Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer.

IMPORTANCE: Novel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC). OBJECTIVE: To investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance. DESIGN, SETTING, AND PARTICIPANTS: In this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years. MAIN OUTCOMES AND MEASURES: Outcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival. RESULTS: A total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P < .001). Similarly, shortly after ACT ctDNA-positive patients were 17 times (HR, 17.5; 95% CI, 5.4-56.5; P < .001) more likely to relapse. All 7 patients who were ctDNA positive after ACT experienced relapse. Monitoring during and after ACT indicated that 3 of the 10 ctDNA-positive patients (30.0%) were cleared by ACT. During surveillance after definitive therapy, ctDNA-positive patients were more than 40 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 43.5; 95% CI, 9.8-193.5 P < .001). In all multivariate analyses, ctDNA status was independently associated with relapse after adjusting for known clinicopathologic risk factors. Serial ctDNA analyses revealed disease recurrence up to 16.5 months ahead of standard-of-care radiologic imaging (mean, 8.7 months; range, 0.8-16.5 months). Actionable mutations were identified in 81.8% of the ctDNA-positive relapse samples. CONCLUSIONS AND RELEVANCE: Circulating tumor DNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, ACT monitoring, and early relapse detection.

Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery.

OBJECTIVE: To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence. DESIGN: We have established efficient procedures for identification of SSVs by next-generation sequencing and subsequent quantification of 3-6 SSVs in plasma. The consequence of intratumour heterogeneity on our approach was assessed. The level of circulating tumour DNA (ctDNA) was quantified in 151 serial plasma samples from six relapsing and five non-relapsing colorectal cancer (CRC) patients by droplet digital PCR, and correlated to clinical findings. RESULTS: Up to six personalised assays were designed for each patient. Our approach enabled efficient temporal assessment of disease status, response to surgical and oncological intervention, and early detection of incipient recurrence. Our approach provided 2-15 (mean 10) months' lead time on detection of metastatic recurrence compared to conventional follow-up. The sensitivity and specificity of the SSVs in terms of detecting postsurgery relapse were 100%. CONCLUSIONS: We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

Postoperative but not preoperative treatment with sorafenib inhibits liver regeneration in rats.

BACKGROUND: Sorafenib, a multikinase inhibitor, has been shown to halt the growth of hepatocellular carcinoma. The aim of the present study was to investigate the effect of Sorafenib on liver regeneration in healthy rats. METHODS: In two substudies we examined the effect of pre- or post-operative treatment with Sorafenib (15 mg/kg/d). Wistar rats (n = 120) received either Sorafenib (S) or placebo (P). After 70% partial hepatectomy, the rats were euthanized on postoperative days 2, 4, or 8. Body weight and liver weight were recorded and regeneration rate (RR) calculated. Hepatocyte proliferation was estimated by immunohistochemistry for Ki-67 antigen using unbiased stereological methods. RESULTS: Eleven animals (9%) died after surgery. In the preoperative substudy, lower body weight gains during the gavage period in the S group were found. No difference between groups S and P regarding liver weight gain, liver RRs, and hepatocyte proliferation on postoperative days 2 and 4 were found. In the postoperative substudy, significantly lower values of liver weight gain, liver RRs, and hepatocyte proliferation were found in the S group. CONCLUSIONS: In our rat model, Sorafenib did not increase posthepatectomy mortality. Postoperative treatment significantly impaired liver regeneration. Preoperative treatment impaired body weight during the gavage period, but was without effect on liver regeneration.

The natural history of liver regeneration in rats: description of an animal model for liver regeneration studies.

