P.J.K. Kuppen

Increased local cytostatic drug exposure by isolated hepatic perfusion: a phase I clinical and pharmacologic evaluation of treatment with high dose melphalan in patients with colorectal cancer confined to the liver

A phase I dose-escalation study was performed to determine whether isolated hepatic perfusion (IHP) with melphalan (L-PAM) allows exposure of the liver to much higher drug concentrations than clinically achievable after systemic administration and leads to higher tumour concentrations of L-PAM. Twenty-four patients with colorectal cancer confined to the liver were treated with L-PAM dosages escalating from 0.5 to 4.0 mg kg–1. During all IHP procedures, leakage of perfusate was monitored. Duration of IHP was aimed at 60 min, but was shortened in eight cases as a result of leakage from the isolated circuit. From these, three patients developed WHO grade 3–4 leukopenia and two patients died due to sepsis. A reversible elevation of liver enzymes and bilirubin was seen in the majority of patients. Only one patient was treated with 4.0 mg kg–1L-PAM, who died 8 days after IHP as a result of multiple-organ failure. A statistically significant correlation was found between the dose of L-PAM, peak L-PAM concentrations in perfusate (R = 0.86, P≤ 0.001), perfusate area under the concentration-time curve (AUC; R = 0.82, P< 0.001), tumour tissue concentrations of L-PAM (R = 0.83, P = 0.011) and patient survival (R = 0.52, P = 0.02). The peak L-PAM concentration and AUC of L-PAM in perfusate at dose level 3.0 mg kg–1(n = 5) were respectively 35- and 13-fold higher than in the systemic circulation, and respectively 30- and 5-fold higher than reported for high dose oral L-PAM (80–157 mg m–2) and autologous bone marrow transplantation. Median survival after IHP (n = 21) was 19 months and the overall response rate was 29% (17 assessable patients; one complete and four partial remissions). Thus, the maximally tolerated dose of L-PAM delivered via IHP is approximately 3.0 mg kg–1, leading to high L-PAM concentrations at the target side. Because of the complexity of this treatment modality, IHP has at present no place in routine clinical practice.

Isolated Hepatic Perfusion with Tumor Necrosis Factor α and Melphalan: Experimental Studies in Pigs and Phase I Data from Humans

We report our experience with isolated hepatic perfusion (IHP) with tumor necrosis factor (TNF) and melphalan in an experimental pig study and of a phase I study in humans. IHP was performed with inflow catheters in the hepatic artery and portal vein and an outflow catheter in eh caval vein. An extracorporeal venovenous bypass was used. IHP consisted of a 60-min perfusion with hyperthermia (> 41 degrees C). For the pig protocol rhTNF alpha 50 micrograms/kg alone (n = 5) or rhTNF alpha 50 micrograms/kg plus melphalan 1 mg/kg (n = 3) were added. In two control pigs no drugs were added. In the phase I study, patients received melphalan 1 mg/kg with 0.4 mg rhTNF alpha (n = 8) or 0.8 mg rhTNF alpha (n = 1). After the perfusion the liver was washed with Macrodex before vascular continuity was restored. All pigs but one survived the procedure. Systemic leakage was less than 0.02%. Transient, mild liver toxicity was seen in all pigs, including controls, as demonstrated by liver enzyme assays and histology. There was no significant hemodynamic, cardiopulmonary hematological, or renal toxicity. In the phase I clinical study there was leakage in one patient (cumulative leakage 20%). There were three perioperative deaths (one possibly drug-related). All patients demonstrated significant hepatotoxicity. Survival ranged from 6 to 26 months (median 10.3 months). All patients demonstrated a tumor response (partial response 5/6, 1/6 stable disease) with a median duration of 18 weeks. In contrast to our pig program, many problems were encountered in the phase I study. By using both the hepatic artery and portal vein for IHP we encountered more toxicity than expected based on data from the pig program, resulting in fatal coagulative disturbances in one patient who received the second rhTNF alpha dose. Furthermore, local control after one IHP with TNF alpha and melphalan is only temporary.

Isolated hepatic perfusion with high-dose melphalan for the treatment of colorectal metastasis confined to the liver†‡

Isolated hepatic perfusion (IHP) involves complete vascular isolation of the liver to allow treatment with doses that would be toxic if delivered systemically. A phase II study of IHP in patients with colorectal metastases confined to the liver was performed.

The phenotypic heterogeneity of human natural killer cells: presence of at least 48 different subsets in the peripheral blood.

Peripheral blood natural killer (NK) cells are usually defined as a homogeneous cell population. However, NK cells show heterogeneous expression of a diversity of cell surface molecules, which might reflect the diversity of NK‐cell functions. Therefore, a more specific phenotypic definition of NK cells is necessary. In this study, we made an inventory of phenotypic subsets that are present within the peripheral blood NK‐cell population of healthy donors based on differential expression of nine cell‐surface markers. Using three‐colour flow cytometric analysis we were able to define at least 48 different CD56+ NK‐cell subsets within the peripheral blood. This phenotypic heterogeneity appeared to be stable among healthy individuals, and was also steady within CD56dim and CD56bright NK populations, indicating a possible role for these subsets in NK‐cell function or differentiation.

Assessment of DNA methylation status in early stages of breast cancer development.

