Delayed gastric emptying after pylorus-preserving pancreatoduodenectomy: Comparison between traditional open surgery and full-robotic approach with da Vinci Xi.

INTRODUCTION: Delayed gastric emptying (DGE) is a frequent complication after pancreatoduodenectomy, especially after pylorus preservation (Pp). We evaluated the effect of a fully robotic approach with da Vinci Xi on DGE after PpPD. METHODS: Open and robotic PDs were performed in 353 and 50 cases, respectively, from January 2009 to March 2022. We compared the clinical outcomes and incidence of clinically relevant DGE between robotic PpPD (R-PpPD) and open PpPD after one-to-one case-control matching. RESULTS: Each group consisted of 30 patients. Clinically relevant DGE was less common after R-PpPD (3/30 [10%] vs. 10/30 cases [33.3%], p = 0.028). The median length of hospital stay (LoS) was significantly lower in the R-PpPD group (10 vs. 15 days, p = 0.013). CONCLUSION: The reduced tissue trauma by the minimally invasive robotic approach is associated with a lower incidence of DGE, reducing the LoS and encouraging PpPD performed using the fully robotic approach.

Immune landscape and oncobiota in HPV-Associated Colorectal Cancer: an explorative study.

Worldwide more than 550,000 new patients suffering from malignant tumors are associated with human papillomaviruses (HPV) infection. However, only a small portion of patients infected progress to cancer, suggesting that other factors other than HPV may play a role. Some studies have investigated HPV infection in colorectal cancer (CRC) with discordant results; moreover, the role of HPV in CRC development is still unknown. We investigated HPV infection in 50 CRC from different regions, excluding the anal one, by immunohistochemistry (IHC), real-time PCR and RNA-seq. For each patient, we studied the tumor microenvironment in neoplastic and matched non-neoplastic samples, and we compared the tumor-infiltrating immune cell phenotypes among HPV-positive and negative samples. Finally, we compared the CRC-associated microbiota in HPV-positive and negative neoplastic samples by 16S rRNA sequencing. HPV infection was identified in 20% of CRC from the right side (caecum, ascending and transverse colon) and in 40% from the left side (descending colon and rectum). In all HPV-positive CRCs we found no expression of p53 and RB, thus suggesting HPV involvement in tumorigenesis. As far as the tumor microenvironment is concerned, in HPV-related cancers we observed a neoplastic environment with a reduced immune surveillance but an enhanced cytotoxic response by lymphocytes. HPV-positive and -negative CRC showed a different microbiota with lack of species normally found in CRC in the HPV-positive ones. Our results support the carcinogenic significance of HPV in CRC, suggesting a role of HPV in modulating the tumor immune microenvironment.

Minimally Invasive Surgery for the Treatment of Moderate to Critical Acute Pancreatitis: A Case-matched Comparison With the Traditional Open Approach Over 10 years.

PURPOSE: The purpose of this study is to compare short-term and midterm outcomes between patients with acute pancreatitis (AP) treated with minimally invasive surgery (MIS) and patients treated with open necrosectomy (ON). MATERIALS AND METHODS: We compared data of all patients who had undergone MIS for AP with a similar group of patients with ON patients between January 2012 and June 2021 using a case-matched methodology based on AP severity and patient characteristics. Inhospital and midterm follow-up variables, including quality-of-life assessment, were evaluated. RESULTS: Starting from a whole series of 79 patients with moderate to critical AP admitted to our referral center, the final study sample consisted of 24 patients (12 MIS and 12 ON). Postoperative (18.7±10.9 vs. 30.3±21.7 d; P =0.05) and overall hospitalization (56.3±17.4 vs. 76.9±39.4 d; P =0.05) were lower in the MIS group. Moreover, the Short-Form 36 scores in the ON group were statistically significantly lower in role limitations because of emotional problems ( P =0.002) and health changes ( P =0.03) at 3 and 6 months and because of emotional problems ( P =0.05), emotional well-being ( P =0.02), and general health ( P =0.007) at 1 year. CONCLUSIONS: MIS for the surgical management of moderate to critical AP seems to be a good option, as it could provide more chances for a better midterm quality of life compared with ON. Further studies are needed to confirm our findings.

Initial 50 consecutive full-robotic pancreatoduodenectomies without conversion by a single surgeon: a learning curve analysis from a tertiary referral high-volume center.

