PD-1 Blockade Combined with Heated Intraperitoneal Chemotherapy Improves Outcome in Experimental Peritoneal Metastases from Colonic Origin in a Murine Model.

BACKGROUND: Heated intraperitoneal chemotherapy (HIPEC) was shown to induce immunogenicity of peritoneal metastases from colorectal cancer (PM-CRC) by induction of immunogenic cell death. We aimed to explore whether the addition of a checkpoint inhibitor would augment the effect of HIPEC in an experimental murine model of PM-CRC. METHODS: PM-CRC was established in C57BL mice by intraperitoneal inoculation of MC38 colon cancer cells. HIPEC was administered using the closed technique with mitomycin C (MMC). Clinical and immunological parameters were compared between animals treated with HIPEC alone and those treated with HIPEC + anti-programmed death receptor-1 (aPD-1). RESULTS: MMC-based HIPEC increased the overall survival of animals compared with sham-treated animals (22.8; 95% confidence interval [CI] 21.14-24.53 vs. 18.9 days; 95% CI 17.6-20.3, p < 0.001). The extent of peritoneal disease as measured by the modified peritoneal carcinomatosis index was also reduced by HIPEC. This clinical benefit was accompanied by increased infiltration of CD8+, CD68+, and CD20+ cells into tumor metastases in HIPEC-treated animals compared with sham-treated animals. We identified heat shock protein (HSP) 90 as a potential immunogenic cell death protein whose expression is increased under HIPEC conditions (fold change: 2.37 ± 1.5 vs. 1 without HIPEC, p < 0.05). Combined HIPEC + PD-1 treatment ameliorated survival compared with HIPEC alone and sham treatment (24.66; 95% CI 20.13-29.2 vs. 19; 95% CI 15.85-22.14 and 14.33 days; 95% CI 9.6-19.04, respectively; p = 0.008). This clinical effect was accompanied by increased CD8+ tumor infiltration. CONCLUSIONS: HIPEC induced the expression of immunogenic cell death signals that can support an anti-tumor immune response. This response can be further exploited by a checkpoint inhibitor.

Lymph Node Metastases from Visceral Peritoneal Colorectal Metastases are Associated with Systemic Recurrence.

BACKGROUND: Visceral peritoneal colorectal metastases (VPCMs) may further metastasize to lymph nodes that drain those organs. The rate of lymph node metastases (LNMs) from VPCMs and their clinical and prognostic significance are unknown. METHODS: This study retrospectively analyzed the authors' institutional databases of 160 patients with peritoneal colorectal metastases who underwent cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. Patients with LNM-VPCM (n = 12) were identified by pathologic reports, and both their short- and long-term outcomes were compared with those of patients without LNM-VPCM. RESULTS: The clinical presentation and primary tumor pathologic characteristics did not differ between the two groups. The patients with LNM-VPCM had a higher tumor burden (measured by the peritoneal carcinomatosis index [PCI]) and visible remnant disease compared with those who had no LNM-VPI (10 vs 5.5 [p = 0.03] vs 33.3% vs 6.8% [p = 0.007], respectively). The postoperative outcomes also were comparable. The patients with LNM-VPCM had a shorter overall survival (OS) than those without LNM-VPCM (median OS, 22.5 months; 95% confidence interval [CI], 15.1-29.9 months vs 40.1 months; 95% CI, 38.1-42 months; p = 0.02). However, only tumor grade and PCI were predictors of OS in the multivariate analysis (hazard ratio [HR], 2.33 [p = 0.001]; 1.77 [p = 0.03], respectively). The study showed that LNM-VPCM was associated with systemic but not peritoneal recurrence compared with non-LNM-VPCM (81.8% vs 51.6% for systemic recurrence, respectively; p = 0.05). CONCLUSION: The small distinct group of patients defined by LNM-VPCM were prone to systemic recurrence. Given its correlation with systemic recurrence, LNM-VPCM may indicate the need for adjuvant treatment.

