Patients, Study design and Follow-up
Between September 2009 and December 2014, patients referred to Oslo University Hospital, Norway with potentially resectable pancreatic and periampullary malignancy were included in a standardised preoperative workup and evaluation in a multidisciplinary tumour board as detailed previously (19). Clinical data were recorded prospectively in an Epi-Info 3.5.3 database (CDC; Atlanta, GA; USA). The follow up included clinical status, computed tomography (CT) of the chest and abdomen as well as CA19-9 assessment twice a year. The mortality was deducted from the Norwegian Cause of Death Registry, provided by the Norwegian Institute of Public Health. The same patient cohort was described previously (20), with the observation period further extended by 25 months to January, 31st 2019. The study was undertaken in accordance to the STROBE(2014) and REMARK(2012) criteria for analysis and reporting.
Detection of CK-positive cells in the bone marrow
BM samples were collected under anaesthesia just prior to surgery, five ml BM from the anterior iliac crest bilaterally in syringes containing 200IE Heparin in 0.5ml NaCl. Samples were processed within 24h at the Micrometastasis Laboratory, Oslo University Hospital, Norway as previously described (21). BM mononuclear cells were isolated by density centrifugation over a Ficoll-Hypaque gradient (Lymphoprep®, STEMCELL Technologies UK Ltd., Cambridge, UK) and cytospin-slides were prepared with 0.5x106 BM-MNC/slide. For each sample 4 slides ( i.e. 2x106 cells) were immunostained for CK-positive cells with a combination of the monoclonal mouse primary antibodies AE1 and AE3 (Prod.# MAB 1612 & 1611, Milipore), with a broad affinity for the acidic and basic cytokeratin types, namely CK 1-8, 10, 14-16 and 19. The APAAP method for detection used a rabbit anti-mouse secondary antibody (Dako, #Z0259) and an alkaline phosphatase-mouse-anti-alkaline phosphatase tertiary antibody(Dako, #D0651), followed by a colour reaction with New Fuchsin, staining positive cells red (22). Nuclear counterstaining was performed with haematoxylin. The cytospins were screened for ICC-positive cells in an automated Ariol SL50 analyser (Leica biosystems). Detected elements were reviewed by a trained research engineer (CBS) and candidate cells were classified by a dedicated pathologist (EB). The cytomorphological evaluation of detected ICC-positive cells was performed in accordance to the ISHAGE consensus guidelines(7,8).This protocol, originally validated for breast cancer samples, has become the de facto standard for DTC reporting, including pancreatic cancer studies(13). Based on this classification, and according to earlier practice(23), the ICC-positive cells are divided into 4 categories: tumour cells (TC), hematopoietic (ie “false positive”) cells (HC), QHC (questionable/probable HC) and uninterpretable cells (UIC). If cells classified as TC or UIC were detected, 4 additional cytospins were incubated with the non-reactive mouse monoclonal antibody MOPC21 (Prod# M9269, Sigma Aldrich) of the same Ig isotype as AE1AE3, and detected by APAAP as above, constituting a negative control for the ICC reaction. Samples harbouring cells classified as TC in AE1/AE3 slides and not in the corresponding negative control slides were classified as “DTC-positive”. Samples harbouring cells classified as TC in AE1/AE3 slides and in the corresponding negative control slides were interpreted as “not evaluable” (n.e.) and excluded from further analysis. Samples harbouring “UIC”, “HC” or “QHC” were interpreted as “DTC-negative”. Results were stored in a database at the Micrometastasis Laboratory, Dept. of Pathology, Oslo University Hospital and were not available to treating clinicians. Following closure of the observation period, the classification of ICC-positive cells was combined with the clinical data using a scripted tool upon import to SPSS.
Characteristics of the patient cohort
Patients were categorised into three distinct clinical groups. Patients who underwent surgical resection did either have confirmed malignancy (resectable cancer) or a non-malignant condition (benign disease). The third group consisted of advanced cancer patients who underwent exploratory laparotomy but did not undergo resection, either due to locally advanced tumour growth or overt metastases (advanced cancer).
In addition to the presence of ICC-positive cells, the following clinical- and pathological parameters were recorded: age, gender, CA19-9, tumour size on CT-scan, AJCC/UICC-stage (7th ed.), pTNM-staging including resection margin, cancer origin, grade of histopathological differentiation, histological subtype (predominantly intestinal or pancreatobiliary), vascular and perineural infiltration. Continuous variables were dichotomized at the following thresholds: CA19-9 ≥ 200kU/L and the size of the lesion on CT-scan ≥ 25mm (for results, see Table 1).
Data were analysed in IBM SPSS, V25 (IBM Cooperation Analytics, Armonk, NY, USA) and STATA 15 (Stata Corp LLC, College Station, Texas, USA). Graphs were prepared in PRISM 8 (GraphPad Software Inc., La Jolla, CA, USA). The primary endpoints of the study were overall survival (OS), defined as survival until death by any cause and disease-free survival (DFS), defined as survival until signs of local relapse or metastasis were detected. Survival analyses were carried out with the Kaplan-Meier method, using the Log-rank test for difference of curve pairs. The association between TC-status and survival was quantified by a hazard ratio (HR) with a 95% confidence interval (CI) using Cox regression analysis. Statistical significance was assumed for p<0.05.