BACKGROUND: Several studies report on a learning curve for robotic pancreatoduodenectomy (R-PD) ranging between 20 and 80 operations, with conversion rates varying between 1.1 and 35%. However, as these publications mostly refer to initial robotic experiences and do not take into account the previous surgical background in pancreatic surgery (PS) and in robotic-assisted surgery (RAS), the center's volume, as well as the platform used, we aimed to perform a surgical outcomes analysis with a particular view to these aspects. METHODS: Intraoperative and perioperative outcomes of the first 50 consecutive R-PD performed with the da Vinci Xi by the same surgeon, within a tertiary referral high-volume center, between January 2018 and March 2022, were analyzed. The surgeon was previously experienced in both PS and RAS. Shewhart control chart and cumulative sum (CUSUM) analysis were used to evaluate the learning curve of R-PD. RESULTS: All the operations were performed with a full-robotic technique, without any conversion to open surgery. Twenty of 50 patients (40%) had a BMI ≥ 25 kg/m2, while 24/50 (48%) had undergone previous abdominal surgery. Mean console time was 276.30 ± 31.16 min. The median post-operative length of hospital stay was 10 days, while 20/50 (40%) patients were discharged within post-operative day 8. Six patients (12%) had major complications (Clavien-Dindo grade 3 or above). There was no 30-day mortality. Shewhart control chart and CUSUM analysis did not show a significant learning curve during the study period. CONCLUSIONS: An extensive prior experience in both PS and RAS, within a tertiary referral high-volume center with availability of the da Vinci Xi platform, can significantly flatten the learning curve and, therefore, enable safe performance of challenging operations, i.e., pancreatoduodenectomies with a minimally invasive approach, with very low risk of conversion to open surgery, even in the first 50 operations.

Surgical treatment of recurrent retroperitoneal sarcoma in its different patterns: A 15-years' two-centers experience.

INTRODUCTION: There is limited data available regarding the role of surgery in the treatment of retroperitoneal sarcoma (RPS) recurrences. We herein report the short- and mid-term outcomes of patients who underwent surgical treatment of RPS recurrences at two Italian centers over a 15-years' experience. MATERIALS AND METHODS: From January 2005 to January 2020, 33 patients underwent surgical treatment of isolated locally recurrent RPS (LR group), locally recurrent RPS associated with the presence of distant recurrence (LR + DM group), and distant-only recurrent RPS (DM group). Only procedures performed to obtain a macroscopically radical treatment with curative intent were included. Data regarding pre-, intra-, post-operative course, and follow-up, collected in an Institutional database, were retrospectively analyzed, and compared. RESULTS: LR-group was composed of 15 patients, LR + DM group of 9 patients, and DM group of 9 patients. During the follow-up, 78.5% of the LR group, 77.8% of the DM group and 100% of the LR + DM group (p = 0.244) experienced a second recurrence. 7/11 (63.6%) patients in the LR group, 2/7 (28.5%) patients in the DM-group, and 0/9 (0.0%) patients in the LR + DM group underwent to almost one further local treatments of their recurrences (p = 0.010). No differences in the mean disease-free survival (p = 0.127), overall survival (OS) (p = 0.165) was reported among the three groups. Repeated surgery was an independent factor affecting survival in multivariate analysis (p = 0.01). CONCLUSIONS: A surgical treatment of RPS recurrences should always be taken into consideration, also in metastatic patients and/or in those who have already undergone surgery for previous RPS recurrence, because this approach may offer survival benefits.

Comparison of different pancreatic stump management strategies during robot-assisted distal pancreatectomy.

BACKGROUND: Postoperative pancreatic fistula (POPF) represents the most feared complication after distal pancreatectomy, and the possible role of robotic assistance in this setting is poorly investigated so far. METHODS: We analysed short-term outcomes of 88 patients who had undergone robot-assisted distal pancreatectomy (RDP), dividing them according to pancreatic stump management: selective Wirsung duct ligation/hand sewn suture (WirsLIG group), use of robotic EndoWrist staplers (RobSTAP group), and use of laparoscopic staplers (LapSTAP group). RESULTS: Mean operative time resulted significantly longer in WirsLIG group (291.1 ± 77.21 min vs. 245 ± 56.22 min in RobSTAP group vs. 221.77 ± 64.64 min in LapSTAP group). No significant differences were found in median hospital stay and in POPF occurrence. CONCLUSIONS: No strategy for pancreatic stump management during RDP has proven superior to the others in reducing POPF rates. The hand-sewn technique resulted more time consuming, nevertheless it remains essential where there is not enough space to insert the stapler.

