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The aim of this study was to investigate the immunological needle (Integra, Rätingen, Germany) advanced into the
features of HCC and CRC metastases patients 12 months tumor. Midazolam (0.5-5 mg) and/or pethidine (25-100 mg)
after RFA. were administered intravenously as necessary. Patients were
monitored by pulse oximetry during the whole procedure.
METHODS
Two mm (14 G) diameter RFA needle applicators and
The study was approved by the Ethical Committee of 15 mm active electrode with microbores were used. During
the University of Erlangen-Nuremberg (Ethikkomitee der HF application (40 W power output) the RFA needle was
Universität Erlangen Nürnberg) and performed according continuously perfused with isotonic saline via the bore
to the declaration of Helsinki. All treated HCC and CRC holes. RF energy was delivered by a computer-assisted
®
metastases were confirmed histologically prior to therapy. radiofrequency generator (Elektrotom 106 HF , Integra,
Rätingen, Germany) and continuous perfusion of the RFA
Selection and description of participants needle was secured by a syringe pump (Pilot C, Fresenius
All enrolled patients took part in a prior trial in which baseline Medical Care, Alzenau, Germany) linked to the RF generator.
data before RFA treatment were recorded according to the Perfusion was adjusted according to impedance by
present protocol. means of an electronic interface between generator and
perfusor, automatically increasing in response to a rise in
Patients with up to 3 tumor nodules within the liver, with impedance (> 400 Ohm). The RF energy was applied for
a maximum diameter of 6 cm per lesion, were enrolled in 10-15 min at each needle position, leading to a coagulation
the study. Prior local ablative therapy (LiTT, RFA, ethanol zone of 30-35 mm in diameter. Tumors larger than 20 mm
injection) or prior chemo-embolization of the malignant were targeted using different applicator positions to create
liver tumor was an exclusion criterion. The possibility overlapping coagulation zones, in order to treat the entire
of curative treatment by resection had to be ruled out. lesion with a safety margin > 5 mm. Larger tumors were
Therefore, all cases were discussed in our tumor conference, treated with up to 3 simultaneous needle insertions arranged
including gastroenterologists, oncologists, surgeons, and in a triangle (2-4 cm) or square (for larger tumors).
radiologists.
Interferon gamma secretion assay and lymphocyte staining
Patients with at least one of the following findings were also Heparinized blood (LI-Heparin 10 mL) was collected
excluded: Karnofsky index < 60, thrombocytes < 50,000/μL, 12 months after RFA. The samples were stored at 4 °C and
prothrombin activity < 50%, partial thromboplastin time > 80 s. tests performed within 12 h after sampling. A liver biopsy
No transfusion of platelets or fresh frozen plasma was performed. of normal and tumor tissue was collected from every patient
Informed consent was obtained from every patient no later directly before RFA and stored at -20 °C.
than 24 h before treatment.
Tissue-lysates were freshly prepared in cold phosphate
The size and number of tumor nodules were determined by buffer (50 mmol/L, pH 7.2) using a glass homogenizer as
ultrasonography and by computed tomography (CT) (dynamic described. The suspension was filtered with a filter tip (pore
[6]
spiral CT with intravenous application of contrast medium) size 1.2 mm) to adjust the fragment size to < 1.2 mm. The
prior to RFA. protein concentration was measured spectrophotometrically
using the Bradford assay, and adjusted to 1 mg/mL.
Five consecutive male patients with 8 tumor nodules in
total (CRC patients with 2 nodules each and HCC patients Autologous test antigens (normal and tumor lysate, 12.5 mg)
with 1 nodule) who met the inclusion criteria were enrolled. were added to 250 μL heparinized blood and cultured in a
Mean patient age was 64 years (range: 59-74 years). Three 15 mL conical polypropylene tube for 16 h at 37 °C under
patients suffered from CRC metastases to the liver while 2 5% CO . A negative control without the addition of antigen
2
suffering from HCC. lysate was included, while staphylococcal enterotoxin
B served as positive control antigen. Thereafter, the
Radiofrequency ablation technique samples were put on ice and washed with ice cold washing
The whole procedure was performed under ultrasound solution [phosphate buffer saline containing 0.5% bovine
guidance (Elegra Advanced , Siemens, Erlangen, Germany) serum albumin and 2 mmol/L ethylenediaminetetraacetic
®
under sterile conditions. The proposed puncture site acid (EDTA), pH 7.4] and the cell suspension centrifuged at
was infiltrated with a local anesthetic (2% mepivacaine 300 g for 10 min at 4 °C. The cell pellet was resuspended
hydrochloride) and the perfused radio-frequency (HF) with 80 μL ice cold culture medium (RPMI1640 containing

Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015 93