Cristina Ridolfi

Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy

Objective Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. Design In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour–stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. Results In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour–stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206+ and IL-10+ TAMs at the tumour–stroma interface. Conclusions Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.

Spatial distribution of B cells predicts prognosis in human pancreatic adenocarcinoma

ABSTRACT B-cell responses are emerging as critical regulators of cancer progression. In this study, we investigated the role of B lymphocytes in the microenvironment of human pancreatic ductal adenocarcinoma (PDAC), in a retrospective consecutive series of 104 PDAC patients and in PDAC preclinical models. Immunohistochemical analysis revealed that B cells occupy two histologically distinct compartments in human PDAC, either scatteringly infiltrating (CD20-TILs), or organized in tertiary lymphoid tissue (CD20-TLT). Only when retained within TLT, high density of B cells predicted longer survival (median survival 16.9 mo CD20-TLThi vs. 10.7 mo CD20-TLTlo; p = 0.0085). Presence of B cells within TLT associated to a germinal center (GC) immune signature, correlated with CD8-TIL infiltration, and empowered their favorable prognostic value. Immunotherapeutic vaccination of spontaneously developing PDAC (KrasG12D-Pdx1-Cre) mice with α-enolase (ENO1) induced formation of TLT with active GCs and correlated with increased recruitment of T lymphocytes, suggesting induction of TLT as a strategy to favor mobilization of immune cells in PDAC. In contrast, in an implanted tumor model devoid of TLT, depletion of B cells with an anti-CD20 antibody reinstated an antitumor immune response. Our results highlight B cells as an essential element of the microenvironment of PDAC and identify their spatial organization as a key regulator of their antitumor function. A mindfully evaluation of B cells in human PDAC could represent a powerful prognostic tool to identify patients with distinct clinical behaviors and responses to immunotherapeutic strategies.

A Partnership Model Between High- and Low-Volume Hospitals to Improve Results in Hepatobiliary Pancreatic Surgery

Objective:To optimize the results of low-volume (LV) centers for hepatopancreaticobiliary (HPB) surgery. Background:High-volume (HV) centers for HPB surgery have lower mortality than LV. Strategies for collaboration between HV and LV centers are not well investigated. Methods:Postoperative outcomes of patients undergoing curative HPB resection were evaluated at an LV hospital before (2006–2008) and during the collaboration (2009–2012) and at 2 hospitals with HV for either liver or pancreatic resection (2009–2012). Itinerant tutor surgeons from the HV centers were involved in the pre-, intra- and postoperative course of HPB patients at the LV hospital. Results:HPB cases at the LV center increased from 18 to 40 patients per year from 2006 to 2012, whereas 6-month postoperative mortality decreased from 17.8% (2006–2008) to 6% (2009–2012), P < 0.05 (liver: 10.3% vs 4.7% and pancreas: 29.4% vs 7.9%). During the collaborative study period, outcomes for hepatectomy were similar for LV and HV (85 vs 507 cases): postoperative Clavien-Dindo scores 4 and 5 were 2% and 0.2% for HV versus 2.4% and 1.2% for LV, respectively. Outcomes for pancreatic procedures (LV 63 vs HV 269 cases) showed better postoperative Clavien-Dindo scores 4 and 5 in the HV (0.7% score 4 and 1.5% score 5 for HV vs 3.2% and 6.3%, respectively, for LV) but the difference disappeared in the last 2 years (2011–2012) and matching the cases. Conclusions:Our partnership model helped improve postoperative outcomes at the LV center. Results at the LV hospital were comparable with the HV centers, although 2 years of partnership were required to achieve this in pancreatic surgery.