BACKGROUND: Rodent models have been used to evaluate aspects of liver regeneration. The aim of the present study was to investigate the natural history of liver regeneration in healthy rats. METHODS: A 70% partial hepatectomy was performed in 64 rats. The animals were randomised into 8 groups and evaluated on postoperative days one to eight. Hepatocyte proliferation was evaluated by immunohistochemistry using unbiased stereological principles. RESULTS: The mean rat body weight was 238 g (211-287). The mean weight of the resected liver was 6.3 g (5.2-7.3) and the estimated mean total liver weight was 8.9 g (7.4-10.4). Both liver weight analysis and regeneration rate showed an ascending curve, with a maximum slope on postoperative days 1-4, reaching a steady state on days 5-8. Hepatocyte proliferation (positive Ki-67 cell profiles pr. mm(2)) was high (250 cell profiles/mm(2)) on postoperative days 1-3 and tapered off on day 5. CONCLUSION: Seventy percent partial hepatectomy in healthy rats induces a rapid regenerative response and PODs 2, 4 and 8 seems optimal for assessing hepatic growth in future studies.

Sorafenib inhibits liver regeneration in rats.

BACKGROUND: Sorafenib is a multikinase inhibitor with antiangiogenic and antiproliferative properties, approved for the treatment of hepatocellular carcinoma. The effect of Sorafenib on liver regeneration in healthy rats was investigated. METHODS: Sixty Wistar rats received either Sorafenib (group S; 15 mg/kg) or placebo for 14 days prior to resection and until sacrifice. After a 70% partial hepatectomy, the rats were euthanized on post-operative days (POD) 2, 4 or 8. Hepatocyte proliferation was estimated by immunohistochemistry for Ki-67 antigen using stereological methods on sections prepared by systematic uniform random sampling. RESULTS: Seven animals (12%) died after surgery. Death rates were similar in treated rats and controls. At hepatectomy, the body weight was significantly lower in group S rats. The liver weight and regeneration rates were lower in group S rats on PODs 2, 4 and 8. Hepatocyte proliferation was significantly lower in group S animals on PODs 2 and 4. Alanine aminotransferase ALAT was significantly higher in the Sorafenib-treated group on PODs 2, 4 and 8. Alkaline phosphatase ALP and bilirubin levels were similar in the two groups, although bilirubin was elevated in group S rats on POD 8. CONCLUSION: In this rat model, Sorafenib did not increase post-hepatectomy mortality, but was associated with a significant impaired liver weight gain, regeneration rates and hepatocyte proliferation.

Quantitative histological assessment of hepatic ischemia-reperfusion injuries following ischemic pre- and post-conditioning in the rat liver.

BACKGROUND: Ischemic preconditioning (IPC) has been shown to protect the liver against ischemia-reperfusion (I/R) injuries. However, ischemic post-conditioning has received little attention. The aim of the present study was to quantify and compare the hepato-protective properties of IPC and IPO, for the first time, using unbiased design-based stereological methods. METHODS: We divided 67 rats into four groups: sham, liver ischemia (LI), IPC, and IPO. Rats were subjected to 60 min LI, followed by 4- or 24-h reperfusion. We performed quantification of (NVR) and apoptotic cell profile number. RESULTS: We observed no significant differences in NVR between ischemic groups after 4 h. After 24-h reperfusion, NVR had increased to 70% in the LI group, compared with 51% (P = 0.02) and 49% (P = 0.01) in the IPC and IPO groups, respectively. After 4-h reperfusion, the apoptotic cell number was significantly higher in all ischemic groups than in the sham group; we detected no difference between ischemic groups. After 24-h reperfusion, we detected a significantly lower number of apoptotic cell profiles in the IPC group than in the LI group (P = 0.02). The mean number of apoptotic cell profiles decreased insignificantly in the IPO group (P = 0.06). Liver parameters were at all time comparable between groups. CONCLUSIONS: After I/R, IPC and IPO reduce the degree of hepatocellular injury. Both methods are equally efficient at preventing hepatocellular necrosis. Furthermore, apoptosis is significantly lower after IPC.

Ischemic pre- and postconditioning has pronounced effects on gene expression profiles in the rat liver after ischemia/reperfusion.

