Nationwide validation of the distal fistula risk score (D-FRS).

PURPOSE: Distal pancreatectomy (DP) is associated with a high complication rate of 30-50% with postoperative pancreatic fistula (POPF) as a dominant contributor. Adequate risk estimation for POPF enables surgeons to use a tailor-made approach. Assessment of the risk of POPF prior to DP can lead to the application of preventive strategies. The current study aims to validate the recently published preoperative and intraoperative distal fistula risk score (D-FRS) in a nationwide cohort. METHODS: This nationwide retrospective Dutch cohort study included all patients after DP for any indication, all of whom were registered in the Dutch Pancreatic Cancer Audit (DPCA) database between 2013 and 2021. The D-FRS was validated by filling in the probability equations with data from this cohort. The predictive capacity of the models was represented by an area under the receiver operating characteristic (AUROC) curve. RESULTS: A total of 896 patients underwent DP of which 152 (17%) developed POPF of whom 144 grade B (95%) and 8 grade C (5%). The preoperative D-FRS, consisting of the variables pancreatic neck thickness and pancreatic duct diameter, showed an AUROC of 0.73 (95%CI 0.68-0.78). The intraoperative D-FRS, comprising pancreatic neck, duct diameter, BMI, operating time, and soft pancreatic aspect, showed an AUROC of 0.69 (95%CI 0.64-0.74). CONCLUSION: The current study is the first nationwide validation of the preoperative and intraoperative D-FRS showing acceptable distinguishing capacity for only the preoperative D-FRS for POPF. Therefore, the preoperative score could improve prevention and mitigation strategies such as drain management, which is currently investigated in the multicenter PANDORINA trial.

Nurses' experiences with continuous vital sign monitoring on the general surgical ward: a qualitative study based on the Behaviour Change Wheel.

BACKGROUND: To support early recognition of clinical deterioration on a general ward continuous vital signs monitoring (CMVS) systems using wearable devices are increasingly being investigated. Although nurses play a crucial role in successful implementation, reported nurse adoption and acceptance scores vary significantly. In-depth insight into the perspectives of nurses regarding CMVS is lacking. To this end, we applied a theoretical approach for behaviour change derived from the Behaviour Change Wheel (BCW). AIM: To provide insight in the capability, opportunity and motivation of nurses working with CMVS, in order to inform future implementation efforts. METHODS: A qualitative study was conducted, including twelve nurses of a surgical ward in a tertiary teaching hospital with previous experience of working with CMVS. Semi-structured interviews were audiotaped, transcribed verbatim, and analysed using thematic analysis. The results were mapped onto the Capability, Opportunity, Motivation - Behaviour (COM-B) model of the BCW. RESULTS: Five key themes emerged. The theme 'Learning and coaching on the job' linked to Capability. Nurses favoured learning about CVSM by dealing with it in daily practice. Receiving bedside guidance and coaching was perceived as important. The theme 'interpretation of vital sign trends' also linked to Capability. Nurses mentioned the novelty of monitoring vital sign trends of patients on wards. The theme 'Management of alarms' linked to Opportunity. Nurses perceived the (false) alarms generated by the system as excessive resulting in feelings of irritation and uncertainty. The theme 'Integration and compatibility with clinical workflow' linked to Opportunity. CVSM was experienced as helpful and easy to use, although integration in mobile devices and the EMR was highly favoured and the management of clinical workflows would need improvement. The theme 'Added value for nursing care' linked to Motivation. All nurses recognized the potential added value of CVSM for postoperative care. CONCLUSION: Our findings suggest all parts of the COM-B model should be considered when implementing CVSM on general wards. When the themes in Capability and Opportunity are not properly addressed by selecting interventions and policy categories, this may negatively influence the Motivation and may compromise successful implementation.

Comparing practice and outcome of laparoscopic liver resection between high-volume expert centres and nationwide low-to-medium volume centres.