Morphohistological Features of Pancreatic Stump Are the Main Determinant of Pancreatic Fistula after Pancreatoduodenectomy

Introduction. Pancreatic surgery is challenging and associated with high morbidity, mainly represented by postoperative pancreatic fistula (POPF) and its further consequences. Identification of risk factors for POPF is essential for proper postoperative management. Aim of the Study. Evaluation of the role of morphological and histological features of pancreatic stump, other than main pancreatic duct diameter and glandular texture, in POPF occurrence after pancreaticoduodenectomy. Patients and Methods. Between March 2011 and April 2013, we performed 145 consecutive pancreaticoduodenectomies. We intraoperatively recorded morphological features of pancreatic stump and collected data about postoperative morbidity. Our dedicated pathologist designed a score to quantify fibrosis and inflammation of pancreatic tissue. Results. Overall morbidity was 59,3%. Mortality was 4,1%. POPF rate was 28,3%, while clinically significant POPF were 15,8%. Male sex (P = 0.009), BMI ≥ 25 (P = 0.002), prolonged surgery (P = 0.001), soft pancreatic texture (P < 0.001), small pancreatic duct (P < 0.001), pancreatic duct decentralization on stump anteroposterior axis, especially if close to the posterior margin (P = 0.031), large stump area (P = 0.001), and extended stump mobilization (P = 0.001) were related to higher POPF rate. Our fibrosis-and-inflammation score is strongly associated with POPF (P = 0.001). Discussion and Conclusions. Pancreatic stump features evaluation, including histology, can help the surgeon in fitting postoperative management to patient individual risk after pancreaticoduodenectomy.

Role of C-Reactive Protein Assessment as Early Predictor of Surgical Site Infections Development after Pancreaticoduodenectomy

Background: Surgical site infections (SSIs) are extremely common in pancreatic surgery and explain its considerable morbidity and mortality, even in tertiary centers. Early detection of these complications, with the help of laboratory assays, improve clinical outcome. The aim of the present study is to evaluate C-reactive protein (CRP) diagnostic accuracy as early predictor of SSIs after pancreaticoduodenectomy (PD). Methodology: We considered 251 consecutive PD. We prospectively recorded preoperative clinical and anthropometric data, intraoperative details and the postoperative outcome. In the first pool of consecutive patients (n = 150), we analyzed CRP levels from postoperative day 1 to 7 and investigated the prediction of SSIs. We then validated the diagnostic accuracy on the following 101 consecutive cases. Results: At multivariate analysis, high BMI and preoperative biliary stenting appeared to be independently associated with SSIs and organ-space SSI development. The CRP cutoff of 17.27 mg/dl on postoperative day 3 (78% sensitivity, 79% specificity) and of 14.72 mg/dl on postoperative day 4 (87% sensitivity, 82% specificity) was in a position to predict the course of 78.2 and 80.2% of patients, respectively. Conclusions: CRP on postoperative days 3 and 4 seems able to predict postoperative course, selecting patients deserving intensification of diagnostic assessment; patients not satisfying these conditions could be reasonably directed toward early discharge.

Occurrence of Tertiary Lymphoid Tissue in Pancreatic Adenocarcinoma

Context Stimulating the patient’s immune system to attack malignant tumor cells is considered a promising alternative therapeutic strategy to treat pancreatic adenocarcinoma (PDAC). Recent data point to the neogenesis of organized and vascularized ectopic (or tertiary) lymphoid tissue (TLT) at the tumor site, where B and T cell responses are efficiently initiated and sustained. Objective To investigate the occurrence of TLT in human PDAC and test whether a protocol of immunotherapy induces formation of TLT in a PDAC murine model. This might represent an alternative approach to target the tumor stroma, by creating a lymphoid like microenvironment, to increase the recruitment and activation of T cells. Methods Occurrence of TLT was evaluated by immunohistochemistry in PDAC tissue specimens from consecutive patients who underwent surgical resection at the Humanitas Clinical and Research Centre. A dendritic-cell (DC) based vaccine was used to immunize mice injected with Panc02 murine cells. Results In human PDAC tissue specimens, we identified organized lymphoid tissue, including compartmentalized T and B cell areas, DCs and high endothelial venules (HEV). In the heterogeneity of PDAC tissue, TLT occurred preferentially in the stromal compartment. The density of TLT correlated to the density of intra-tumor CD8 T cells, which displayed a phenotype indicating a defective activation status. In a murine model of PDAC, vaccination with DCs loaded with apoptotic PDAC cells occasionally induced formation of TLT. Conclusion Here we report the occurrence of lymphoid tissue in human PDAC, in the context of the desmoplastic stromal reaction and the correlation with CD8+ T cell infiltration. Immunotherapeutic approaches might induce formation of TLT and be exploited as alternative strategies to modify PDAC stroma and induce an anti-tumor immune response.