Ischemic pre (IPC)- and postconditioning (IPO) protect the liver against ischemia/reperfusion injuries (IRI). Conditioning involves several different trigger factors, mediators, and effectors, many of which are affected during the early phase of reperfusion, ultimately resulting in decreased liver injuries. The aim of the present study was to investigate the genomic response induced by IPC and IPO in ischemia/reperfusion-damaged rat liver biopsies. Forty-eight male Wistar rats were divided into five groups: sham (n = 8), IRI (n = 10), IPC (n = 10), IPO (n = 10), and IPC + IPO (n = 10). The rat livers were subjected to 30 min of ischemia. Liver biopsies and blood samples were taken after 30 min of reperfusion. The biopsies were analyzed using cDNA microarrays with validation by quantitative RT-PCR. The significance analysis of microarray was used to identify genes with changed expression levels. A comparison analysis of the intervention groups showed a highly increased number of genes, with significantly different expression in the conditioned groups compared with the IRI group. A total of 172 genes were identified as the most highly affected, and these genes showed similar patterns with regard to the up- and downregulated expression levels within the conditioned groups. Pathway analysis of the 172 genes identified four networks that were involved in increased gene expression, cellular growth, and proliferation. In conclusion, the present study demonstrated that IPC, IPO, and IPC + IPO had pronounced effects on the expression levels of a large number of genes during early reperfusion. IPC, IPO, and IPC + IPO seem to mediate their protective effects by regulating the same genes and genetic networks. These identified networks are known to be involved in maintaining cellular homeostasis.

Effects of ischemic pre- and postconditioning on HIF-1α, VEGF and TGF-ß expression after warm ischemia and reperfusion in the rat liver.

BACKGROUND: Ischemic pre- and postconditioning protects the liver against ischemia/reperfusion injuries. The aim of the present study was to examine how ischemic pre- and postconditioning affects gene expression of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor A (VEGF-A) and transforming growth factor ß (TGF-ß) in liver tissue. METHODS: 28 rats were randomized into five groups: control; ischemia/reperfusion; ischemic preconditioning (IPC); ischemic postconditioning (IPO); combined IPC and IPO. IPC consisted of 10 min of ischemia and 10 min of reperfusion. IPO consisted of three cycles of 30 sec. reperfusion and 30 sec. of ischemia. RESULTS: HIF-1α mRNA expression was significantly increased after liver ischemia compared to controls (p = 0.010). HIF-1α mRNA expression was significantly lower in groups subjected to IPC or combined IPC and IPO when compared to the ischemia/reperfusion group (p = 0.002). VEGF-A mRNA expression increased in the ischemia/reperfusion or combined IPC and IPO groups when compared to the control group (p < 0.05). CONCLUSION: Ischemic conditioning seems to prevent HIF-1α mRNA induction in the rat liver after ischemia and reperfusion. This suggests that the protective effects of ischemic conditioning do not involve the HIF-1 system. On the other hand, the magnitude of the HIF-1α response might be a marker for the degree of I/R injuries after liver ischemia. Further studies are needed to clarify this issue.

Expression of genes involved in rat liver angiogenesis after ischaemia and reperfusion: effects of ischaemic pre- and post-conditioning.

BACKGROUND: During surgery, ischaemic pre- (IPC) and post-conditioning (IPO) protects the liver against ischaemia/reperfusion injuries (I/R-injuries). The impact of ischaemic conditioning on liver regeneration has been less well studied. Angiogenesis is an important part of liver regeneration after hepatectomy. The aim of the present study was to investigate the effect of ischaemia/reperfusion and ischaemic conditioning on the expression of genes with angiogenic potential in a model of rat liver ischaemia. METHODS: A model of total liver ischaemia (30 min) and reperfusion (30 min) was employed using Wistar rats. Rats were randomized into five groups: (C) control (IRI) ischaemic, IPC, IPO and IPC + IPO. Liver enzymes were sampled at the end of reperfusion. Liver biopsies were analysed using cDNA microarrays. RESULTS: Alanine aminotransferase (ALT) increased significantly in all the ischaemic groups compared with controls (P= 0.000). Searching databases 99 genes involved in rat liver angiogenesis were identified. Compared with group (C) the number of genes significantly up-regulated was as follows: IRI (n= 5), IPC (n= 24), IPO (n= 33) and IPC + IPO (n= 18). No genes were down-regulated in the four groups compared with controls. CONCLUSION: Ischaemic conditioning, as demonstrated in the present study, seems to be potent activators of angiogenic genes. This might be favourable to the regenerating liver.

