of 124 patients with advanced UC who were treated with first-line platinum-based chemotherapy at Osaka City University Hospital between April 2009 and January 2020 were retrospectively reviewed. RESULTS: Of the 124 patients, clinical information regarding overall survival was available for 115 patients. The median age was 72 years (range, 43-95 years). Only 59 patients (51.3 %) were treated with gemcitabine and cisplatin, and 52 patients (45.2 %) were treated with gemcitabine and carboplatin. The median number of cycles was three (1-8), and the percentage of patients who discontinued chemotherapy due to progressive disease was 80.3%, 64.0%, and 86.4% in those receiving one to three, four, and five or more cycles, respectively. Moreover, no difference in overall survival was observed between patients who received four cycles and those who received five or more cycles at both univariate and multivariate levels. CONCLUSIONS: The present study shows that five or more cycles of first-line platinum-based chemotherapy did not prolong overall survival compared with four cycles, suggesting that four cycles of chemotherapy might be sufficient, considering the new treatment strategy involving switch maintenance with avelumab.

Pilot experience of simultaneous robotic-assisted partial nephrectomy for bilateral renal tumors-single center analysis.

INTRODUCTION: Bilateral renal tumors accounts for approximately 3% of renal tumors. However, surgical treatment methods for bilateral renal tumors have not yet been established. It is imperative to balance the need for curative surgery with the goal of maximal functional preservation in patients with bilateral synchronous renal tumors. Therefore, partial nephrectomy may be the optimal surgical treatment for bilateral synchronous renal tumors. METHODS: We conducted a retrospective, observational study to analyze the clinical outcome of simultaneous robotic-assisted partial nephrectomy (RAPN) for bilateral renal tumors at our institution between 2016 and 2019. A total of eight patients were enrolled and the number of renal masses in the 16 kidneys was 18. RESULTS: There was no positive surgical margin after RAPN in our case series and no local recurrence or metastasis during the follow-up period. The only complication of simultaneous RAPN in the present case series was that one patient experienced acute kidney injury after operation without need for dialysis therapy. CONCLUSION: Our study suggests that simultaneous RAPN for bilateral renal tumors might be feasible both for the preservation of renal function and for oncological outcome such as negative surgical margin.

Combination therapy with androgen receptor N-terminal domain antagonist EPI-7170 and enzalutamide yields synergistic activity in AR-V7-positive prostate cancer.

Resistance of castration-resistant prostate cancer (CRPC) to enzalutamide and abiraterone involves the expression of constitutively active, truncated androgen receptor (AR) splice variants (AR-Vs) that lack a C-terminal ligand-binding domain (LBD). Both full-length AR and truncated AR-Vs require a functional N-terminal domain (NTD) for transcriptional activity thereby providing rationale for the development of ralaniten (EPI-002) as a first-in-class antagonist of the AR-NTD. Here, we evaluated the antitumor effect of a next-generation analog of ralaniten (EPI-7170) as a monotherapy or in combination with enzalutamide in prostate cancer cells that express AR-V7 that were resistant to enzalutamide. EPI-7170 had 8-9 times improved potency compared to ralaniten. Enzalutamide increased levels of AR-V7 and expression of its target genes. Knockdown of AR-V7 restored sensitivity to enzalutamide, indicating a role for AR-V7 in the mechanism of resistance. EPI-7170 inhibited expression of genes transcriptionally regulated by full-length AR and AR-V7. A combination of EPI-7170 and enzalutamide resulted in synergistic inhibition of proliferation of enzalutamide-resistant cells that was consistent with results from cell cycle and clonogenic assays. In addition, this drug enhanced the antitumor effect of enzalutamide in enzalutamide-resistant CRPC preclinical models. Thus, a combination therapy targeting both the NTD and LBD of AR, and thereby blocking both full-length AR and AR-Vs, has potential for the treatment of enzalutamide-resistant CRPC.

Ralaniten Sensitizes Enzalutamide-Resistant Prostate Cancer to Ionizing Radiation in Prostate Cancer Cells that Express Androgen Receptor Splice Variants.

