Prostaglandin E2-EP2/EP4 signaling induces immunosuppression in human cancer by impairing bioenergetics and ribosome biogenesis in immune cells.

While prostaglandin E2 (PGE2) is produced in human tumor microenvironment (TME), its role therein remains poorly understood. Here, we examine this issue by comparative single-cell RNA sequencing of immune cells infiltrating human cancers and syngeneic tumors in female mice. PGE receptors EP4 and EP2 are expressed in lymphocytes and myeloid cells, and their expression is associated with the downregulation of oxidative phosphorylation (OXPHOS) and MYC targets, glycolysis and ribosomal proteins (RPs). Mechanistically, CD8+ T cells express EP4 and EP2 upon TCR activation, and PGE2 blocks IL-2-STAT5 signaling by downregulating Il2ra, which downregulates c-Myc and PGC-1 to decrease OXPHOS, glycolysis, and RPs, impairing migration, expansion, survival, and antitumor activity. Similarly, EP4 and EP2 are induced upon macrophage activation, and PGE2 downregulates c-Myc and OXPHOS in M1-like macrophages. These results suggest that PGE2-EP4/EP2 signaling impairs both adaptive and innate immunity in TME by hampering bioenergetics and ribosome biogenesis of tumor-infiltrating immune cells.

SMARCA4-deficient uterine tumors in young women: response to immune checkpoint inhibitors.

SMARCA4-deficient tumors have been reported in various organs and are associated with a poor prognosis. SMARCA4-deficient undifferentiated uterine sarcoma (SDUS) was first described in 2018. Conversely, loss of SMARCA4 (BRG1) expression, as observed by immunostaining, has been observed in several cases of undifferentiated endometrial carcinoma. SDUS has considerable morphologic overlap with undifferentiated endometrial carcinoma, while there are differences in their clinicopathological features. Here, we present two cases of SMARCA4-deficient uterine tumors in patients in their 20 s: SDUS (Case 1) and undifferentiated endometrial carcinoma without SMARCA4 nuclear expression (Case 2). Using comprehensive genome profiling, we found that both cases had SMARCA4 mutations, with tumor mutation burdens of 0 and 68 Muts/Mb, respectively. Case 1 had multiple lung metastases 9 months after surgery. We treated the patients with combination of an immune checkpoint inhibitor (pembrolizumab) and a multikinase inhibitor (lenvatinib), and the response to the treatment was stable. This study presents the first report on the response to immune checkpoint inhibitor and multikinase inhibitor in SDUS. Supplementary Information: The online version contains supplementary material available at 10.1007/s13691-024-00721-2.

A case of grade1 follicular lymphoma diagnosed by laparoscopic lymph node resection: differentiating from late lymph node recurrence of endometrial cancer.

Follicular lymphoma is a common hematologic malignancy; however, it is less common among all malignant diseases and is difficult to suspect in advance due to the lack of specific clinical findings. Here, we report a case in which a late recurrence of corpus cancer was first suspected and finally diagnosed as follicular lymphoma. A 67-year-old female presented to our department with enlarged pelvic lymph nodes. She was diagnosed with breast cancer (HER2-posiotive with lymph node metastasis) and corpus cancer (endometrioid carcinoma grade 2, stage IA) 16 years prior, received definitive therapy and was followed up. A positron emission tomography scan was performed, and an accumulation of 18F-fluorodeoxyglucose (FDG) was detected in multiple lymph nodes, including the lymph nodes with no change in size or enlargement. We performed laparoscopic resection of the enlarged and FDG-accumulated lymph nodes and a pathological examination. The patient was diagnosed with follicular lymphoma (FL) grade 1 and is currently under observation at the Department of Hematology. FL can be considered when there is a discrepancy between the change in lymph node size and the degree of FDG accumulation. A pathological examination is useful for accurate diagnosis. Therefore, it is important to consider tissue collection; however, care must be taken to minimize the invasiveness of the procedure for the patient.

A case report of mucinous borderline ovarian tumor with recurrence as invasive carcinoma with high copy number alterations.