Association between a polymorphic variant in the CDKN2B-AS1/ANRIL gene and pancreatic cancer risk.

Genes carrying high-penetrance germline mutations may also be associated with cancer susceptibility through common low-penetrance genetic variants. To increase the knowledge on genetic pancreatic ductal adenocarcinoma (PDAC) aetiology, the common genetic variability of PDAC familial genes was analysed in our study. We conducted a multiphase study analysing 7745 single nucleotide polymorphisms (SNPs) from 29 genes reported to harbour a high-penetrance PDAC-associated mutation in at least one published study. To assess the effect of the SNPs on PDAC risk, a total of 14 666 PDAC cases and 221 897 controls across five different studies were analysed. The T allele of the rs1412832 polymorphism, that is situated in the CDKN2B-AS1/ANRIL, showed a genome-wide significant association with increased risk of developing PDAC (OR = 1.11, 95% CI = 1.07-1.15, P = 5.25 × 10-9 ). CDKN2B-AS1/ANRIL is a long noncoding RNA, situated in 9p21.3, and regulates many target genes, among which CDKN2A (p16) that frequently shows deleterious somatic and germline mutations and deregulation in PDAC. Our results strongly support the role of the genetic variability of the 9p21.3 region in PDAC aetiopathogenesis and highlight the importance of secondary analysis as a tool for discovering new risk loci in complex human diseases.

Robotic Hepatectomy plus Biliary Reconstruction for Bismuth Type III and Type IV Hilar Cholangiocarcinoma: State of the Art and Literature Review.

BACKGROUND: In Bismuth type III and IV Hilar Cholangiocarcinoma (III-IV HC), surgical resection is the only chance for long-term survival. As the surgical procedure is complex and Robotic-Assisted Surgery (RAS) may be particularly suitable in this setting, the aim of this study is to evaluate the potential benefits of RAS in III-IV HC in terms of post-operative outcomes. METHODS: We conducted a systematic review using the PRISMA checklist for article selection. We searched the PubMed database and included only studies with clinical data about the treatment of III-IV HC using RAS. RESULTS: A total of 12 papers involving 50 patients were included. All cases were Bismuth IIIa (n = 18), IIIb (n = 27) or IV type (n = 5) and underwent hepatectomy with biliary confluence resection and reconstruction. The mean operative time was 500 minutes with a conversion rate of 4%. The mean hospital stay was 12.2 days, and the morbidity and 30-day mortality rate were 61.9% and 2%, respectively. Over a mean follow up period of 10.1 months, 9/18 cases experienced recurrence (50%). CONCLUSIONS: RAS for III-IV HC is safe and feasible, at least if performed by experienced surgeons on selected cases. The oncological outcomes appear acceptable, given the aggressiveness of this pathology, but further studies are needed to fully elucidate the exact role of robotics in this setting.

Surgery combined with intra-operative microwaves ablation for the management of colorectal cancer liver metastasis: A case-matched analysis and evaluation of recurrences.

Background: Hepatic resection is the only chance of cure for a subgroup of patients with colorectal cancer liver metastasis. As the oncologic outcomes of intra-operative microwaves ablation combined with hepatic resection still remain uncertain in this setting, we aimed to compare this approach with surgery alone in patient's candidate to metastases resection with radical intent. Methods: Using a case-matched methodology based on age, gender, American Society of Anesthesiology score, Body Mass Index, and burden that take in consideration the number and maximum size of lesions, 20 patients undergoing hepatic resection plus intra-operative microwaves (SURG + IMW group) and 20 patients undergoing hepatic resection alone (SURG group), were included. Relapse-free Survival and post-resection Overall Survival were compared between patients of two groups. Results: At the median follow up of 22.4 ± 17.8, 12/20 patients (60%) in SURG +IMW group and 13/20 patients (65%) in the SURG group experienced liver metastasis recurrence (p=0.774). None of them had recurrence at the same surgical or ablation site of the first hepatic treatment. 7/12 patients in the SURG+IMW group and 7/13 patients in the SURG group underwent at least one further surgical treatment after relapse (p = 1.000). No difference was reported between the two groups in terms of Relapse-free Survival (p = 0.685) and post-resection Overall Survival (p = 0.151). The use of intra-operative microwaves was not an independent factor affecting Relapse-free Survival and post-resection Overall Survival at univariate and multivariate analysis. Conclusions: Patients with colorectal cancer liver metastasis undergoing surgery plus intra-operative microwaves have similar post-operative results compared with surgery alone group. The choice between the two approaches could be only technical, depending on the site, number, and volume of the metastases. This approach could also be used in patients with liver metastasis relapse who have already undergone hepatic surgery.