Background:Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development.Methods:To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARβ2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed.Results:Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARβ2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics.Conclusion:DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.

Near-Infrared Fluorescence Imaging in Patients Undergoing Pancreaticoduodenectomy

Background: Intraoperative visualization of pancreatic tumors has the potential to improve radical resection rates. Intraoperative visualization of the common bile duct and bile duct anastomoses could be of added value. In this study, we explored the use of indocyanine green (ICG) for these applications and attempted to optimize injection timing and dose. Methods: Eight patients undergoing a pancreaticoduodenectomy were injected intravenously with 5 or 10 mg ICG. During and after injection, the pancreas, tumor, common bile duct and surrounding organs were imaged in real time using the Mini-FLARE™ near-infrared (NIR) imaging system. Results: No clear tumor-to-pancreas contrast was observed, except for incidental contrast in 1 patient. The common bile duct was clearly visualized using NIR fluorescence, within 10 min after injection, with a maximal contrast between 30 and 90 min after injection. Patency of biliary anastomoses could be visualized due to biliary excretion of ICG. Conclusion: No useful tumor demarcation could be visualized in pancreatic cancer patients after intravenous injection of ICG. However, the common bile duct and biliary anastomoses were clearly visualized during the observation period. Therefore, these imaging strategies could be beneficial during biliary surgery in cases where the surgical anatomy is aberrant or difficult to identify.

The microscopic anatomy of experimental rat CC531 colon tumour metastases: consequences for immunotherapy?

The colon adenocarcinoma cell line CC531 was adopted as a model for immunotherapeutical treatment of experimental colorectal metastases in a syngeneic rat model. We studied the presence and localization of T and natural killer cells, vessels and matrix proteins in in vivo growing CC531 tumours by immunohistochemistry. CC531 tumours were induced either in the lungs by injecting CC531 tumour cells into a tail vein or in the liver by injection of CC531 tumour cells under the liver capsule or into a mesenteric vein. All 3 tumour types were composed of islets of tightly apposed tumour cells surrounded by abundantly present tumour-stroma which contained tumour vessels and matrix proteins. Some of these matrix proteins, especially laminin and collagen IV formed a basal membrane-like structure around the tumour nodules. This structure was most pronounced in mesenteric vein-induced liver tumours and less prominent in subcapsular-induced liver tumours and tail vein-induced lung tumours. Tumour-infiltrating lymphocytes of both T and natural killer cell origin were found in the tumours, but predominantly in the tumour stroma, separated from the islets of tumour cells by the basal membrane-like structure. We hypothesize that the matrix proteins of these tumours play an ambivalent role: they may provide a substratum for migration of effector cells into the tumour stroma but may also provide a barrier preventing direct contact between tumour target cells and immune effector cells.

Caspase-3 Activity Predicts Local Recurrence in Rectal Cancer

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy. Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay. Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001). Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

The prognostic role of TGF-β signaling pathway in breast cancer patients

BACKGROUND The transforming growth factor-β (TGF-β) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-β signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-β signaling biomarkers. PATIENTS AND METHODS The expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-β receptors I and II (TβRI and TβRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-β receptors. RESULTS Tumors with high expression of TβRII, TβRI and TβRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-β receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390-6.658] and 2.20 (95% CI 1.464-3.307), respectively, for disease relapse. CONCLUSIONS Combining TGF-β biomarkers provides prognostic information for patients with stage I-III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.BACKGROUND The transforming growth factor-β (TGF-β) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-β signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-β signaling biomarkers. PATIENTS AND METHODS The expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-β receptors I and II (TβRI and TβRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-β receptors. RESULTS Tumors with high expression of TβRII, TβRI and TβRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-β receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390-6.658] and 2.20 (95% CI 1.464-3.307), respectively, for disease relapse. CONCLUSIONS Combining TGF-β biomarkers provides prognostic information for patients with stage I-III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.

Epigenetic status of LINE-1 predicts clinical outcome in early-stage rectal cancer

Background:We evaluated the clinical prognostic value of methylation of two non-coding repeat sequences, long interspersed element 1 (LINE-1) and Alu, in rectal tumour tissues. In addition to DNA methylation, expression of histone modifications H3K27me3 and H3K9Ac was studied in this patient cohort.Methods:LINE-1 and Alu methylation were assessed in DNA extracted from formalin-fixed paraffin-embedded tissues. A pilot (30 tumour and 25 normal tissues) and validation study (189 tumour and 53 normal tissues) were performed. Histone modifications H3K27me3 and H3K9Ac were immunohistochemically stained on tissue microarrays of the study cohort.Results:In early-stage rectal cancer (stage I-II), hypomethylation of LINE-1 was an independent clinical prognostic factor, showing shorter patient survival (P=0.014; HR: 4.6) and a higher chance of tumour recurrence (P=0.001; HR: 9.6). Alu methylation did not show any significant correlation with clinical parameters, suggesting an active role of LINE-1 in tumour development. Expression of H3K27me3 (silencing gene expression) and H3K9Ac (activating gene expression) in relation to methylation status of LINE-1 and Alu supported this specific role of LINE-1 methylation.Conclusion:The epigenetic status of LINE-1, but not of Alu, is prognostic in rectal cancer, indicating an active role for LINE-1 in determining clinical outcome.