BACKGROUND: Based on excellent outcomes from high-volume centres, laparoscopic liver resection is increasingly being adopted into nationwide practice which typically includes low-medium volume centres. It is unknown how the use and outcome of laparoscopic liver resection compare between high-volume centres and low-medium volume centres. This study aimed to compare use and outcome of laparoscopic liver resection in three leading European high-volume centres and nationwide practice in the Netherlands. METHOD: An international, retrospective multicentre cohort study including data from three European high-volume centres (Oslo, Southampton and Milan) and all 20 centres in the Netherlands performing laparoscopic liver resection (low-medium volume practice) from January 2011 to December 2016. A high-volume centre is defined as a centre performing >50 laparoscopic liver resections per year. Patients were retrospectively stratified into low, moderate- and high-risk Southampton difficulty score groups. RESULTS: A total of 2425 patients were included (1540 high-volume; 885 low-medium volume). The median annual proportion of laparoscopic liver resection was 42.9 per cent in high-volume centres and 7.2 per cent in low-medium volume centres. Patients in the high-volume centres had a lower conversion rate (7.4 versus 13.1 per cent; P < 0.001) with less intraoperative incidents (9.3 versus 14.6 per cent; P = 0.002) as compared to low-medium volume centres. Whereas postoperative morbidity and mortality rates were similar in the two groups, a lower reintervention rate (5.1 versus 7.2 per cent; P = 0.034) and a shorter postoperative hospital stay (3 versus 5 days; P < 0.001) were observed in the high-volume centres as compared to the low-medium volume centres. In each Southampton difficulty score group, the conversion rate was lower and hospital stay shorter in high-volume centres. The rate of intraoperative incidents did not differ in the low-risk group, whilst in the moderate-risk and high-risk groups this rate was lower in high-volume centres (absolute difference 6.7 and 14.2 per cent; all P < 0.004). CONCLUSION: High-volume expert centres had a sixfold higher use of laparoscopic liver resection, less conversions, and shorter hospital stay, as compared to a nationwide low-medium volume practice. Stratification into Southampton difficulty score risk groups identified some differences but largely outcomes appeared better for high-volume centres in each risk group.

Radiofrequency ablation and chemotherapy versus chemotherapy alone for locally advanced pancreatic cancer (PELICAN): study protocol for a randomized controlled trial.

BACKGROUND: Approximately 80% of patients with locally advanced pancreatic cancer (LAPC) are treated with chemotherapy, of whom approximately 10% undergo a resection. Cohort studies investigating local tumor ablation with radiofrequency ablation (RFA) have reported a promising overall survival of 26-34 months when given in a multimodal setting. However, randomized controlled trials (RCTs) investigating the effect of RFA in combination with chemotherapy in patients with LAPC are lacking. METHODS: The "Pancreatic Locally Advanced Unresectable Cancer Ablation" (PELICAN) trial is an international multicenter superiority RCT, initiated by the Dutch Pancreatic Cancer Group (DPCG). All patients with LAPC according to DPCG criteria, who start with FOLFIRINOX or (nab-paclitaxel/)gemcitabine, are screened for eligibility. Restaging is performed after completion of four cycles of FOLFIRINOX or two cycles of (nab-paclitaxel/)gemcitabine (i.e., 2 months of treatment), and the results are assessed within a nationwide online expert panel. Eligible patients with RECIST stable disease or objective response, in whom resection is not feasible, are randomized to RFA followed by chemotherapy or chemotherapy alone. In total, 228 patients will be included in 16 centers in The Netherlands and four other European centers. The primary endpoint is overall survival. Secondary endpoints include progression-free survival, RECIST response, CA 19.9 and CEA response, toxicity, quality of life, pain, costs, and immunomodulatory effects of RFA. DISCUSSION: The PELICAN RCT aims to assess whether the combination of chemotherapy and RFA improves the overall survival when compared to chemotherapy alone, in patients with LAPC with no progression of disease following 2 months of systemic treatment. TRIAL REGISTRATION: Dutch Trial Registry NL4997 . Registered on December 29, 2015. ClinicalTrials.gov NCT03690323 . Retrospectively registered on October 1, 2018.

Total neoadjuvant FOLFIRINOX versus neoadjuvant gemcitabine-based chemoradiotherapy and adjuvant gemcitabine for resectable and borderline resectable pancreatic cancer (PREOPANC-2 trial): study protocol for a nationwide multicenter randomized controlled trial.