Mucinous borderline ovarian tumors (MBOTs) have a very low recurrence rate and a good prognosis, especially in the early stages, but some MBOTs occasionally recur with the progression to mucinous ovarian carcinomas (MOCs). Here, we present a case of MBOT that recurred as invasive MOC within 3 years. To examine the reason for the progression from MBOT to MOC, whole-exome sequencing of our case identified identical mutations and copy number alterations in KRAS, CDKN2A, and TP53 in both the MBOT and recurrent MOC. The recurrent MOC had a greater copy number alteration burden compared to the primary MBOT. These findings suggest that MBOT may have progressed to MOC via recurrence, wherein the increased burden of copy number alterations could be its key driver. It was also suggested that TP53 mutations already present in MBOT may contribute to the increased copy number alterations leading to MOC. Supplementary Information: The online version contains supplementary material available at 10.1007/s13691-024-00722-1.

Vaginal Cuff Infection Caused by Ureaplasma parvum After Hysterectomy for Uterine Cervical Cancer: A Case Report.

Ureaplasma parvum is one of the most common endemic mycoplasmas in the genitourinary tract and can cause amniotic fluid infection leading to preterm labor. We report a rare case of Ureaplasma parvum infection ascending from the vagina to the abdominal cavity after hysterectomy, causing vaginal cuff infection, postoperative peritonitis, and small bowel obstruction. A 29-year-old nulliparous woman presented with infected uterine cervical cancer. After radical hysterectomy for uterine cervical cancer, the patient had paralytic ileus with ascites and fever. Peritonitis was suspected; however, all cultures were negative, making it difficult to identify the causative organism. Polymerase chain reaction (PCR) of the ascites revealed Ureaplasma parvum, which could be treated with levofloxacin (LVFX). Open drainage to control the infection revealed a necrotic tissue around vaginal cuff and the small intestine encased in cocoon-like fibers like sclerosing encapsulating peritonitis. After the infection was improved, the bowel obstruction was also improved. Ureaplasma spp. can be difficult to culture. PCR testing for Ureaplasma infection should be considered when urogenital infection is suspected in patients prone to opportunistic infections, such as those with malignant tumors.

Niraparib in Japanese patients with heavily pretreated, homologous recombination-deficient ovarian cancer: final results of a multicenter phase 2 study.

OBJECTIVE: To evaluate the long-term efficacy and safety of niraparib in Japanese women with heavily pretreated ovarian cancer. METHODS: This was the follow-up analysis of a phase 2, multicenter, open-label, single-arm study in Japanese women with homologous recombination-deficient, platinum-sensitive, relapsed, high-grade serous epithelial ovarian, fallopian tube, or primary peritoneal cancer who had completed 3-4 lines of chemotherapy and were poly(ADP-ribose) polymerase inhibitor naïve. Participants received niraparib (starting dose, 300 mg) once daily in continuous 28-day cycles until objective disease progression, unacceptable toxicity, or consent withdrawal. The primary endpoint was confirmed objective response rate (ORR), as assessed using Response Evaluation Criteria in Solid Tumors version 1.1. Safety evaluations included treatment-emergent adverse events (TEAEs). RESULTS: 20 patients were enrolled in the study and included in both efficacy and safety analyses. Median total study duration was 759.5 days. Median dose intensity was 201.3 mg/day. Confirmed ORR was 60.0% (90% confidence interval [CI]=39.4-78.3); 2 patients had complete response and 10 patients had partial response. Median duration of response was 9.9 months (95% CI=3.9-26.9) and the disease control rate was 90.0% (95% CI=68.3-98.8). The most common TEAEs were anemia (n=15), nausea (n=12), and decreased platelet count (n=11). TEAEs leading to study drug dose reduction, interruption, or discontinuation were reported in 16 (80.0%), 15 (75.0%), and 2 patients (10.0%), respectively. CONCLUSION: The long-term efficacy and safety profile of niraparib was consistent with previous findings in the equivalent population in non-Japanese patients. No new safety signals were identified. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03759600.

Origin of Peritoneal Cancer With Features of High-grade Serous Carcinoma: A Detailed Molecular Analysis.

