We conducted a retrospective cohort study of patients diagnosed with PDAC undergoing PD in our centre during the period 2006 to 2013 with subsequent follow-ups until November 2018.
Consecutive patients undergoing PD with a diagnosis of adenocarcinoma of the head of the pancreas, who had sufficient histological material to make the stains, were included. Patients older than 18 years old, in whom a multislice computed tomography (CT) was diagnostic of resectable PDAC, who had not received preoperative chemotherapy or radiotherapy, and who had an American Society of Anaesthesiologist classification (ASA) of I, II, or III, were included. Patients with unresectable intraoperative pancreatic tumours, in whom surgical resection was not performed or whose final pathology was not adenocarcinoma were excluded.
Demographic and staging parameters.
For each patient, the following data were extracted from the institutional pancreas database: demographic parameters (age, gender, body mass index [BMI], ASA, comorbidities and symptoms at diagnosis), postoperative complications (reported as per the Clavien-Dindo classification(19)), staging parameters (tumour size [T], lymph node status [N], lymph node ratio 20 [LNR20], defined as the ratio between affected and total retrieved lymph nodes being higher than 20%, tumour differentiation, stage, vascular, perineural and lymphatic invasion, positive resection margin rate [R] and recurrence), and survival parameters (disease-free survival [DFS] and overall survival [OS], measured from surgical intervention to recurrence or death respectively).
For all patients included in the study, prior to surgical intervention, the resectability of the tumour was assessed during the Multidisciplinary Team Meeting of Digestive Surgery at our centre. All the patients included in the study underwent a 64-slice CT (Siemens Somaton Sensation 64 ® - Siemens Healthcare © - Erlangen, Germany). The lesions were considered resectable in the absence of extended disease or vascular infiltration (presence of fatty planes around the vessels) or contact with the superior mesenteric vein or portal vein ≤ 180º in the absence of vein contour irregularity(20).
All patients underwent a classic Whipple procedure performed by experienced pancreatic surgeons. End-to-side duct-to-mucosa pancreaticojejunostomy or dunking pancreaticojejunostomy was performed at the surgeons’ discretion, depending on the pancreas consistency. End-to-side hepaticojejunostomy and transmesocolic end-to-side, double layer gastrojejunostomy was the standard technique used in our unit.
Each surgical piece was assessed with a ultraView Universal DAB v1.02.0018 detection kit (Ventana Medical Systems, Tucson, Arizona). For diagnostic purposes, 5 µm-thick paraffin sections were stained with hematoxylin and eosin. All the histological preparations were reviewed by two pathologists without previous knowledge of the prognostic factors and/or clinical evolution. Our GLUT-1 protocol consisted of heating the slides to 65ºC and incubating them for 12 minutes in a stove. Afterwards, dewaxing was carried out at 72ºC for 8 minutes with xylene, 99º ethanol and 96ºC ethyl. Then cellular conditioning was performed at 95ºC for 8 minutes. The buffer was subsequently washed at 36° for 4 minutes. After washing, the sections were incubated with a drop of UV INHIBITOR (cell inhibitor) for 4 minutes at 36 °C, then with a drop of GLUT-1 (rabbit polyclonal antibody, Roche) for 32 minutes and afterwards with a drop of universal multimeric antibody for 8 minutes. Next, a drop of chromogen diaminobenzidine was used as the substrate in the colour development reaction and incubated for 8 minutes. The sections were then counterstained with hematoxylin and incubated for 12 minutes. Finally, a drop of Bluing reagent was applied to the counterstained tile and incubated for 4 minutes.
A sample corresponding to the surgical specimen after PD of each patient was analysed by two independent investigators who were unaware of the outcome of the patients. The expression of GLUT-1 was considered positive when it was present in the membrane, the cytoplasm, or both. The erythrocytes and perineurium were considered as internal positive controls for GLUT-1 staining. Each preparation was evaluated at 4, 10, and 40 magnification. The staining was evaluated by two parameters: intensity and extension. The intensity was classified as weak or strong, defining strong as the same intensity as that of the positive controls; the extension was defined by the percentage of cells that presented the strongest staining (cytoplasmic, membranous, or both), which were subsequently grouped in low extension (< 80% of cells stained) versus high extension (≥ 80% of cells stained), thus identifying two groups for later comparison as reported previously in the literature.
Descriptive data were expressed as counts and proportions for categorical variables; continuous variables were presented as a median within an interquartile range. Statistical analysis was performed using the exact Fisher test for the comparison of categorical variables and the Student t test or the Mann-Whitney U test for continuous variables. Survival was analysed using the Kaplan-Meier method and compared with the Log-rank test. The differences were considered significant at a two-sided p value of < 0.05. All statistical analyses were performed using SPSS® 23.0 for Windows (SPSS Inc., Chicago, IL, USA).