BACKGROUND: Neoadjuvant therapy has several potential advantages over upfront surgery in patients with localized pancreatic cancer; more patients receive systemic treatment, fewer patients undergo futile surgery, and R0 resection rates are higher, thereby possibly improving overall survival (OS). Two recent randomized trials have suggested benefit of neoadjuvant chemoradiotherapy over upfront surgery, both including single-agent chemotherapy regimens. Potentially, the multi-agent FOLFIRINOX regimen (5-fluorouracil with leucovorin, irinotecan, and oxaliplatin) may further improve outcomes in the neoadjuvant setting for localized pancreatic cancer, but randomized studies are needed. The PREOPANC-2 trial investigates whether neoadjuvant FOLFIRINOX improves OS compared with neoadjuvant gemcitabine-based chemoradiotherapy and adjuvant gemcitabine in resectable and borderline resectable pancreatic cancer patients. METHODS: This nationwide multicenter phase III randomized controlled trial includes patients with pathologically confirmed resectable and borderline resectable pancreatic cancer with a WHO performance score of 0 or 1. Resectable pancreatic cancer is defined as no arterial and ≤ 90 degrees venous involvement; borderline resectable pancreatic cancer is defined as ≤90 degrees arterial and ≤ 270 degrees venous involvement without occlusion. Patients receive 8 cycles of neoadjuvant FOLFIRINOX chemotherapy followed by surgery without adjuvant treatment (arm A), or 3 cycles of neoadjuvant gemcitabine with hypofractionated radiotherapy (36 Gy in 15 fractions) during the second cycle, followed by surgery and 4 cycles of adjuvant gemcitabine (arm B). The primary endpoint is OS by intention-to-treat. Secondary endpoints include progression-free survival, quality of life, resection rate, and R0 resection rate. To detect a hazard ratio of 0.70 with 80% power, 252 events are needed. The number of events is expected to be reached after inclusion of 368 eligible patients assuming an accrual period of 3 years and 1.5 years follow-up. DISCUSSION: The PREOPANC-2 trial directly compares two neoadjuvant regimens for patients with resectable and borderline resectable pancreatic cancer. Our study will provide evidence on the neoadjuvant treatment of choice for patients with resectable and borderline resectable pancreatic cancer. TRIAL REGISTRATION: Primary registry and trial identifying number: EudraCT: 2017-002036-17 . Date of registration: March 6, 2018. Secondary identifying numbers: The Netherlands National Trial Register - NL7094 , NL61961.078.17, MEC-2018-004.

The treatment and survival of elderly patients with locally advanced pancreatic cancer: A post-hoc analysis of a multicenter registry.

BACKGROUND: The treatment options for patients with locally advanced pancreatic cancer (LAPC) have improved in recent years and consequently survival has increased. It is unknown, however, if elderly patients benefit from these improvements in therapy. With the ongoing aging of the patient population and an increasing incidence of pancreatic cancer, this patient group becomes more relevant. This study aims to clarify the association between increasing age, treatment and overall survival in patients with LAPC. METHODS: Post-hoc analysis of a multicenter registry including consecutive patients with LAPC, who were registered in 14 centers of the Dutch Pancreatic Cancer Group (April 2015-December 2017). Patients were divided in three groups according to age (<65, 65-74 and ≥75 years). Primary outcome was overall survival stratified by primary treatment strategy. Multivariable regression analyses were performed to adjust for possible confounders. RESULTS: Overall, 422 patients with LAPC were included; 162 patients (38%) aged <65 years, 182 patients (43%) aged 65-74 and 78 patients (19%) aged ≥75 years. Chemotherapy was administered in 86%, 81% and 50% of the patients in the different age groups (p<0.01). Median overall survival was 12, 11 and 7 months for the different age groups (p<0.01).Patients treated with chemotherapy showed comparable median overall survival of 13, 14 and 10 months for the different age groups (p=0.11). When adjusted for confounders, age was not associated with overall survival. CONCLUSION: Elderly patients are less likely to be treated with chemotherapy, but when treated with chemotherapy, their survival is comparable to younger patients.

Preoperative imaging for colorectal liver metastases: a nationwide population-based study.