Zebrafish Patient-Derived Xenograft Model to Predict Treatment Outcomes of Colorectal Cancer Patients.

The use of zebrafish embryos for personalized medicine has become increasingly popular. We present a co-clinical trial aiming to evaluate the use of zPDX (zebrafish Patient-Derived Xenografts) in predicting the response to chemotherapy regimens used for colorectal cancer patients. zPDXs are generated by xenografting tumor tissues in two days post-fertilization zebrafish embryos. zPDXs were exposed to chemotherapy regimens (5-FU, FOLFIRI, FOLFOX, FOLFOXIRI) for 48 h. We used a linear mixed effect model to evaluate the zPDX-specific response to treatments showing for 4/36 zPDXs (11%), a statistically significant reduction of tumor size compared to controls. We used the RECIST criteria to compare the outcome of each patient after chemotherapy with the objective response of its own zPDX model. Of the 36 patients enrolled, 8 metastatic colorectal cancer (mCRC), response rate after first-line therapy, and the zPDX chemosensitivity profile were available. Of eight mCRC patients, five achieved a partial response and three had a stable disease. In 6/8 (75%) we registered a concordance between the response of the patient and the outcomes reported in the corresponding zPDX. Our results provide evidence that the zPDX model can reflect the outcome in mCRC patients, opening a new frontier to personalized medicine.

Pattern of recurrence and survival after D2 right colectomy for cancer: is there place for a routine more extended lymphadenectomy?

BACKGROUND: Conventional Right Colectomy with D2 lymphadenectomy (RC-D2) currently represent the most common surgical treatment of right-sided colon cancer (RCC). However, whether it should be still considered a standard of care, or replaced by a routine more extended D3 lymphadenectomy remains unclear. In the present study, we aim to critically review the patterns of relapse and the survival outcomes obtained from our 11-year experience of RC-D2. METHODS: Clinical data of 489 patients who underwent RC-D2 for RCC at two centres, from January 2009 to January 2020, were retrospectively reviewed. Patients with synchronous distant metastases and/or widespread nodal involvement at diagnosis were excluded. Post-operative clinical-pathological characteristics and survival outcomes were evaluated including the pattern of disease relapse. RESULTS: We enrolled a total of 400 patients with information follow-up. Postoperative morbidity was 14%. The median follow-up was 62 months. Cancer recurrence was observed in 55 patients (13.8%). Among them, 40 patients (72.7%) developed systemic metastases, and lymph-node involvement was found in 7 cases (12.8%). None developed isolated central lymph-node metastasis (CLM), in the D3 site. The estimated 3- and 5-year relapse-free survival were 86.1% and 84.4%, respectively. The estimated 3- and 5-year cancer-specific OS were 94.5% and 92.2%, respectively. CONCLUSIONS: The absence of isolated CLM, as well as the cancer-specific OS reported in our series, support the routine use of RC-D2 for RCC. However, D3 lymphadenectomy may be recommended in selected patients, such as those with pre-operatively known CLM, or with lymph-node metastases close to the origin of the ileocolic vessels.

A "tailored" interventional and surgical management for moderate to critical acute pancreatitis in late phase: a cohort study.