Frequent Ineffectiveness of Cefazolin as Perioperative Prophylaxis in Pancreatic Surgery

Context Cefazolin is widely accepted as routine antimicrobial prophylaxis in hepatobiliary and pancreatic surgery. Microbial resistance to this drug is rapidly increasing, nullifying the expected beneficial effects of its administration and exposing patients to potentially severe infective complications. Objective Analysis of bile culture results and comparison with postoperative outcomes in patients undergoing pancreatic surgery in a referral centre; definition of an alternative prophylaxis schedule. Methods Between Jan 2010 and Jan 2013 we performed 170 consecutive major resections requiring abdominal bile exposure, of which 156 pancreaticoduodenectomies and 14 total pancreatectomies. We routinely performed culture on bile samples obtained intraoperatively and on drain liquid samples taken on fifth postoperative day. We then prospectively recorded data about postoperative septic complications and analyzed them consulting our referral infectious disease specialist. Results Among the 170 patients, 95 (56%) had positive bile cultures, of which 73 (43%) with multiple microbes. The great majority of microbes obtained belonged to Enterococcus Spp . ( E. Faecalis 29%, E. Facium 18%), while other well represented germs were E. Coli (18%), Enterobacter Spp . (18%), Klebsiella Spp . (12%). Eighty patients (47%) received preoperative biliary stenting, which resulted strongly associated with bile infection (100% vs . 17%, P<0.001). Bile infection was associated with higher morbidity both in patient with stent (72% vs . 56%, P=0.037), and without stent (87% vs . 56%, P=0.026). Forty-seven (49%) of infected bile samples harbored microbes resistant to cefazolin; in these patient postoperative infective complication rate was significantly higher (61% v s . 39%, P=0.016) than in patients that did not harbor. The rate of cefazolin resistant microbe identification was even higher on drain samples (58%), which shared microbes with previous bile sample in 80% of cases, even though it was not associated with higher morbidity. Analyzing antimicrobial susceptibility of these germs we identify the association ampicillin-sulbactam as a new possible drug for perioperative prophylaxis. Conclusions Infective complications in pancreatic surgery could lead to severe consequences. Adequate antimicrobial prophylaxis, selected using epidemiological evidence from patients samples, is mandatory. Ampicillin-sulbactam could be a viable alternative to cefazolin in surgical procedures involving abdomen bile exposure, although further studies are necessary to validate this drug benefits.

Morphological Features of Pancreatic Stump, Other than Wirsung Diameter and Pancreatic Texture, Influence Pancreatic Fistula Rate After Pancreaticoduodenectomy