Primary peritoneal cancer has characteristics similar to high-grade serous carcinomas of ovarian and fallopian tube origin. However, the relationship between endometriosis and primary peritoneal cancer is not well understood. This study focuses on a case of peritoneal cancer in a patient who had undergone hysterectomy and bilateral salpingo-oophorectomy 5 yr before. In addition to morphology, there was a positive for TP53 in immunohistochemistry and homologous recombination deficiency test, which were similar to high-grade serous carcinomas. However, WT1 was negative in the tumor, and extensive endometriosis coexisted. To reveal the clonal relationship between tumor and endometriosis, we dissected 3 sites each from the tumor and endometriosis and performed whole-exome sequencing analysis. As a result, we found that the tumors were of identical origin. Contrarily, no shared mutations were found in the 3 endometriosis sites. Furthermore, several shared mutations were found between the tumor and 1 endometriosis tissue, showing that the tumor and 1 ectopic endometrial gland originated from the same clone. This study indicates that several peritoneal cancers may be derived from endometriosis. We should consider the possibility of more diverse origins of peritoneal cancer than we speculated before.

Real-world application of comprehensive genomic profiling for gynecological malignancies: a multicenter observational study.

BACKGROUND: The actual status of comprehensive genomic profiling (CGP) applications in Japan has not been clarified. We conducted a multicenter study to investigate the real-world application of CGP in gynecological malignancies. METHODS: Nine designated cancer hospitals participated in this study. Patients who underwent CGP in 2020-2021 were assigned to the CGP group (n = 134). For the population that would have been eligible for CGP, patients who received initial treatment in 2015-2016 and were either alive with disease or died of disease at 5 years follow up were included in the control group (n = 316). We compared clinicopathological characteristics including tumor type (cervix, corpus, ovary, and others including sarcoma) and age. We also investigated the context of CGP-recommended treatment. RESULTS: The CGP group had significantly fewer cervical cases and more others cases (cervix/corpus/ovary/others: CGP, 22/44/56/12; control, 89/79/142/6; p = 0.0003). The CGP group was significantly younger than the control group (median: CGP, 54.0; control, 65.0; p < 0.0001). Subgroup analyses revealed that patients with cervical and ovarian cancers were significantly younger in the CGP group. Among the CGP group, 17 patients (12.7%) received CGP-recommended treatments, 15 of which were not covered by public insurance. The survival time after CGP in 17 patients was longer than in the other 117 cases (median 21 vs. 11 months). CONCLUSION: There was significant selection bias in tumor type and age for the application of CGP for gynecological malignancies in clinical practice in Japan. While CGP often recommended drugs not covered by public insurance, prognosis can be improved by use of CGP.

Lynch syndrome screening in patients with young-onset extra-colorectal Lynch syndrome-associated cancers.

BACKGROUND: Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in mismatch repair (MMR) genes, which predisposes to various types of cancers showing deficient MMR (dMMR). Identification of LS probands is crucial to reduce cancer-related deaths in affected families. Although universal screening is recommended for colorectal and endometrial cancers, and age-restricted screening is proposed as an alternative, LS screening covering a broader spectrum of cancer types is needed. In the current study, we elucidated the rate of dMMR tumors and evaluated the outcome of LS screening in young-onset extra-colorectal LS-associated cancers. METHODS: Immunohistochemistry for MMR proteins were retrospectively performed in a total of 309 tissue samples of endometrial, non-mucinous ovarian, gastric, urothelial, pancreatic, biliary tract, and adrenal cancers in patients < 50 years of age. Clinicopathological information and the results of genetic testing were obtained from medical charts. RESULTS: There were 24 dMMR tumors (7.8%) including 18 endometrial, three ovarian, two urothelial, and one gastric cancer. Co-occurrence of colorectal cancer and family history of LS-associated cancers was significantly enriched in patients with dMMR tumors. Among the 16 patients with dMMR tumors who were informed of the immunohistochemistry results, five with endometrial and one with urothelial cancer were diagnosed as LS with positive pathogenic variants in MMR genes. CONCLUSIONS: We report the outcome of immunohistochemistry for MMR proteins performed in multiple types of young-onset extra-colorectal LS-associated cancers. Our study demonstrates the feasibility of a comprehensive LS screening program incorporating young-onset patients with various types of extra-colorectal LS-associated cancers.

Investigation of the clinical implications of anterior cervical invasion in locally advanced cervical squamous cell carcinoma.