PURPOSE: Several interventional procedures are available to treat moderate-to-critical acute pancreatitis (AP) in its late phase. The ongoing debate on these options, together with the scarcity of reported quality of life (QoL) information in the Literature, prompted us to conduct a review of our experience. METHODS: All the patients treated at our referral Center for moderate-to-critical AP according to Determinant-Based Classification (DBC) were retrospectively reviewed. Patients treated conservatively or operated within 4 weeks were excluded. The included patients were managed following a "tailored" interventional-surgical approach, which did not exclude the possibility to skip one or more steps of the classic "step-up" approach, based on the patient's clinical course, and divided into four groups, according to the first procedure performed: percutaneous drainage (PD), endoscopic approach (END), internal derivation (INT), and necrosectomy (NE). In-hospital and mid-term follow-up variables were analyzed. RESULTS: The study sample consisted in 47 patients: 11 patients were treated by PD, 11 by END, 13 by INT, and 12 by NE. A significant distribution of the DBC severity (p = 0.029) was registered among the four groups. Moreover, the NE group had statistically significant reduced SF-36 scores in the domain of social functioning at 3 months (p = 0.011), at 1 year (p = 0.002), and at 2 years (p = 0.001); role limitations due to physical health at 6 months (p = 0.027); and role limitations due to emotional problems at 1 year (p = 0.020). CONCLUSIONS: In the "late phase" of moderate to critical AP requiring an invasive management, PD, END, INT, and NE are all effective options, depending on patents' status and necrosis location. A "tailored" interventional-surgical management could be pursued, but up-front more invasive approaches are at higher risk of worse QoL. TRIAL REGISTRATION: The manuscript was registered at clinicaltrials.gov in 04/2021 and identified with NCT04870268.

In Pancreatic Adenocarcinoma Alpha-Synuclein Increases and Marks Peri-Neural Infiltration.

α-Synuclein (α-syn) is a protein involved in neuronal degeneration. However, the family of synucleins has recently been demonstrated to be involved in the mechanisms of oncogenesis by selectively accelerating cellular processes leading to cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a specifically high neurotropism. The molecular bases of this biological behavior are currently poorly understood. Here, α-synuclein was analyzed concerning the protein expression in PDAC and the potential association with PDAC neurotropism. Tumor (PDAC) and extra-tumor (extra-PDAC) samples from 20 patients affected by PDAC following pancreatic resections were collected at the General Surgery Unit, University of Pisa. All patients were affected by moderately or poorly differentiated PDAC. The amount of α-syn was compared between tumor and extra-tumor specimen (sampled from non-affected neighboring pancreatic areas) by using in situ immuno-staining with peroxidase anti-α-syn immunohistochemistry, α-syn detection by using Western blotting, and electron microscopy by using α-syn-conjugated immuno-gold particles. All the methods consistently indicate that each PDAC sample possesses a higher amount of α-syn compared with extra-PDAC tissue. Moreover, the expression of α-syn was much higher in those PDAC samples from tumors with perineural infiltration compared with tumors without perineural infiltration.

Contrast enhanced ultrasound for the diagnosis of bilateral renal lymphangiectasia: literature review and contrast enhanced ultrasound findings.

Renal lymphangiectasia (RLmp) is a rare benign lymphatic malformation which should be distinguished from other more common pathologies. Ultrasound (US) examination can define the first diagnostic suspicion, but the definitive diagnosis is usually reached with a second level imaging such as computed tomography (CT) or magnetic resonance imaging (MRI). We herein describe for the first time in literature, the use of the contrast enhanced ultrasound (CEUS) in disclosing the nature of peri-renal anechoic lesions in a 27-years old woman, that were initially confused with parenchymal cysts at B-Mode US. The diagnosis of RLmp may be particularly demanding due to its rarity and different clinical presentations. We suggest a possible role of CEUS as a real time, cost saving and easily accessible second level diagnostic tool, that can represent an appealing alternative in the diagnostic work up of suspected RLmp, respect to other imaging modalities.

Minimally invasive distal pancreatectomy: a case-matched cost-analysis between robot-assisted surgery and direct manual laparoscopy.