Head-to-head comparison of [68Ga]Ga-FAPI-04 and [18F]-FDG PET/CT in evaluating the extent of disease in gastric adenocarcinoma.

BACKGROUND: [18F]-Fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET/CT) may sometimes be suboptimal for imaging gastric adenocarcinoma. The recently introduced [68Ga]Ga-FAPI-04 (FAPI) PET/CT targets tumor stroma and has shown considerable potential in evaluating the extent of disease in a variety of tumors. METHODS: We performed a head-to-head prospective comparison of FAPI and FDG PET/CT in the same group of 13 patients with gastric adenocarcinoma who presented for either initial staging (n = 10) or restaging (n = 3) of disease. Lesion detection and maximum standardized uptake value (SUVmax) were compared between the two types of radiotracers. RESULTS: All ten primary gastric tumors were FAPI-positive (100% detection rate), whereas only five were also FDG-positive (50%). SUVmax was not significantly different, but the tumor-to-background ratio was higher for FAPI (mean, median, and range of 4.5, 3.2, and 0.8-9.7 for FDG and 12.9, 11.9, and 2.2-23.9 for FAPI, P = 0.007). The level of detection of regional lymph node involvement was comparable. FAPI showed a superior detection rate for peritoneal carcinomatosis (100% vs. none). Two patients with widespread peritoneal carcinomatosis underwent a follow-up FAPI scan after chemotherapy: one showed partial remission and the other showed progressive disease. CONCLUSIONS: The findings of this pilot study suggest that FAPI PET/CT outperforms FDG PET/CT in detecting both primary gastric adenocarcinoma and peritoneal carcinomatosis from gastric cancer. FAPI PET/CT also shows promise for monitoring response to treatment in patients with peritoneal carcinomatosis from gastric cancer; however, larger trials are needed to validate these preliminary findings.

Severe Postoperative Complications are Associated with Impaired Survival in Primary but not in Recurrent Retroperitoneal Sarcoma.

BACKGROUND: Retroperitoneal sarcoma (RPS) surgery entails multivisceral resection, which may cause postoperative complications. We assessed the effects of complications on survival to identify their predisposing factors in primary (PRPS) and recurrent (RRPS) RPS. METHODS: We retrospectively analyzed our institutional database. Severe postoperative complications (SC) were defined as Clavien-Dindo classification ≥ 3. Predisposing factors for complications were investigated, as was their effect on long-term outcomes. RESULTS: In total, 154 RPS resections (78 PRPS and 76 RRPS) performed between January 2008 and December 2018 were included. Neoadjuvant chemotherapy and multifocal tumors were more common in RRPS than PRPS (34.2% vs. 11.3%, P = 0.001 and 42.1% vs. 10.3%, P < 0.001, respectively). Although surgical extent in RRPS was limited compared with PRPS (weighted organ score 1 vs. 2, P = 0.01; transfusion requirement 23.6% vs. 35.8%, P = 0.04), SC and mortality rates were comparable. SC rates were 30.1% and 35.5% for PRPS and RRPS, respectively. NACT rate tended to be higher in PRPS patients with SC (20.8% vs. 7.4%, P = 0.09), whereas weighted organ score and transfusion requirement were increased in RRPS patients with SC (2 vs. 1, P = 0.01; 40.7% vs. 14.3%, P = 0.009, respectively). PRPS patients with SC had decreased overall survival (35 months, 95% confidence interval [CI] 12.2-57.7) compared with those without SC (90 months, 95% CI 71.4-108.5, P = 0.01). CONCLUSIONS: Postoperative complications are associated with impaired outcomes in PRPS but not in RRPS. The negative effects of complications on outcomes should be factored to perioperative management.

Postoperative Morbidity After Resection of Recurrent Retroperitoneal Sarcoma: A Report from the Transatlantic Australasian RPS Working Group (TARPSWG).