PURPOSES: This study investigates the clinical significance of the anterior parametrical invasion in surgically treated patients with cervical squamous cell carcinoma (SCC). METHODS: We included patients diagnosed with cervical SCC with local lesions classified as T2b, who were treated at our department between January 2006 and December 2020. We evaluated the degree of anterior invasion using pretreatment magnetic resonance imaging and divided patients into three groups: partial, equivocal, and full invasion. The frequency of recurrence within 3 years (early recurrence) and overall prognosis were assessed. RESULTS: There were 12, 24, and 46 cases in the partial equivocal, and full invasion groups, respectively. Neoadjuvant chemotherapy followed by surgery and adjuvant chemotherapy was the mainstay of treatment across all groups (7, 17, and 27 cases, respectively). Although the frequency of early recurrence tended to be worse in the full group (partial; 2/7 cases, equivocal; 3/17 cases and full; 9/27 cases), all early local recurrence cases in the full group (four cases) responded well to the subsequent treatment. As for overall survival, the full invasion group had the best prognosis among the three groups. CONCLUSIONS: In surgical treatment, although full anterior invasion may increase the risk of early local recurrence, it was considered to have little prognostic impact.

Artificial Intelligence-Based Histopathological Subtyping of High-Grade Serous Ovarian Cancer.

Four subtypes of ovarian high-grade serous carcinoma (HGSC) have previously been identified, each with different prognoses and drug sensitivities. However, the accuracy of classification depended on the assessor's experience. This study aimed to develop a universal algorithm for HGSC-subtype classification using deep learning techniques. An artificial intelligence (AI)-based classification algorithm, which replicates the consensus diagnosis of pathologists, was formulated to analyze the morphological patterns and tumor-infiltrating lymphocyte counts for each tile extracted from whole slide images of ovarian HGSC available in The Cancer Genome Atlas (TCGA) data set. The accuracy of the algorithm was determined using the validation set from the Japanese Gynecologic Oncology Group 3022A1 (JGOG3022A1) and Kindai and Kyoto University (Kindai/Kyoto) cohorts. The algorithm classified the four HGSC-subtypes with mean accuracies of 0.933, 0.910, and 0.862 for the TCGA, JGOG3022A1, and Kindai/Kyoto cohorts, respectively. To compare mesenchymal transition (MT) with non-MT groups, overall survival analysis was performed in the TCGA data set. The AI-based prediction of HGSC-subtype classification in TCGA cases showed that the MT group had a worse prognosis than the non-MT group (P = 0.017). Furthermore, Cox proportional hazard regression analysis identified AI-based MT subtype classification prediction as a contributing factor along with residual disease after surgery, stage, and age. In conclusion, a robust AI-based HGSC-subtype classification algorithm was established using virtual slides of ovarian HGSC.

Niraparib in Japanese patients with platinum-sensitive recurrent ovarian cancer: final results of a multicenter phase 2 study.

OBJECTIVE: This study evaluated the long-term safety and efficacy of niraparib in Japanese patients with platinum-sensitive recurrent ovarian cancer. METHODS: This was a follow-up analysis of a phase 2, multicenter, open-label, single-arm study in Japanese women with platinum-sensitive, relapsed ovarian cancer. Participants received niraparib (starting dose 300 mg) once daily in continuous 28-day cycles. The primary endpoint was the incidence of Grade 3 or 4 thrombocytopenia-related events (defined as the overall incidence of the MedDRA Preferred Terms "thrombocytopenia" and "platelet count decreased") occurring in the 30 days after initial administration of niraparib, and secondary endpoints included evaluation of treatment-emergent adverse events and progression-free survival. RESULTS: Nineteen patients (median age, 62 years; median body weight, 53.9 kg) were enrolled. As previously reported, the incidence of Grade 3 or 4 thrombocytopenia-related events during the first 30 days of treatment was 31.6%. At data cutoff, median (range) treatment exposure was 504.0 (56-1,054) days and mean ± standard deviation dose intensity was 154.4±77.5 mg/day. The most common treatment-emergent adverse events were nausea (n=14, 73.7%), decreased platelet count (n=12, 63.2%), decreased neutrophil count (n=11, 57.9%), anemia, vomiting, and decreased appetite (all n=9, 47.4%). One patient was diagnosed with treatment-related leukemia, which resulted in death. Median (95% confidence interval) progression-free survival was 18.0 (5.6-26.7) months. CONCLUSION: Overall, the safety profile of niraparib was considered manageable in this study population of Japanese patients with platinum-sensitive, relapsed ovarian cancer and was consistent with that observed in studies of non-Japanese patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03759587.