Blocking androgen receptor (AR) transcriptional activity by androgen deprivation therapy (ADT) improves the response to radiotherapy for intermediate and high risk prostate cancer. Unfortunately, ADT, antiandrogens, and abiraterone increase expression of constitutively active splice variants of AR (AR-Vs) which regulate DNA damage repair leading to resistance to radiotherapy. Here we investigate whether blocking the transcriptional activities of full-length AR and AR-Vs with ralaniten leads to enhanced sensitivity to radiotherapy. Combination therapies using ralaniten with ionizing radiation were evaluated for effects on proliferation, colony formation, cell cycle, DNA damage, and Western blot analyses in human prostate cancer cells that express both full-length AR and AR-Vs. Ralaniten and a potent next-generation analog (EPI-7170) decreased expression of DNA repair genes whereas enzalutamide had no effect. FACS analysis revealed a dose-dependent decrease of BrdU incorporation with increased accumulation of γH2AX with a combination of ionizing radiation with ralaniten. An additive inhibitory effect on proliferation of enzalutamide-resistant cells was achieved with a combination of ralaniten compounds with ionizing radiation. Ralaniten and EPI-7170 sensitized prostate cancer cells that express full-length AR and AR-Vs to radiotherapy whereas enzalutamide had no added benefit.

Androgen Receptor Splice Variant 7 Drives the Growth of Castration Resistant Prostate Cancer without Being Involved in the Efficacy of Taxane Chemotherapy.

Expression of androgen receptor (AR) splice variant 7 (AR-V7) has been identified as the mechanism associated with the development of castration-resistant prostate cancer (CRPC). However, a potential link between AR-V7 expression and resistance to taxanes, such as docetaxel or cabazitaxel, has not been unequivocally demonstrated. To address this, we used LNCaP95-DR cells, which express AR-V7 and exhibit resistance to enzalutamide and docetaxel. Interestingly, LNCaP95-DR cells showed cross-resistance to cabazitaxel. Furthermore, these cells had increased levels of P-glycoprotein (P-gp) and their sensitivity to both docetaxel and cabazitaxel was restored through treatment with tariquidar, a P-gp antagonist. Results generated demonstrated that P-gp mediated cross-resistance between docetaxel and cabazitaxel. Although the LNCaP95-DR cells had increased expression of AR-V7 and its target genes (UBE2C, CDC20), the knockdown of AR-V7 did not restore sensitivity to docetaxel or cabazitaxel. However, despite resistance to docetaxel and carbazitaxel, EPI-002, an antagonist of the AR amino-terminal domain (NTD), had an inhibitory effect on the proliferation of LNCaP95-DR cells, which was similar to that achieved with the parental LNCaP95 cells. On the other hand, enzalutamide had no effect on the proliferation of either cell line. In conclusion, our results suggested that EPI-002 may be an option for the treatment of AR-V7-driven CRPC, which is resistant to taxanes.

Carbonic anhydrase 2 is a novel invasion-associated factor in urinary bladder cancers.

Rat bladder cancer is nearly always papillary non-invasive urothelial carcinoma (UC). To establish an animal model mimicking invasive UC that arises from papillary non-invasive UC in the bladder, male human c-Ha-ras proto-oncogene transgenic rats (Hras128) were treated with 0.05% N-butyl-N-(hydroxybutyl)nitrosameine (BBN) in their drinking water and/or 0.1% phenylethyl isothiocyanate (PEITC) in their diet as follows: BBN (8 weeks)→PEITC (8 weeks); PEITC (8 weeks)→BBN (8 weeks); BBN alone (16 weeks); PEITC alone (16 weeks); and no treatment. At the end of week 16, the highest incidence of invasive UC was observed in the BBN→PEITC group. Therefore, we used Hras128 rats treated with BBN followed by PEITC as a model of invasive bladder cancer to identify invasion-associated proteins. Proteome analysis was performed to compare the protein profiles of invasive and non-invasive UC in Hras128 rats. We identified 49 proteins that were either overexpressed or underexpressed in invasive UC but not in non-invasive UC. Immunohistochemical analysis of carbonic anhydrase 2 (CA2), an overexpressed protein, showed that the relative number of CA2-positive UC was significantly higher for invasive UC compared to non-invasive UC in rats. Moreover, the incidence of CA2-positive cancers was also significantly higher for human muscle-invasive bladder cancer (MIBC) compared to non-MIBC (NMIBC) and was positively associated with the progression of NMIBC. Our findings indicate that CA2 is an invasion-associated factor and suggest that it could serve as a potential therapeutic molecular target for bladder cancers.

Anti-PD-L1 treatment enhances antitumor effect of everolimus in a mouse model of renal cell carcinoma.