BACKGROUND: In patients with colorectal liver metastases (CRLM) preoperative imaging may include contrast-enhanced (ce) MRI and [18 F]fluorodeoxyglucose (18 F-FDG) PET-CT. This study assessed trends and variation between hospitals and oncological networks in the use of preoperative imaging in the Netherlands. METHODS: Data for all patients who underwent liver resection for CRLM in the Netherlands between 2014 and 2018 were retrieved from a nationwide auditing database. Multivariable logistic regression analysis was used to assess use of ceMRI, 18 F-FDG PET-CT and combined ceMRI and 18 F-FDG PET-CT, and trends in preoperative imaging and hospital and oncological network variation. RESULTS: A total of 4510 patients were included, of whom 1562 had ceMRI, 872 had 18 F-FDG PET-CT, and 1293 had combined ceMRI and 18 F-FDG PET-CT. Use of ceMRI increased over time (from 9·6 to 26·2 per cent; P < 0·001), use of 18 F-FDG PET-CT decreased (from 28·6 to 6·0 per cent; P < 0·001), and use of both ceMRI and 18 F-FDG PET-CT 16·9 per cent) remained stable. Unadjusted variation in the use of ceMRI, 18 F-FDG PET-CT, and combined ceMRI and 18 F-FDG PET-CT ranged from 5·6 to 100 per cent between hospitals. After case-mix correction, hospital and oncological network variation was found for all imaging modalities. DISCUSSION: Significant variation exists concerning the use of preoperative imaging for CRLM between hospitals and oncological networks in the Netherlands. The use of MRI is increasing, whereas that of 18 F-FDG PET-CT is decreasing.

ANTECEDENTES: En pacientes con metástasis hepáticas colorrectales (colorrectal liver metastases, CRLM), los estudios de imagen preoperatorios pueden incluir resonancia magnética con contraste (ce)MRI y 18 F-FDG-PET-CT. Este estudio evaluó las tendencias y la variación entre los hospitales y las redes oncológicas en el uso de estudios de imagen preoperatorios en los Países Bajos. MÉTODOS: Todos los pacientes que se sometieron a una resección hepática por CRLM en los Países Bajos entre 2014 y 2018 fueron seleccionados a partir de una base de datos a nivel nacional auditada. El análisis de regresión logística multivariable se utilizó para evaluar el uso de ceMRI, de 18 F-FDG-PET-CT y de ceMRI combinado con 18 F-FDG-PET-CT, así como para determinar las tendencias en los estudios de imagen preoperatorios y las variaciones hospitalarias y de la red oncológica. RESULTADOS: En total, se incluyeron 4.510 pacientes, de los cuales 1.562 se sometieron a ceMRI, 872 a 18 F-FDG-PET-CT y 1.293 a ceMRI combinado con 18 F-FDG-PET-CT. El uso de ceMRI aumentó con el tiempo del 9,6% al 26,2% (P < 0,001), el uso de 18 F-FDG-PET-CT disminuyó (25% a 6,0%, P < 0,001) y el uso de ceMRI y 18 F-FDG-PET- CT (17%) se mantuvo estable. La variación no ajustada entre hospitales en el uso de ceMRI, 18 F-FDG-PET-CT y la combinación de ceMRI y 18 F-FDG-PET-CT oscilaba del 5% al 10%. Después de la corrección por case-mix, la variación hospitalaria y de la red oncológica persistía en todas las pruebas de imagen. CONCLUSIÓN: En los Países Bajos existe una variación significativa entre hospitales y redes oncológicas respecto al uso de pruebas de imagen preoperatorias para el CRLM. El uso de MRI está aumentando, mientras que el uso de 18 F-FDG-PET-CT está disminuyendo.

Impact of nationwide enhanced implementation of best practices in pancreatic cancer care (PACAP-1): a multicenter stepped-wedge cluster randomized controlled trial.