The influence of preconditioning on metabolic changes in the pig liver before, during, and after warm liver ischemia measured by microdialysis.

PURPOSE: Ischemia-reperfusion injury induced by the Pringle maneuver is a well-known problem after liver surgery. The aim of this study was to monitor metabolic changes in the pig liver during warm ischemia and the following reperfusion preceded by ischemic preconditioning (IPC). METHODS: Eight Landrace pigs underwent laparotomy. Two microdialysis catheters were inserted in the liver, one in the left lobe and another in the right lobe. A reference catheter was inserted in the right biceps femoris muscle. Microdialysis samples were collected every 30 min during the study. After 2 h of baseline measurement, IPC was performed by subjecting pigs to 10 min of ischemia, followed by 10 min of reperfusion. Total ischemia for 60 min was followed by 3 h of reperfusion. The samples were analyzed for glucose, lactate, pyruvate, and glycerol. Blood samples were drawn three times to determine standard liver parameters. RESULTS: All parameters remained stable during baseline. Glycerol and glucose levels increased significantly during ischemia, followed by a decrease from the start of reperfusion. During the ischemic period, lactate levels increased significantly and decreased during reperfusion. The lactate-pyruvate ratio increased significantly during ischemia and decreased rapidly during reperfusion. Only minor changes were observed in standard liver parameters. CONCLUSIONS: The present study demonstrated profound metabolic changes before, during, and after warm liver ischemia under the influence of IPC. Compared with a similar study without IPC, the metabolic changes seem to be unaffected by preconditioning.

T-tube drainage stimulates the healing of choledocho-choledochostomies. An experimental study in pigs.

BACKGROUND/PURPOSE: We investigated the effect of T-tube drainage on the healing of choledocho-choledochostomies in pigs. METHODS: Twenty pigs with a median weight of 56 kg were used for the experiments. The pigs were randomized to two groups of ten. In all pigs the gallbladder was removed and the common bile duct was transected. In both groups continuity was re-established by standardized single-line, interrupted, and inverted sutures. In one group a T-tube for decompression was inserted. On postoperative day 6, a laparotomy was performed. Pigs were investigated for signs of cholascos, and an intraoperative cholangiography was performed. The excised anastomosis was examined for breaking strength and collagen content. Blood samples were drawn prior to the first and the final operations. RESULTS: In both groups standard liver parameters were unaffected by surgery, and cholangiography showed no signs of extrahepatic stenosis or intrahepatic dilatation. The T-tube-drained choledocho-choledochostomies showed a significantly higher breaking strength (P = 0.035) compared to the group which had no drainage. Collagen content per volume was unaffected by T-tube drainage. CONCLUSIONS: T-tube drainage had a significant stimulatory effect on the breaking strength of choledocho-choledochostomies in pigs on postoperative day 6, but was without effect on collagen content.

Spondylodiscitis. A retrospective study of 163 patients.