Immunotherapy based on blockade of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis has shown promising clinical activity for renal cell carcinoma (RCC) patients; however, the most effective use of these agents in combination with conventional targeted therapy remains to be resolved. Here we evaluated the therapeutic efficacy of the combination of the mTOR inhibitor everolimus (EVE) and anti-PD-L1 using an immunocompetent mouse model of RCC. We first assessed the in vitro effect of EVE on PD-L1 expression in the human 786-O and mouse RENCA RCC cell lines and found that EVE upregulated PD-L1 expression in these RCC cell lines. We then treated RENCA tumor-bearing mice with EVE and found that PD-L1 expression was also increased in tumor cells after EVE treatment. To determine the antitumor effects of EVE alone, anti-PD-L1 alone, and EVE in combination with anti-PD-L1, we evaluated their antitumor effects on RENCA tumor-bearing mice. A significant decrease in the tumor burden was observed in the EVE alone but not in the anti-PD-L1 alone treatment group compared with the control group. Importantly, the combination of EVE with anti-PD-L1 significantly reduced tumor burden compared with the EVE alone treatment, increasing tumor infiltrating lymphocytes (TILs) and the ratio of cytotoxic CD8+ T cells to TILs. The results of the present study demonstrated that anti-PD-L1 treatment enhanced the antitumor effect of EVE in a mouse model, supporting a direct translation of this combination strategy to the clinic for the treatment of RCC.

Role of deltaNp63(pos)CD44v(pos) cells in the development of N-nitroso-tris-chloroethylurea-induced peripheral-type mouse lung squamous cell carcinomas.

The role of cells expressing stem cell markers deltaNp63 and CD44v has not yet been elucidated in peripheral-type lung squamous cell carcinoma (pLSCC) carcinogenesis. Female A/J mice were painted topically with N-nitroso-tris-chloroethylurea (NTCU) for induction of pLSCC, and the histopathological and molecular characteristics of NTCU-induced lung lesions were examined. Histopathologically, we found atypical bronchiolar hyperplasia, squamous metaplasia, squamous dysplasia, and pLSCCs in the treated mice. Furthermore, we identified deltaNp63(pos)CD44v(pos)CK5/6(pos)CC10(pos) clara cells as key constituents of early precancerous atypical bronchiolar hyperplasia. In addition, deltaNp63(pos)CD44v(pos) cells existed throughout the atypical bronchiolar hyperplasias, squamous metaplasias, squamous dysplasias, and pLSCCs. Overall, our findings suggest that NTCU induces pLSCC through an atypical bronchiolar hyperplasia-metaplasia-dysplasia-SCC sequence in mouse lung bronchioles. Notably, Ki67-positive deltaNp63(pos)CD44v(pos) cancer cells, cancer cells overexpressing phosphorylated epidermal growth factor receptor and signal transducer and activator of transcription 3, and tumor-associated macrophages were all present in far greater numbers in the peripheral area of the pLSCCs compared with the central area. These findings suggest that deltaNp63(pos)CD44v(pos) clara cells in mouse lung bronchioles might be the origin of the NTCU-induced pLSCCs. Our findings also suggest that tumor-associated macrophages may contribute to creating a tumor microenvironment in the peripheral area of pLSCCs that allows deltaNp63(pos)CD44v(pos) cancer cell expansion through activation of epidermal growth factor receptor signaling, and that exerts an immunosuppressive effect through activation of signal transducer and activator of transcription 3 signaling.

[A case of castration-refractory prostate cancer showing marked decrease of serum PSA level after zoledronic acid treatment with estramustine phosphate and prednisolone].

A 66-year-old man was referred to our outpatient clinic for an elevated serum prostatic-specific antigen (PSA 4,319 ng/ mL). Magnetic resonance imaging (MRI) showed multiple metastatic lesions in the bones. The patient had received androgen deprivation therapy, but six months after treatment, he was diagnosed as having prostate cancer refractory to hormones. Combined treatment with docetaxel (DOC 30 mg/m²/week )and estramustine phosphate (EMP 560 mg/day) was initiated as first-line chemotherapy, but the treatment was discontinued because of side effects. Then, treatment with zoledronic acid was started(4 mg/4 weeks)and the PSA level decreased dramatically from 457.2 ng/mL to 5.5 ng/mL. Seven months after the diagnosis of CRPC, MRI showed a decrease ofbone metastases, and the PSA levels continued to decrease, eventually reaching 0.3 ng/mL. Zoledronic acid appears to not only show efficacy in preventing skeletal-related events, but has a potential antitumor effect in patients with metastatic CRPC.