BACKGROUND: This study aimed to evaluate perioperative morbidity after surgery for first locally recurrent (LR1) retroperitoneal sarcoma (RPS). Data concerning the safety of resecting recurrent RPS are lacking. METHODS: Data were collected on all patients undergoing resection of RPS-LR1 at 22 Trans-Atlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG) centers from 2002 to 2011. Uni- and multivariable logistic models were fitted to study the association between major (Clavien-Dindo grade ≥ 3) complications and patient/surgery characteristics as well as outcome. The resected organ score, a method of standardizing the number of organs resected, as previously described by the TARPSWG, was used. RESULTS: The 681 patients in this study had a median age of 59 years, and 51.8% were female. The most common histologic subtype was de-differentiated liposarcoma (43%), the median resected organ score was 1, and 83.3% of the patients achieved an R0 or R1 resection. Major complications occurred for 16% of the patients, and the 90-day mortality rate was 0.4%. In the multivariable analysis, a transfusion requirement was found to be a significant predictor of major complications (p < 0.001) and worse overall survival (OS) (p = 0.010). However, having a major complication was not associated with a worse OS or a higher incidence of local recurrence or distant metastasis. CONCLUSIONS: A surgical approach to recurrent RPS is relatively safe and comparable with primary RPS in terms of complications and postoperative mortality when performed at specialized sarcoma centers. Because alternative effective therapies still are lacking, when indicated, resection of a recurrent RPS is a reasonable option. Every effort should be made to minimize the need for blood transfusions.

Analysis of Differentiation Changes and Outcomes at Time of First Recurrence of Retroperitoneal Liposarcoma by Transatlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG).

BACKGROUND: Local recurrence following resection of retroperitoneal liposarcoma (RLPS) is common. Well-differentiated (WD) and dedifferentiated (DD) RLPS are distinct entities with differing outcomes. A few reports suggest that WDLPS can recur as DDLPS and that DDLPS can recur as WDLPS. This study evaluates whether this change in differentiation from the primary tumor to the first local recurrence impacts long-term outcomes. METHODS: Retrospective review from 22 sarcoma centers identified consecutive patients who underwent resection for a first locally recurrent RLPS from January 2002 to December 2011. Outcomes measured included overall survival, local recurrence, and distant metastasis. RESULTS: A total of 421 RPLS patients were identified. Of the 230 patients with primary DDLPS, 34 (15%) presented WDLPS upon recurrence (DD → WD); and of the 191 patients with primary WDLPS, 54 (28%) presented DDLPS upon recurrence (WD → DD). The 6-year overall survival probabilities (95% CI) for DD → DD, DD → WD, WD → WD, and WD → DD were 40% (32-48%), 73% (58-92%), 76% (68-85%), and 56% (43-73%) (p < 0.001), respectively. The 6-year second local recurrence incidence was 66% (59-73%), 63% (48-82%), 66% (57-76%), and 77% (66-90%), respectively. The 6-year distant metastasis incidence was 13% (9-19%), 3% (0.4-22%), 5% (2-11%), and 4% (1-16%), respectively. On multivariable analysis, DD → WD was associated with improved overall survival when compared with DD → DD (p < 0.001). Moreover, WD → DD was associated with a higher risk of LR (p = 0.025) CONCLUSION: A change in RLPS differentiation from primary tumor to first local recurrence appears to impact survival. These findings may be useful in counseling patients on their prognosis and subsequent management.

5-Hydroxymethylcytosine as a clinical biomarker: Fluorescence-based assay for high-throughput epigenetic quantification in human tissues.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.

Patterns of recurrence and survival probability after second recurrence of retroperitoneal sarcoma: A study from TARPSWG.