BACKGROUND: Pancreatic cancer has a very poor prognosis. Best practices for the use of chemotherapy, enzyme replacement therapy, and biliary drainage have been identified but their implementation in daily clinical practice is often suboptimal. We hypothesized that a nationwide program to enhance implementation of these best practices in pancreatic cancer care would improve survival and quality of life. METHODS/DESIGN: PACAP-1 is a nationwide multicenter stepped-wedge cluster randomized controlled superiority trial. In a per-center stepwise and randomized manner, best practices in pancreatic cancer care regarding the use of (neo)adjuvant and palliative chemotherapy, pancreatic enzyme replacement therapy, and metal biliary stents are implemented in all 17 Dutch pancreatic centers and their regional referral networks during a 6-week initiation period. Per pancreatic center, one multidisciplinary team functions as reference for the other centers in the network. Key best practices were identified from the literature, 3 years of data from existing nationwide registries within the Dutch Pancreatic Cancer Project (PACAP), and national expert meetings. The best practices follow the Dutch guideline on pancreatic cancer and the current state of the literature, and can be executed within daily clinical practice. The implementation process includes monitoring, return visits, and provider feedback in combination with education and reminders. Patient outcomes and compliance are monitored within the PACAP registries. Primary outcome is 1-year overall survival (for all disease stages). Secondary outcomes include quality of life, 3- and 5-year overall survival, and guideline compliance. An improvement of 10% in 1-year overall survival is considered clinically relevant. A 25-month study duration was chosen, which provides 80% statistical power for a mortality reduction of 10.0% in the 17 pancreatic cancer centers, with a required sample size of 2142 patients, corresponding to a 6.6% mortality reduction and 4769 patients nationwide. DISCUSSION: The PACAP-1 trial is designed to evaluate whether a nationwide program for enhanced implementation of best practices in pancreatic cancer care can improve 1-year overall survival and quality of life. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03513705. Trial opened for accrual on 22th May 2018.

Volume-outcome relationship of liver surgery: a nationwide analysis.

BACKGROUND: Evidence for an association between hospital volume and outcomes for liver surgery is abundant. The current Dutch guideline requires a minimum volume of 20 annual procedures per centre. The aim of this study was to investigate the association between hospital volume and postoperative outcomes using data from the nationwide Dutch Hepato Biliary Audit. METHODS: This was a nationwide study in the Netherlands. All liver resections reported in the Dutch Hepato Biliary Audit between 2014 and 2017 were included. Annual centre volume was calculated and classified in categories of 20 procedures per year. Main outcomes were major morbidity (Clavien-Dindo grade IIIA or higher) and 30-day or in-hospital mortality. RESULTS: A total of 5590 liver resections were done across 34 centres with a median annual centre volume of 35 (i.q.r. 20-69) procedures. Overall major morbidity and mortality rates were 11·2 and 2·0 per cent respectively. The mortality rate was 1·9 per cent after resection for colorectal liver metastases (CRLMs), 1·2 per cent for non-CRLMs, 0·4 per cent for benign tumours, 4·9 per cent for hepatocellular carcinoma and 10·3 per cent for biliary tumours. Higher-volume centres performed more major liver resections, and more resections for hepatocellular carcinoma and biliary cancer. There was no association between hospital volume and either major morbidity or mortality in multivariable analysis, after adjustment for known risk factors for adverse events. CONCLUSION: Hospital volume and postoperative outcomes were not associated.

ANTECEDENTES: La asociación entre el volumen hospitalario y los resultados de la cirugía hepática no está clara. Según la recomendación actual de las guías holandesas se requiere un volumen mínimo de 20 procedimientos anuales por centro. El objetivo de este estudio fue analizar la asociación entre el volumen hospitalario con los resultados postoperatorios en la auditoría hepatobiliar obligatoria holandesa a nivel nacional. MÉTODOS: Se realizó un estudio a nivel nacional en los Países Bajos. Se incluyeron todas las resecciones hepáticas registradas en la auditoría hepatobiliar holandesa entre 2014 y 2017. El volumen anual del centro se calculó y se clasificó en categorías de 20 procedimientos por año. Los objetivos principales fueron la morbilidad de mayor grado (Clavien-Dindo grado IIIA o superior) y la mortalidad hospitalaria o la mortalidad a los 30 días. RESULTADOS: Se realizaron un total de 5.590 resecciones en 34 centros con una mediana (rango intercuartílico) de volumen anual de 35 procedimientos (20-69). La tasa global de morbilidad mayor fue del 11% y la mortalidad del 2%. La mortalidad fue de 1,9% después de la resección por metástasis hepáticas colorrectales (colorectal liver metastases, CRLM), 1,2% para no CRLM, 0,4% para tumores benignos, 4,9% para carcinoma hepatocelular, y 10,3% para tumores biliares. Los centros de mayor volumen realizaron más resecciones hepáticas mayores y más resecciones por carcinoma hepatocelular y cáncer biliar. En el análisis multivariable después de ajustar por factores de riesgo conocidos de eventos adversos, no se observó ninguna asociación entre el volumen hospitalario y la morbilidad o mortalidad mayor. CONCLUSIÓN: No hubo asociación entre el volumen hospitalario y los resultados postoperatorios de la cirugía hepática en los Países Bajos.