Context Pancreaticoduodenectomy (PD) is still characterized by high morbidity rates even in high volume centers, mainly represented by postoperative pancreatic fistula (POPF) and its associated further consequences. Wirsung diameter and pancreatic texture are well known features related to increased POPF risk. However, limited information about the relationship between morphological features of pancreatic stump and POPF onset is available. Objective Aim of the study was to evaluate the relationship between pancreatic stump morphology, intra-operatively assessed, and the occurrence of POPF in patients undergoing PD in an high volume center. Methods Between January 2010 and June 2012 we performed 136 PDs for benign and malignant disease; in a subgroup composed by 72 cases we realized a prospective study recording intra-operative measurements of the remnant pancreatic stump, recording surgeon’s judgment about pancreatic texture, and highlighting the caliber of main pancreatic duct, its position and the whole area. Between March 2011 and June 2012, in a consecutive series of 72 PD, we prospectively recorded the following characteristics of the pancreatic stump: surgeon’s judgment about pancreatic texture, diameter of main pancreatic duct, the whole area of pancreatic section (approximately calculated as elliptic), the distance between Wirsung duct and cranio-caudal and antero-posterior margins. In all patients, post-operative complications were recorded. Results Pancreatic fistula occurred in 19 cases (23.8%), 10 of them clinically significant (grade B and C according to ISGPF classification). In univariate analysis, mean Wirsung diameter resulted significantly smaller in patients with POPF (3.18 vs . 4.48 mm; P=0.007) and in patients with clinically significant POPF (P=0.015); searching for a cut off value, Wirsung diameter smaller than 4 mm demonstrated an association with higher POPF incidence (37.5% vs . 4.35%; P=0.003). Similarly, soft pancreas texture resulted associated with an increasing POPF rate (75% vs . 7%; P<0.001). Analyzing pancreatic stump morphology, we observed a relationship between larger stump areas and POFP development (206 mm 2 in POPF cases vs . 131 mm 2 in patients without fistula; P=0.017); moreover, main pancreatic duct decentralization appeared significantly associated with a lower POPF risk (more evident on antero-posterior axis than on cranio-caudal; P=0.019 and P=0.144, respectively). In multivariate analysis, only pancreatic stump soft texture and Wirsung diameter smaller than 4 mm were associated with POPF. Conclusion This study confirms small Wirsung diameter and soft pancreatic texture as the main determinants for POPF development. Moreover, an higher risk of failure of pancreatic anastomosis is also observed in large pancreatic stumps and when main pancreatic duct is centrally located.

Enucleation of Pancreatic Head Tumors Combined With Pre-Operative Wirsung Stenting, Intra-Operative Ultrasounds and Absorbable Fibrin Sealant Patch Application: Experience on 13 Cases

Context Benign/low malignant tumors or metastasis from renal cancer of the pancreatic head can be treated by enucleation; this procedure is technically demanding and it is often followed by high morbidity rates. Objective To analyze the peri-operative outcome of our series of enucleations for tumors of the pancreatic head. Methods From January 2010 to June 2012 29 out of 149 resected periampullary tumors were cystic neoplasms, 15 were neuroendocrine tumors (NET), 1 was metastasis from renal cancer and 1 was pseudopapillary solid tumor; in 13 of them (28% of 46 patients with pancreatic diseases eligible for atypical resection) an enucleation was performed. Enucleation was contraindicated when the lesion was deeply embedded in gland’s parenchyma involving Wirsung or originating from main pancreatic duct itself (main duct type IPMN), and in case of overt malignant appearance. We analyze the features of patients who underwent enucleation. Endoscopic ultrasound was performed preoperatively in 10 patients. ERCP with Wirsung stenting was realized when the distance between the main pancreatic duct and the lesion was less than 3 mm (4 cases). In patients submitted to stent positioning the mean distance between the lesion and the Wirsung duct was 1.9 mm while in the other group of patients it was 5 mm (P<0.05, t test). Intra-operative US was performed in all patients. At the end an absorbable fibrin sealant patch (Tachosil ® ) was placed on the area of enucleation. Results The mean diameter of the lesion was 1.9±1.1 cm (range: 1-4.5 cm); histology revealed 2 benign side branch IPMNs, 9 G1 NET, 1 metastasis from renal cancer and 1 pseudopapillary solid tumor. Mean operative time was 209.9±60.3 minutes and mean blood loss was 97.7±63.8 mL. No mortality was observed. Overall morbidity was 46% (including POPF rate of 31%): 4 POPF (2 grade B and 2 grade A). Mean length of hospital stay was 8.5±2.7 days. The reoperation rate was nihil. Readmission regarded only 1 patient. Conclusion Enucleation is a good alternative to pancreaticoduodenectomy in benign/low malignant tumors of the pancreatic head. Pre-operative Wirsung stenting, intra-operative ultrasounds and Tachosil ® application appear related to a satisfactory postoperative outcome.