YAP1 Suppression by ZDHHC7 Is Associated with Ferroptosis Resistance and Poor Prognosis in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC), which has unique clinical characteristics, arises from benign endometriotic cysts, forming an oxidative stress environment because of excess iron accumulation, and exhibits poor prognosis, particularly in advanced stages owing to resistance to conventional therapeutics. Ferroptosis is an iron-dependent form of programmed cell death induced by lipid peroxidation and controlled by Hippo signaling. We hypothesized that overcoming ferroptosis resistance is an attractive strategy because OCCC acquires oxidative stress resistance during its development and exhibits chemoresistant features indicative of ferroptosis resistance. This study aimed to determine whether OCCC is resistant to ferroptosis and clarify the mechanism underlying resistance. Unlike ovarian high-grade serous carcinoma cells, OCCC cells were exposed to oxidative stress. However, OCCC cells remained unaffected by lipid peroxidation. Cell viability assays revealed that OCCC cells exhibited resistance to the ferroptosis inducer erastin. Moreover, Samroc analysis showed that the Hippo signaling pathway was enriched in OCCC cell lines and clinical samples. Furthermore, patients with low expression of nuclear yes-associated protein 1 (YAP1) exhibited a significantly poor prognosis of OCCC. Moreover, YAP1 activation enhanced ferroptosis in OCCC cell lines. Furthermore, suppression of zinc finger DHHC-type palmitoyltransferase 7 (ZDHHC7) enhanced ferroptosis by activating YAP1 in OCCC cell lines. Mouse xenograft models demonstrated that ZDHHC7 inhibition suppressed tumor growth via YAP1 activation by erastin treatment. In conclusion, YAP1 activation regulated by ZDHHC7 enhanced ferroptosis in OCCC. Thus, overcoming ferroptosis resistance is a potential therapeutic strategy for OCCC.

Clinical Significance of Serum SCC Levels Before Treatment for Locally Advanced Cervical Squamous Cell Carcinoma.

BACKGROUND/AIM: Although serum squamous cell carcinoma (SCC) antigen values are known to be useful in predicting the prognosis of cervical SCC, they have only been examined in a cursory manner. This study aimed to meticulously investigate the clinical significance of serum SCC antigen levels in patients with locally advanced cervical squamous cell carcinoma (LACSC). PATIENTS AND METHODS: The study included patients who were diagnosed with local stage (T-stage) 1b3/2/3 LACSC and underwent initial treatment at our institute between January 2006 and December 2016 (T-1b3: n=30; T-2: n=75; T-3: n=34). The patients were divided into three groups based on pre-treatment SCC values, and differences in clinical background, laboratory and pathology findings, and prognosis were examined. RESULTS: No significant difference in the SCC distribution was observed among the T-1b3/2/3 cases with elevated SCC levels. In stages T-1b3, T-2, and T-3, most recurrences in the SCC-High group were distant (T-1b3: three out of five recurrences; T-2: six out of seven recurrences; T-3: four out of eight recurrences), while most recurrences in the SCC-Low group were pelvic (T-1b3: two out of three recurrences; T-2: eight out of eight recurrences; T-3: three out of three recurrences). CONCLUSION: In LACSC, serum SCC antigen levels before treatment correlate strongly with the recurrence site. Patients with low levels should be closely monitored for local recurrence, whereas those with high levels warrant vigilance for distant recurrence.

A Deep Learning-Based Assessment Pipeline for Intraepithelial and Stromal Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Carcinoma.

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, TIL evaluation has not been used in routine clinical practice because of reproducibility issues. The current study developed two convolutional neural network models to detect TILs and to determine their spatial location in whole slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8+ T cells and immune gene expressions. Patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival and progression-free survival in multiple cohorts. On the basis of the density of intraepithelial and stromal TILs, patients were classified into three immunophenotypes: immune inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity and T-cell-inflamed gene-expression profile scores, whereas the immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor signaling pathway. The established evaluation method provided detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin-stained slides. It has potential for clinical application for personalized treatment of patients with ovarian cancer.

Clinical significance of initial symptoms in endometriosis-associated ovarian cancer.