BACKGROUND: In this series from the Transatlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG), the authors examined longitudinal outcomes of patients with a second recurrence of retroperitoneal sarcoma (RPS) after complete resection of a first local recurrence (LR). METHODS: Data from patients undergoing resection of a first LR from January 2002 to December 2011were collected from 22 sarcoma centers. The primary outcome was overall survival (OS) after second recurrence. RESULTS: Second recurrences occurred in 400 of 567 patients (70.5%) after an R0/R1 resection of a first locally recurrent RPS. Patterns of disease recurrence were LR in 323 patients (80.75%), distant metastases (DM) in 55 patients (13.75%), and both LR and DM in 22 patients (5.5%). The main subtype among the LR group was liposarcoma (77%), whereas DM mainly were leiomyosarcomas (43.6%). In patients with a second LR only, a total of 200 patients underwent re-resection (61.9%). The 5-year OS rate varied significantly based on the pattern of failure (P < .001): 45.6% for the LR group, 25.5% for the DM group, and 0% for the group with LR and DM. The only factors found to be associated with improved OS on multivariable analysis were both time between second surgery and the development of the second recurrence (32 months vs 8 months: hazard ratio, 0.44 [P < .001]) and surgery for second recurrence (yes vs no: hazard ratio, 3.25 [P < .001]). The 5-year OS rate for patients undergoing surgery for a second LR was 59% versus 18% in the patients not deemed suitable for surgical resection. CONCLUSIONS: Survival rates after second recurrence of RPS varied based on patterns of disease recurrence and treatment. Durable disease-free survivors were identified after surgery for second LR in patients selected for this intervention.

Increased Rate of Complete Pathologic Response After Neoadjuvant FOLFIRINOX for BRCA Mutation Carriers with Borderline Resectable Pancreatic Cancer.

BACKGROUND: Neoadjuvant FOLFIRINOX is a standard-of-care treatment for BRPC patients. Patients with gBRCAm who have demonstrated improved response to platinum-based chemotherapy may have impaired homologous repair deficiency. This study aimed to describe the pathologic complete response rate and long-term survival for patients with germline BRCA1 or BRCA2 mutation (gBRCAm) and borderline resectable pancreatic cancer (BRPC) treated with neoadjuvant FOLFIRINOX. METHODS: A dual-center retrospective analysis was performed. Patients who had BRPC treated with neoadjuvant FOLFIRINOX followed by curative resection were identified from clinical databases. Pathologic complete response was defined as no viable tumor cells present in the specimen. Common founder Jewish germline BRCA1 or BRCA2 mutation was determined for available patients. RESULTS: The 61 BRPC patients in this study underwent resection after neoadjuvant FOLFIRINOX. Analysis of BRCA mutation was performed for 39 patients, and 9 patients were found to be BRCA2 germline mutation carriers. The pathologic complete response rate was 44.4% for the gBRCAm patients and 10% for the BRCA non-carriers (p = 0.009). The median disease-free survival was not reached for the gBRCAm patients and was 7 months for the BRCA non-carriers (p = 0.03). The median overall survival was not reached for the gBRCAm patients and was 32 months for the BRCA non-carriers (p = 0.2). After a mean follow-up period of 33.7 months, all eight patients with pathologic complete response were disease-free. CONCLUSIONS: The study showed that gBRCAm patients with BRPC have an increased chance for pathologic complete response and prolonged survival after neoadjuvant FOLFIRINOX. The results support the benefit of exposing gBRCAm patients to platinum-based chemotherapy early in the course of the disease. Neoadjuvant FOLFIRINOX should be considered for BRCA carriers who have resectable pancreatic cancer.

Innate inflammatory markers for predicting survival in retroperitoneal sarcoma.

INTRODUCTION: Existing prognostic tools for retroperitoneal sarcomas (RPS) utilize parameters that can be accurately determined only postoperatively. This study evaluated the application of the neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) levels for predicting prognosis in primary RPS. MATERIALS AND METHODS: We retrospectively analyzed our database of patients with primary RPS operated between 2008 and 2018. The NLR was calculated from preoperative blood tests and its association with outcomes was determined. RESULTS: The NLR values of 78 suitable patients were analyzed. Patients were classified in the NLR-high group if the NLR was ≥2.1. High-grade tumors were more common in the NLR-high group (71.6% vs 48%, P = .02). NLR-high patients had impaired overall survival (OS) and progression-free survival (PFS) compared to NLR-low patients (median OS not reached vs 74 months 95% confidence interval [CI]: 21.6-126.4, P = .03; median PFS not reached vs 48 months 95% CI: 6.5-98.6, P = .06, respectively). Multivariate analysis showed statistical significance only for PFS but not for OS (hazard ratio [HR] = 4.1, P = .03; HR = 2.3, P = .3). Patients with low CRP levels had improved OS and PFS. CONCLUSIONS: The NLR may serve as a preoperative, easily derived marker for prognosis in RPS. Serum biomarkers may prove useful in these large and spatially heterogeneous tumors.