The role of abdominal drainage in pancreatic resection - A multicenter validation study for early drain removal.

BACKGROUND: Abdominal drainage and the timing of drain removal in patients undergoing pancreatic resection are under debate. Early drain removal after pancreatic resection has been reported to be safe with a low risk for clinical relevant postoperative pancreatic fistula (CR-POPF) when drain amylase on POD1 is < 5000U/L. The aim of this study was to validate this algorithm in a large national cohort. METHODS: Patients registered in the Dutch Pancreatic Cancer Audit (2014-2016) who underwent pancreatoduodenectomy, distal pancreatectomy or enucleation were analysed. Data on post-operative drain amylase levels, drain removal, postoperative pancreatic fistulae were collected. Univariate and multivariate analysis using a logistic regression model were performed. The primary outcome measure was grade B/C pancreatic fistula (CR-POPF). RESULTS: Among 1402 included patients, 433 patients with a drain fluid amylase level of <5000U/L on POD1, 7% developed a CR-POPF. For patients with an amylase level >5000U/L the CR-POPF rate was 28%. When using a cut-off point of 2000U/L or 1000U/L during POD1-3, the CR-POPF rates were 6% and 5% respectively. For patients with an amylase level of >2000U/L and >1000UL during POD 1-3 the CR-POPF rates were 26% and 22% respectively (n = 223). Drain removal on POD4 or thereafter was associated with more complications (p = 0.004). Drain amylase level was shown to be the most statistically significant predicting factor for CR-POPF (Wald = 49.7; p < 0.001). CONCLUSION: Our data support early drain removal after pancreatic resection. However, a cut-off of 5000U/L drain amylase on POD1 was associated with a relatively high CR-POPF rate of 7%. A cut-off point of 1000U/L during POD1-3 resulted in 5% CR-POPF and might be a safer alternative.

[An adrenal incidentaloma in a trauma patient; a plea for expectant management]. / Een incidentaloom van de bijnier bij een traumapatiënt; pleidooi voor een expectatief beleid.

An adrenal incidentaloma was detected in 45-year-old female driver after a car-versus-car collision. The mass, which had a diameter of 5.4 cm, was not a hormone-producing tumour, but because of its size laparoscopic adrenalectomy was performed 2 months later. Pathology examination revealed an old haematoma situated centrally in the right adrenal gland. Posttraumatic adrenal haematoma is found in 25% of autopsies of traumatized patients. Most adrenal haematomas have an ovoid appearance on CT and have a slight hyperattenuation. Follow-up CT of an adrenal haematoma shows a decrease in size and attenuation. It is therefore proposed that adrenal incidentalomas detected during trauma screening should be evaluated by repeating CT after 3 months. If the mass has diminished and its density decreased, it is most probably an adrenal haematoma, in which case unnecessary surgery may be avoided.

Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro.

We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

Hepatic gene therapy using adeno-associated virus vectors.

Recombinant adeno-associated virus vectors have been shown to safely transduce a number of tissues in preclinical animal studies. The level of gene transfer is sufficient to successfully treat a large number of medical disorders. Moreover, the long-term persistence of the vector sequences in animals makes it likely that this vector will be useful for genetic diseases requiring life-long therapy. This review outlines the biological principles of the vector, as well as its advantages and current limitations as it relates to use in hepatic gene therapy.

Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice.

Recombinant retroviral vectors are an attractive means of transferring genes into the liver because they integrate into the host cell genome and result in permanent gene expression. However, efficient in vivo gene transfer is limited by the requirement of active cell division for integration. Traditional approaches to induce liver proliferation have the disadvantage of inducing hepatocellular injury by delivery of toxins or by surgical partial hepatectomy. As a nontraumatic alternative, we show that exogenous hepatocyte growth factor (HGF) is a powerful and safe mitogen for the mature intact murine liver when delivered continuously into the portal vein. A 5-day infusion of human HGF (5 mg/kg/d) resulted in > 140% increase in relative liver mass, which returned to normal in 4 to 5 weeks. This clearly shows that an exogenous growth factor can induce robust liver proliferation in vivo. In addition, we show that the HGF-induced proliferation was independent of interleukin-6, an essential cytokine involved in liver regeneration after partial hepatectomy. When recombinant retroviral vectors were infused in combination with HGF, 30% of hepatocytes were stably transduced with no indication of hepatic injury or histopathology. These results show the ability to obtain a clinically relevant transduction efficiency with retroviral vectors in vivo without the prior induction of liver injury. The level of hepatic gene transfer achieved has the potential to be curative for a large number of genetic liver diseases.