BACKGROUND: Few studies have reported a structured cost analysis of robotic distal pancreatectomy (RDP), and none have compared the relative costs between the robotic-assisted surgery (RAS) and the direct manual laparoscopy (DML) in this setting. The aim of the present study is to address this issue by comparing surgical outcomes and costs of RDP and laparoscopic distal pancreatectomies (LDP). METHODS: Eighty-eight RDP and 47 LDP performed between January 2008 and January 2020 were retrospectively analyzed. Three comparable groups of 35 patients each (Si-RDP-group, Xi-RDP group, LDP-group) were obtained matching 1:1 the RDP-groups with the LDP-group. Overall costs, including overall variable costs (OVC) and fixed costs were compared using generalized linear regression model adjusting for covariates. RESULTS: The conversion rate was significantly lower in the Si-RDP-group and Xi-RDP-group: 2.9% and 0%, respectively, versus 14.3% in the LDP-group (p = 0.045). Although not statistically significant, the mean operative time was lower in Xi-RDP-group: 226 min versus 262 min for Si-RDP-group and 247 min for LDP-group. The overall post-operative complications rate and the length of hospital stay (LOS) were not significantly different between the three groups. In LDP-group, the LOS of converted cases was significantly longer: 15.6 versus 9.8 days (p = 0.039). Overall costs of LDP-group were significantly lower than RDP-groups, (p < 0.001). At multivariate analysis OVC resulted no longer statistically significantly different between LDP-group and Xi-RDP-group (p = 0.099), and between LDP-group and the RDP-groups when the spleen preservation was indicated (p = 0.115 and p = 0.261 for Si-RDP-group and Xi-RDP-group, respectively). CONCLUSIONS: RAS is more expensive than DML for DP because of higher acquisition and maintenance costs. The flattening of these differences considering only the variable costs, in a high-volume multidisciplinary center for RAS, suggests a possible optimization of the costs in this setting. RAS might be particularly indicated for minimally invasive DP when the spleen preservation is scheduled.

Robot-assisted pancreatoduodenectomy with the da Vinci Xi: can the costs of advanced technology be offset by clinical advantages? A case-matched cost analysis versus open approach.

BACKGROUND: Robot-assisted pancreatoduodenectomy (RPD) has shown some advantages over open pancreatoduodenectomy (OPD) but few studies have reported a cost analysis between the two techniques. We conducted a structured cost-analysis comparing pancreatoduodenectomy performed with the use of the da Vinci Xi, and the traditional open approach, and considering healthcare direct costs associated with the intervention and the short-term post-operative course. MATERIALS AND METHODS: Twenty RPD and 194 OPD performed between January 2011 and December 2020 by the same operator at our high-volume multidisciplinary center for robot-assisted surgery and for pancreatic surgery, were retrospectively analyzed. Two comparable groups of 20 patients (Xi-RPD-group) and 40 patients (OPD-group) were obtained matching 1:2 the RPD-group with the OPD-group. Perioperative data and overall costs, including overall variable costs (OVCs) and fixed costs, were compared. RESULTS: No difference was reported in mean operative time: 428 min for Xi-RPD-group versus 404 min for OPD, p = 0.212. The median overall length of hospital stay was significantly lower in the Xi-RPD-group: 10 days versus 16 days, p = 0.001. In the Xi-RPD-group, consumable costs were significantly higher (€6149.2 versus €1267.4, p < 0.001), while hospital stay costs were significantly lower: €5231.6 versus €8180 (p = 0.001). No significant differences were found in terms of OVCs: €13,483.4 in Xi-RPD-group versus €11,879.8 in OPD-group (p = 0.076). CONCLUSIONS: Robot-assisted surgery is more expensive because of higher acquisition and maintenance costs. However, although RPD is associated to higher material costs, the advantages of the robotic system associated to lower hospital stay costs and the absence of difference in terms of personnel costs thanks to the similar operative time with respect to OPD, make the OVCs of the two techniques no longer different. Hence, the higher costs of advanced technology can be partially compensated by clinical advantages, particularly within a high-volume multidisciplinary center for both robot-assisted and pancreatic surgery. These preliminary data need confirmation by further studies.

Detailing the ultrastructure's increase of prion protein in pancreatic adenocarcinoma.