Prognostic significance of pancreatic fistula and postoperative complications after pancreaticoduodenectomy in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The influence of postoperative complications, specifically, pancreatic fistula (PF), on long-term oncologic outcome in patients with pancreatic ductal adenocarcinoma (PDAC) is unclear. METHODS: Prospectively collected data of patients who underwent pancreaticoduodenectomy (PD) for PDAC between 2008 and 2016 were retrospectively reviewed and analyzed. Deaths within 90 days were excluded. Median follow-up time was 22 months for the entire cohort (range 2-102 months). PF was graded as biochemical leak, grade B, or grade C according to the criteria of the International Study Group on Pancreatic Fistula. Postoperative complications were graded according to the Clavien-Dindo classification (CDC). Data on clinical and pathological characteristics as well as on recurrence and survival were collected. RESULTS: Twenty-nine of the 148 identified patients (19%) developed PF, of whom 17 (11.4%) had a PF grade B or C. 29 patients developed a postoperative complication CDC grade 3 or 4. The respective 3-year disease-free survival was 15.5% and 19.2% (P = 0.725), and the 5-year overall survival was 20% and 16% (P = 0.914) in patients with and without PF. On multivariate analysis, the use of adjuvant chemotherapy, lymph node involvement, surgical margin involvement, and tumor grade were associated with patient survival. PF and postoperative complications CDC grade 3 or 4 were not associated with decreased long-term survival, disease-free survival or local recurrence rate. CONCLUSIONS: While acknowledging the limited sample size, no association was seen between PF or postoperative complications and overall or disease-free survival in patients undergoing PD for PDAC.

Rare Primary Retroperitoneal Neoplasms.

BACKGROUND: Primary retroperitoneal neoplasms (PRN) arise from diverse retroperitoneal tissues. Soft tissue sarcomas (STS) comprise the majority and are well studied. Other non-sarcomatous PRN are very rare and less familiar. OBJECTIVES: To evaluate the clinicopathologic and radiologic features of non-sarcomatous PRN, as well as the outcome of complete tumor resection (TR). METHODS: Retrospective data were collected on consecutive patients (June 2006 to January 2015) who underwent resection of retroperitoneal lesions at our department. Final pathology of non-sarcomatous PRN was included. RESULTS: The study population included 36 patients (26% with PRN). PRN were neurogenic (17%), fat-containing (3%), and cystic (6%). The preoperative diagnosis was correct in only 28%. All patients underwent TR via laparotomy (72%) or laparoscopy (28%), for mean operative time of 120 ± 46 minutes. En bloc organ resection was performed in 11%. Complete TR was achieved in 97%. Intra-operative spillage occurred in 8%. Intra-operative, 90-day postoperative complications, and mortality rates were 11%, 36%, and 0%, respectively. The mean length of stay was 6.5 ± 5.5 days. The median overall survival was 53 ± 4.9 months. CONCLUSIONS: Familiarity with radiologic characteristics of PRN is important for appropriate management. Counter to STS, other PRN are mostly benign and have an indolent course. Radical surgery is not required, as complete TR confers good prognosis. Expectant management is reserved for small, asymptomatic, benign neoplasms.

Surgical Approach to Abdominal Tumors Involving the Inferior Vena Cava.