BACKGROUND AND PURPOSE: Spondylodiscitis may be a serious disease due to diagnostic delay and inadequate treatment. There is no consensus on when and how to operate. We therefore retrospectively analyzed the outcome of a large series of patients treated either nonoperatively or surgically. PATIENTS AND METHODS: Between 1992 and 2000, 163 patients (101 males) were hospitalized due to spondylodiscitis. The mean age was 56 (1-83) years. The infection was located in the cervical spine in 13 patients (8%), in the thoracic spine in 62 patients (38%), at the thoracolumbar junction in 10 patients (6%), and in the lumbo-sacral spine in 78 patients (48%). In 67 patients (41%), no microorganisms were detected. Most of the other patients had Staphylococcus aureus infection (53 patients) and/or Mycobacterium tuberculosis (22 patients). The patients were divided into 3 groups: (A) 70 patients who had nonoperative treatment, (B) 56 patients who underwent posterior decompression alone, and (C) 37 patients who underwent decompression and stabilization. RESULTS: At 12-month follow-up, nonoperative treatment (A) had failed in 8/70 patients, who had subsequently been operated. 24/56 and 6/37 had been reoperated in groups B and C, respectively. Group A patients had no neurological symptoms. In group B, 11 had neurological deficits and surgery was beneficial for 5 of them; 4 remained unchanged and 2 deteriorated (1 due to cerebral abscess). 11 patients in group C had altered neurogical deficits, which improved in 9 of them. 20 patients had died during the 1-year follow-up, 3 in hospital, directly related with infection. INTERPRETATION: Nonoperative treatment was effective in nine-tenths of the patients. Decompression alone had high a reoperation rate compared to decompression and internal stabilization.

[Combined liver resection and radiofrequency ablation for colorectal liver metastases]. / Kombineret leverresektion og radiofrekvensablation af kolorektale levermetastaser.

INTRODUCTION: Liver resection in combination with radiofrequency ablation (RFA) is a novel approach in patients with colorectal liver metastases who are otherwise unresectable. MATERIALS AND METHODS: Eighteen patients with colorectal liver metastases were treated with surgical resection combined with RFA. All patients were followed prospectively with CT-scanning of thorax and abdomen during a 5-year period, 1, 4, 8 and 12 months after treatment and every 6 months thereafter for the following 4 years. Main outcome was recurrence and survival. RESULTS: Eleven male and 7 female patients with a median age of 65 years (41-78) were treated with combined surgery and RFA. 76 metastases were treated, 34 of these with resection. The median survival rate in the entire group was 35 months. 5-years overall survival was 34%. 12 patients had recurrence and were treated with multimodality therapy. CONCLUSION: Combined surgery and RFA treatment was a good alternative in a group of patients with colorectal liver metastases that were otherwise only suitable for systemic chemotherapy.

Metabolic changes in the pig liver during warm ischemia and reperfusion measured by microdialysis.

AIM: Portal triad clamping can cause ischemia-reperfusion injury. The aim of the study was to monitor metabolic changes by microdialysis before, during, and after warm ischemia in the pigliver. MATERIAL AND METHODS: Eight pigs underwent laparotomy followed by ischemia by Pringle's maneuver. One microdialysis catheter was placed in each of four liver lobes. A reference catheter was placed in a muscle. Microdialysis samples were collected at intervals of 30 min starting 2 h before 1 h of total ischemia followed by 3 h of reperfusion. Glucose, lactate, pyruvate, and glycerol concentrations were measured. Blood samples were drawn for determination of alanine aminotransferase, alkaline phosphatase, and bilirubin together with total leukocytes and prothrombin time. RESULTS: All parameters were stable during the baseline period. During the ischemic period, lactate levels increased significantly (P < 0.05) followed by a rapid decrease after reperfusion. A transient increase was observed for glucose and glycerol. Pyruvate showed a slight increase from the time of ischemia. The lactate-pyruvate ratio increased rapidly after initiating ischemia and decreased immediately after reperfusion. A slight increase in transaminase levels was observed. CONCLUSIONS: During and after warm ischemia, there were profound metabolic changes in the pigliver observed with an increase in lactate, glucose, glycerol, and the lactate-pyruvate ratio. There were no differences between the four liver lobes, indicating the piglivers homogeneity.