High-efficiency retrovirus-mediated gene transfer into the livers of mice.

Recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they integrate in the host cell genome and result in permanent gene expression. However, efficient gene transfer is limited by the requirement of active cell division for integration. Surgical partial hepatectomy has been the traditional method of inducing hepatocellular proliferation, but this invasive approach would be difficult to justify in clinical gene therapy. As an alternative, we used a recombinant adenovirus expressing a nonsecreted form of urokinase plaminogen activator (Ad.PGKmuPA), which results in liver regeneration over a period of 8 days. When a high-titer retroviral vector was continuously infused into the portal vein of mice during this period of hepatocyte proliferation, 33.5% of hepatocytes were stably transduced. In addition, high-level expression of a secreted transgene reporter was sustained for at least 48 weeks (length of experiment). We investigated the influence of vector titer on the in vivo transduction efficiency in our system, and found that the total number of infectious retroviral particles delivered per target cell is a critical factor. The results presented here demonstrate the ability to obtain a high gene transfer efficiency and long-term gene expression in hepatocytes in vivo without the need for surgical hepatectomy. The two-vector system described here may be of clinical relevance, as the level of hepatic gene transfer achieved has potential to be curative for a large number of genetic liver diseases.

Method for continuous infusion into the portal vein of mice.

Recombinant retroviral vectors are attractive for in vivo gene transfer into the liver because they integrate into the host-cell genome, resulting in permanent gene expression. Gene-transfer efficiency can be improved by increasing the number of retroviral particles delivered to hepatocytes. For this purpose, we report a mouse model for continuous infusion into the portal circulation permitting large-volume vector administration, which will allow marked increase in gene-transfer efficiency. Continuous saline infusion was evaluated, using various parameters, and an infusion rate of 6 ml/24 h was found safe and well tolerated for at least 2 weeks. No significant changes in liver and kidney function and electrolyte balance were observed during the infusion. In addition to providing a valuable method for in vivo hepatic gene therapy, this model has a number of other potential applications, including mouse studies of hepatic tumor therapy, pharmacology, toxicology, and liver biology.

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

Expansion of donor hepatocytes after recombinant adenovirus-induced liver regeneration in mice.

Hepatocyte transplantation is a potential form of therapy for patients with genetic hepatodeficiency disorders. Unfortunately, hepatocellular transplantation has been limited because of the relatively low numbers of donor cells that can ultimately take up residence in the host liver. To give the donor cells a proliferative stimulus, a recombinant adenovirus vector that expresses a nonsecreted urokinase (urokinase-type plasminogen activator) was transduced into the livers of recipient animals before transplantation. Because urokinase production in hepatocytes causes the slow turnover of hepatocytes, 2 days after adenovirus-mediated gene transfer into the livers of recipient mice, 2 x 10(6) congenic donor cells tagged with beta-galactosidase (beta-Gal) reporter were implanted via the portal vein. As a result, on average, 8.6% of the recipient hepatocytes in the livers were derived from donor cells--a 20-fold increase compared with control animals in which no proliferative stimulus was present.

Adenovirus-mediated hepatic gene transfer in mice: comparison of intravascular and biliary administration.

Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues. In an attempt to restrict adenovirus-mediated gene transfer to the liver, we developed a microsurgical method that allowed for vector administration directly into the biliary tract of a mouse. We demonstrate that gene transfer was 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular infusion. Intravascular infusion of recombinant adenovirus elicits a powerful immune response that limits gene expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser immune response as determined by the lower neutralizing antibody titers directed against the vector compared with animals treated by intravascular infusion. There was no difference in the persistence of gene expression, suggesting a similar cell-mediated immune response against the vector containing cells in animals administered vector by either method. As future-generation adenovirus vectors that are safer and less immunogenic become available, the more liver specific gene transfer via the biliary tract may offer advantages over intravenous infusion for hepatic gene therapy.