BACKGROUND: Abdominal tumors invading the inferior vena cava (IVC) present significant challenges to surgeons and oncologists. OBJECTIVES: To describe a surgical approach and patient outcomes. METHODS: The authors conducted a retrospective analysis of surgically resected tumors with IVC involvement by direct tumor encasement or intravascular tumor growth. Patients were classified according to level of IVC involvement, presence of intravascular tumor thrombus, and presence of hepatic parenchymal involvement. RESULTS: Study patients presented with leiomyosarcomas (n=5), renal cell carcinoma (n=7), hepatocellular carcinoma (n=1), cholangiocarcinoma (n=2), Wilms tumor (n=1), neuroblastoma (n=1), endometrial leiomyomatosis (n=1), adrenocortical carcinoma (n=1), and paraganglioma (n=1). The surgeries were conducted between 2010 and 2019. Extension of tumor thrombus above the hepatic veins required a venovenous bypass (n=3) or a full cardiac bypass (n=1). Hepatic parenchymal involvement required total hepatic vascular isolation with in situ hepatic perfusion and cooling (n=3). Circular resection of IVC was performed in five cases. Six patients had early postoperative complications, and the 90-day mortality rate was 10%. Twelve patients were alive, and six were disease-free after a mean follow-up of 1.6 years. CONCLUSIONS: Surgical resection of abdominal tumors with IVC involvement can be performed in selected patients with acceptable morbidity and mortality. Careful patient selection, and multidisciplinary involvement in preoperative planning are key for optimal outcome.

Early reoperation following pancreaticoduodenectomy: impact on morbidity, mortality, and long-term survival.

BACKGROUND: Reoperation following PD is a surrogate marker for a complex post-operative course and may lead to devastating consequences. We evaluate the indications for early reoperation following PD and analyze its effect on short- and long-term outcome. METHODS: Four hundred and thirty-three patients that underwent PD between August 2006 and June 2016 were retrospectively analyzed. RESULTS: Forty-eight patients (11%; ROp group) underwent 60 reoperations within 60 days from PD. Forty-two patients underwent 1 reoperation, and 6 had up to 6 reoperations. The average time to first reoperation was 10.1 ± 13.4 days. The most common indications were anastomotic leaks (22 operations in 18 patients; 37.5% of ROp), followed by post-pancreatectomy hemorrhage (PPH) (14 reoperations in 12 patients; 25%), and wound complications in 10 (20.8%). Patients with cholangiocarcinoma had the highest reoperation rate (25%) followed by ductal adenocarcinoma (12.3%). Reoperation was associated with increased length of hospital stay and a high post-operative mortality of 18.7%, compared to 2.6% for the non-reoperated group. For those who survived the post-operative period, the overall and disease-free survival were not affected by reoperation. CONCLUSIONS: Early reoperations following PD carries a dramatically increased mortality rate, but has no impact on long-term survival.

T-Helper 1 Immune Response in Metastatic Lymph Nodes of Pancreatic Ductal Adenocarcinoma: A Marker For Prolonged Survival.

BACKGROUND: Although lymph node (LN) metastases is considered a grave prognostic sign in pancreatic ductal adenocarcinoma (PDAC), patients with positive lymph nodes (PLN) constitute a heterogeneous group. Our purpose was to identify morphological and immune parameters in the primary tumor and in PLN of resected PDAC patients, which could further stratify these patients to different subgroups. METHODS: We retrospectively evaluated histological and immunohistochemical characteristics of 66 patients with PDAC who were operated at our institution. These were subsequently correlated to clinical outcome. RESULTS: Mean patient age and number of LN harvested was 65.5 ± 10.3 and 12.3 ± 6.5 years, respectively. Tumor size (T stage) and perineural invasion had no effect on clinical outcome. High-grade tumor was associated with decreased survival [overall survival (OS) = 19.6 ± 2.7 months for poorly differentiated PDAC vs. 31.2 ± 4 for well and moderately differentiated, p = 0.03]. Patients with ≥ 8 PLN had significantly worse outcome (OS = 7.3 ± 0.8 months for PLN ≥ 8 vs. OS = 30.1 ± 3.2 months for PLN < 8, p < 0.0001). T helper (Th) 1 immune response was measured both by its effector cells (CD8+) and expression of its main transcription factor, T-bet. CD8+ high patients had significantly increased OS compared with CD8+ low (OS = 36.8 ± 5.3 months for CD8 + high vs. OS = 24.3 ± 3.5 for CD8 + low, p = 0.03) Similarly, Th1 predominant immune response measured by T-bet expression was associated with improved OS compared with non-Th1 (OS = 32.8 ± 3.2 vs. OS = 19.5 ± 2.9, p < 0.0001). CONCLUSIONS: Our data indicate an association between Th1-type immune response and increased survival. Future research is needed to exploit Th1 immune response as a biological marker for immunotherapy.

