Introduction

Malignant melanoma(MM) is a malignant neoplasm that arises from melanocyte and typically occur in the skin but may rarely occur in other locations and can give rise to primary melanoma in unexpected locations as peritoneum. It metastasizes to all organs of the body and often demonstrates unpredictable metastatic behavior. However, the prognosis of the patients with metastatic disease is dismal with a 5-year survival of less than 5%, and a median survival duration of 6-9 months^1-3 . On the other hand the incidence of late recurrence of MM ranges from 0.98% to 6.7% of all cases¹. Dacarbazine (DTIC) has been used as the standard of chemotherapy for metastatic melanoma for long since 1970s, and there are many studies of DTIC for advanced melanoma.

Advances in understanding of melanoma biology, immunotherapy and target therapy (as Nivolumab), alone or in combination, have shown promising results in the treatment of advanced melanoma. Nivolumab is a human monoclonal antibody that blocks the interaction between programmed death-1(PD-1), programmed cell death ligand 1( PD-L1) and programmed cell death ligand 2 (PD-L2) and inhibits T cell proliferation and cytokine production. In recent years, nivolumab has been used in metastatic MM, non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, Hodgkin’s lymphoma and urothelial carcinoma. Additionaly, in 2017 National Comprehensive Cancer Network (NCCN) guideline, Nivolumab is used as a first line and second line treatment for inoperable or metastatic melanoma ^4,5. Nivolumab has never been used in peritoneal tumor involvement until now and the results of local applications in peritoneal cancers are not known. In our study, we tried to demonstrate the efficacy of nivolumab on peritoneal malignant melanoma cells in mouse models. Additionally, the effects of DTIC versus Nivolumab treatment on melanoma cells were also compared in this study,

Methods

This study was established at the Experimental Research Center of Cukurova University after obtaining the ethical committee approval of Cukurova University Medical Faculty (Approval number: 2015/10). Fourty five male Swiss Albino mouse , weighing 20-25g were used in the present study. The animals were maintained at 2◦C, humidity at 40-60% with a 12hr light/dark cycle and allowed free access to water and standard chow during the study. This research was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, 1985).

Results

Two mice died in each Group 2 and Group 3 and 1 mouse died in Group 1 during the follow-up. These cases were excluded from the study. There is no significant difference in baseline weight values between the groups. Although the mean body weights were reduced in all groups during the study period, there was no statistical difference among the groups in terms of body weight changes. No local or systemic complications related to chemotherapeutic agent were observed.

In Group 1, small bowel metastases observed in 5 mouse and liver metastasis in 1 mouse. In Group 2 and 3, no metastasis was detected. In the 15th day exploration, all animals developed extensive intraperitoneal tumor growth in group 1 (Figure 1). Macroscopic and microscopic minor tumor implants were detected in Group 2 and pathologic complete response was seen in Group 3 (Figure 2). In group 3, the median tumor size was less than other groups. In group 2 and 3 median tumor size was 0.7±0.3cm and 0.3 ±0.2cm respectively (p: 0.023). PCI were significantly lower in group 3 than other groups (p: 0.019). The lowest total tumor nodules in group 3 were 4 ± 2. The PGRS score was found to be lower in the Nivolumab group than in the DTIC and control groups (p: 0.03). Lymphocytic response rate was found to be higher in the Nivolumab group than in the DTIC and control group (Table 1).

Figure 1 Macroscopic views of each group after treatment. A. Control group: Solid growth of malign melanoma cells (visible at lowest magnification). B. Dacarbazine group: Limited tumor implantation. C. Nivolumab group: Complete response. No tumor cells.  

Figure 2 Histologic response of each group after treatment. A. Control group: No regressive changes and histological response. B. Dacarbazine group: Tumor cells predominant over fibrosis with acellular mucin pools. C. Nivolumab group: Complete response. No tumor cells.  

Discussion

Malignant melanoma is an aggressive and invasive cancer originating from melanocytes. The most dangerous aspect of melanoma is its ability, in later stages, to metastasize to other parts of the body. It effects many parts of region such as peritoneum, eyes, oropharengeal mucosa, gastrointestinal tracts,peritoneum, meninges, respiratory system and gynecological organs⁷. 5-year survival less than 5% patients with metastatic disease, and a median survival is 3-8 months^8-10. Melanoma cells resistant to chemotherapy, but DTIC is the standard chemotherapeutic agent for years with 15% avarage response and extend survival to 10 months¹¹. Despite current NCCN guidelines, many oncology units still apply DTCI alone or combination with other agents in clinical practice¹².

Due to advances in understanding of melanoma biology, immunotherapy and target therapy, new generation of chemotherapeutic agents that can directly affect cancer cells has begun to be used in treatment. Nivolumab is a human monoclonal antibody that blocks the interaction between PD-1, PD-L1 and PD-L2¹³. Binding of these ligands to the PD-1 receptor found on T cells, inhibits T cell proliferation and cytokine production. Upregulation of the PD-1 ligands occurs in some tumors and signaling through this pathway can contribute to inhibition of active T-cell immune surveillance of tumors. According to the 2017 NCCN guidelines, Nivolumab is used as a first line and second line treatment for inoperable or metastatic melanoma. Nivolumab has never been used in peritoneal tumor involvement until now and the results of local applications in peritoneal cancers are not known.

Although nivolumab is recommended in the current treatment of MM, recent controlled studies have shown that it is useful with prolong survival in advanced metastatic MM. Topalian et al. found median overall survival 16.8 months and 2-year overall survival rate 43% in advanced-stage malignant melanoma¹⁴. In another multicenter, randomized, controlled Phase III study, CheckMate 066, DTIC and Nivolumab compared in 418 untreated BRAF wild-type unresectable stage III or IV melanoma patients and 1 year overall survival rate in the Nivolumab and DTIC groups were 73% and 42%, respectively ⁴. Additionally, they found 7.6% complete responses with nivolumab and 1% in DTİC group. However, no study has been conducted on the efficacy of nivolumab in peritoneal carcinomatosis of MM. In this study, we obtained promising results with pathological evidence when applied Nivolumab in combination with cytoreductive surgery+HIPEC in the treatment of MM peritoneal spread in mouse. At the same time, comparison of DTIC and Nivolumab in the peritoneal spread of MM was also performed for the first time in this study.

Radiologic and histopathologic parameters is frequently used in clinical practice and researches to evaluate oncologic treatment responses. However, radiological evaluation especially in peritoneal malignancies is inadequate in showing and measuring tumor implants of 1 cm and smaller size.Therefore histopathological findings are more important in evaluating tumor regression. In the histopathological evaluation of tumor regression in abdminal malignancies, different evaluations (Mandard and Becker grading in upper gastrointestinal carcinomas, Dworak or the Rödel grading system for rectal cancer) have been used in previous studies although they are not specific for peritoneal carcinomatosis^15,16. For this reason, a new standard sampling and histopathological classification system for peritoneal malignancies of Solas et al. was used in our study. In this study, tumor regression was defined in four groups and it was found to be very useful in the evaluation of peritoneal carcinamatosis. In our study, we found that the tumor regression score was significantly higher in the Nivolumab group than in the DTIC and control group. Additionally, Nivolumab group significantly better results from DTIC and control group according to the tumor size, PCI index and lymphocyte response.

Conclusions

Nivolumab is significantly better than DTIC on peritoneal metastases of malign melanoma in mouse models. Nivolumab treatment gives promising results with pathological evidence in the treatment of metastatic disease of malignant melanoma.