BACKGROUND: Recent evidences have shown a relationship between prion protein (PrPc) expression and pancreatic ductal adenocarcinoma (PDAC). Indeed, PrPc could be one of the markers explaining the aggressiveness of this tumor. However, studies investigating the specific compartmentalization of increased PrPc expression within PDAC cells are lacking, as well as a correlation between ultrastructural evidence, ultrastructural morphometry of PrPc protein and clinical data. These data, as well as the quantitative stoichiometry of this protein detected by immuno-gold, provide a significant advancement in understanding the biology of disease and the outcome of surgical resection. AIM: To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data. METHODS: Between June 2018 and December 2020, samples from pancreatic tissues of 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were collected. When the frozen section excluded a PDAC diagnosis, or the nodules were too small for adequate sampling, patients were ruled out from the present study. Western blotting was used to detect, quantify and compare the expression of PrPc in PDAC and control tissues, such as those of non-affected neighboring pancreatic tissue of the same patient. To quantify the increase of PrPc and to detect the subcellular compartmentalization of PrPc within PDAC cells, immuno-gold stoichiometry within specific cell compartments was analyzed with electron microscopy. Finally, an analysis of quantitative PrPc expression according to prognostic data, such as cancer stage, recurrence of the disease at 12 mo after surgery and recurrence during adjuvant chemotherapy was made. RESULTS: The amount of PrPc within specimen from 38 out of 45 patients was determined by semi-quantitative analysis by using Western blotting, which indicates that PrPc increases almost three-fold in tumor pancreatic tissue compared with healthy pancreatic regions [242.41 ± 28.36 optical density (OD) vs 95 ± 17.40 OD, P < 0.0001]. Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirms an increased PrPc expression in PDAC ductal cells of all patients and allows to detect a specific compartmentalization of PrPc within tumor cells. In particular, the number of immuno-gold particles of PrPc was significantly higher in PDAC cells respect to controls, when considering the whole cell (19.8 ± 0.79 particles vs 9.44 ± 0.45, P < 0.0001). Remarkably, considering PDAC cells, the increase of PrPc was higher in the nucleus than cytosol of tumor cells, which indicates a shift in PrPc compartmentalization within tumor cells. In fact, the increase of immuno-gold within nuclear compartment exceeds at large the augment of PrPc which was detected in the cytosol (nucleus: 12.88 ± 0.59 particles vs 5.12 ± 0.32, P < 0.0001; cytosol: 7.74. ± 0.44 particles vs 4.3 ± 0.24, P < 0.0001). In order to analyze the prognostic impact of PrPc, we found a correlation between PrPc expression and cancer stage according to pathology results, with a significantly higher expression of PrPc for advanced stages. Moreover, 24 patients with a mean follow-up of 16.8 mo were considered. Immuno-blot analysis revealed a significantly higher expression of PrPc in patients with disease recurrence at 12 mo after radical surgery (360.71 ± 69.01 OD vs 170.23 ± 23.06 OD, P = 0.023), also in the subgroup of patients treated with adjuvant CT (368.36 ± 79.26 OD in the recurrence group vs 162.86 ± 24.16 OD, P = 0.028), which indicates a correlation with a higher chemo-resistance. CONCLUSION: Expression of PrPc is significantly higher in PDAC cells compared with control, with the protein mainly placed in the nucleus. Preliminary clinical data confirm the correlation with a poorer prognosis.

Zebrafish Patient-Derived Xenografts Identify Chemo-Response in Pancreatic Ductal Adenocarcinoma Patients.

It is increasingly evident the necessity of new predictive tools for the treatment of pancreatic ductal adenocarcinoma in a personalized manner. We present a co-clinical trial testing the predictiveness of zPDX (zebrafish patient-derived xenograft) for assessing if patients could benefit from a therapeutic strategy (ClinicalTrials.gov: XenoZ, NCT03668418). zPDX are generated xenografting tumor tissues in zebrafish embryos. zPDX were exposed to chemotherapy regimens commonly used. We considered a zPDX a responder (R) when a decrease ≥50% in the relative tumor area was reported; otherwise, we considered them a non-responder (NR). Patients were classified as Responder if their own zPDX was classified as an R for the chemotherapy scheme she/he received an adjuvant treatment; otherwise, we considered them a Non-Responder. We compared the cancer recurrence rate at 1 year after surgery and the disease-free survival (DFS) of patients of both groups. We reported a statistically significant higher recurrence rate in the Non-Responder group: 66.7% vs. 14.3% (p = 0.036), anticipating relapse/no relapse within 1 year after surgery in 12/16 patients. The mean DFS was longer in the R-group than the NR-group, even if not statistically significant: 19.2 months vs. 12.7 months, (p = 0.123). The proposed strategy could potentially improve preclinical evaluation of treatment modalities and may enable prospective therapeutic selection in everyday clinical practice.