Optimization of liposomal indocyanine green for imaging of the urinary pathways and a proof of concept in a pig model.

BACKGROUND: Iatrogenic ureteral injury is an increasing concern in the laparoscopic era, affecting both patient morbidity and costs. Current techniques enabling intraoperative ureteral identification require invasive procedures or radiations. Our aim was to develop a real-time, non-invasive, radiation-free method to visualize ureters, based on near-infrared (NIR) imaging. For this purpose, we interfered with the biliary excretion pathway of the indocyanine green (ICG) fluorophore by loading it into liposomes, enabling renal excretion. In this work, we studied various parameters influencing ureteral imaging. METHODS: Fluorescence intensity (FI) of various liposomal ICG sizes and doses were characterized in vitro and subsequently tested in vivo in mice and pigs. Quantification was performed by measuring FI in multiple points and applying the ureteral/retroperitoneum ratio (U/R). RESULTS: The optimal liposomal ICG loading dose was 20%, for the different liposomes' sizes tested (30, 60, 100 nm). Higher concentration of ICG decreased FI. In vivo, the optimal liposome size for ureteral imaging was 60 nm, which yielded a U/R of 5.2 ± 1.7 (p < 0.001 vs. free ICG). The optimal ICG dose was 8 mg/kg (U/R = 2.1 ± 0.4, p < 0.05 vs. 4 mg/kg). Only urine after liposomal ICG injection had a measurable FI, and not after free ICG injection. Using a NIR-optimized laparoscopic camera, ureters could be effectively imaged in pigs, from 10 min after injection and persisting for at least 90 min. Ureteral peristaltic waves could be clearly identified only after liposomal ICG injection. CONCLUSIONS: Optimization of liposomal ICG allowed to visualize enhanced ureters in animal models and seems a promising fluorophore engineering, which calls for further developments.

Intraoperative Localization of Rectal Tumors Using Liposomal Indocyanine Green.

BACKGROUND: Tumor localization may pose a significant challenge during minimally invasive rectal resection. Near-infrared (NIR) imaging can penetrate biological tissue and afford tumor localization from the external surface of the rectum. Our aim was to develop an NIR-based tool for rectal tumor imaging that can be administered intravenously. METHODS: We prepared indocyanine-green (ICG)-loaded liposomes by sonication. Liposomes were evaluated for their size and morphology. We then used an endoscopically induced rectal cancer in mice as a model for rectal cancer. After intravenous administration, tumors were evaluated for their fluorescence intensity. Tumor intensity was expressed in relation to the background signal, that is, tumor to background ratio (TBR). RESULTS: Liposomes in various sizes could be prepared by adjusting sonication time. We selected 100-nm-sized liposomes for further experiments. Transmission electron microscopy showed spherical particles and confirmed the size measurements. The liposomes could be lyophilized and then rehydrated again before use without compromising their structure or signal. Fluorescence intensity was kept for 24 hours after solubilization. Testing the optimal time course for rectal tumor imaging revealed that early time course (up to 3 hours) yielded nonspecific imaging, whereas after long time course (24 hours), a very weak signal remained in the tissue. The optimal time window for imaging was after 12 hours from injection, with TBR = 8.1 ± 3.6 ( P = .002). Free ICG could not achieve similar results. CONCLUSIONS: The liposomal ICG can be reproducibly prepared and kept in lyophilized form. Liposomal ICG could serve as a tool for intraoperative tumor localization.