Objective: Endometriosis is associated with various symptoms, but their severity varies from case to case. In this study, we investigated the reality of symptoms presented by patients with clinically early-stage endometriosis-associated ovarian cancer (EAOC) and explored the relationship between symptoms and laboratory/imaging findings, pathological findings, and prognosis. Materials and Methods: This was a retrospective case-control study of patients who received initial surgical treatment and were diagnosed with clinically early-stage EAOC, including ovarian endometrioid carcinoma (OEC), ovarian clear cell carcinoma (OCCC), and seromucinous borderline tumor (SMBT). Patients with OEC/OCCC diagnosed between 2006 and 2016 and those with SMBT diagnosed between 2006 and 2020 were included. Chi-square and Kaplan-Meier estimates were used for statistical analyses. Results: One hundred-seven patients (OEC, n=31; OCCC, n=39; SMBT, n=37) were included. Fifty-nine (55.1%) patients presented with symptoms, and the proportion of patients with OEC who presented with symptoms was significantly higher than that of others (OEC, 77.4%; OCCC, 43.6%; SMBT, 48.6%). The details of symptoms differed significantly among the pathological types (lower abdominal pain/abdominal discomfort/abnormal bleeding, OEC: 11/8/9; OCCC: 6/12/1; SMBT: 15/5/3). Only in the OEC group did symptomatic patients show significantly higher white blood cell (WBC) count and neutrophil/lymphocyte (N/L) ratio (symptomatic vs. asymptomatic, median: WBC count: 7250 vs. 5000, p=0.008; N/L ratio: 4.6 vs. 1.7, p=0.013). None of the asymptomatic patients showed recurrence during follow-up. Conclusion: Patients with EAOC show varying symptoms depending on the histological type of the tumor. Laboratory findings underlying symptoms also vary by histopathological type, which may reflect differences in the carcinogenesis process.

Neuroendocrine carcinoma of the uterine cervix with extensive pagetoid spread pattern.

There have been no reported cases of neuroendocrine carcinoma (NEC) of the cervix with pagetoid spread (Pag-S). A 44-year-old woman came to our department because of abnormal cytology that persisted immediately after a radical hysterectomy for NEC of the cervix. A mapping biopsy in a large area from the vaginal wall to the vulva revealed that synaptophysin/Ki-67-positive tumor cells were scattered within the epithelium in multiple areas, suggesting a wide Pag-S of NEC. Because tumor cells were found beyond the vaginal wall, the anterior pelvic exenteration was performed. Since we could pathologically confirm the complete resection and no distant metastases were detected, no adjuvant therapy was performed. Four years have passed since the initial treatment without any tumor recurrence. It is known that the prognosis of NEC of the cervix that invades beyond the cervix is poor; however, if there is a Pag-S pattern, a radical surgical treatment can be considered.

Investigation of the Effect of Puncture Order and Position on the Difficulty of Lower and Middle Abdominal Port Placement.

Objectives: Port placements at the mid-abdomen (mainstay of robotic surgery [Rob]) appear to be difficult compared to that at lower abdomen (mainstay of conventional laparoscopy [Con-Lap]). We hypothesized that the reason for this may be the difference in port puncture places. Materials and Methods: We examined how the differences between the place and puncture order of ports affected Con-Lap cases with ports mainly placed in the lower abdomen and Rob cases with ports mainly placed in the middle abdomen. The trocar time was measured from the time when the puncture position and skin incision were determined and initiated, respectively, to the time when the port was punctured and fixed and used as the indicator of difficulty. Results: In the Con-Lap group analysis, the trocar time of the left lower port was longer (right lower: 77 s, middle lower: 117.5 s, and left lower: 138 s, P < 0.0001). In the Rob group analysis, the trocar time of the left most port was significantly longer (right-most: 89.0 s, right-middle: 92.5 s, left-middle: 121.0 s, and left-most: 197.0 s; P < 0.0001). In addition, the total trocar time was significantly longer in the first puncture at the right-middle port in the Rob group (right-most first: 8.4 min, right-middle first: 12.4 min, and left-middle first: 8.5 min, P = 0.0063). Conclusion: In the mid-abdomen port placement, mainstay of Rob cases, the puncture order, and port site have a significant impact on the difficulty of the procedure. It is preferable to avoid initially puncturing the right-middle port in case of the Rob.