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Page 2 of 9 Lugaresi et al. Hepatoma Res 2018;4:67 I http://dx.doi.org/10.20517/2394-5079.2018.88

the small number of patients. All patients with 12 completed cycles showed an improvement of 9 parameters or
more.

Conclusion: Endolymphatic administration of immunotherapy appeared safe, easy to perform and effective in
terms of survival. This study should encourage future large scale studies in order to reach a firmer conclusion and
define uniform inclusion criteria.

Keywords: Hepatocellular carcinoma, endolymphatic, immunotherapy, survival

INTRODUCTION
Hepatocellular carcinoma (HCC) is the fifth most common malignancy globally and the third leading cause of
malignancy - related mortality worldwide. The incidence of HCC is still higher in some African and Eastern
[1,2]
Asian regions. This cancer represents 3%-6% of all solid tumours in the USA and Europe . Hepatitis B
virus (HBV)/hepatitis C virus (HCV) infection and alcohol abuse seem to be the main causes of the spread
[3]
of HCC in Western countries . Despite the established efficacy of screening programs for at-risk individuals,
the diagnosis is usually performed at later stages of disease, wherein the tumour characteristics or liver
[4,5]
disease progressions do not allow for curative therapeutic approach . Many criteria have been proposed for
the staging of HCC, combining different prognostic factors. The current treatment of HCC is based on the
[6,7]
Barcelona Clinic Liver Cancer (BCLC) classification including stages of the disease, macroscopic features
of the lesion and liver function parameters as identified by Child-Pugh scoring system. Curative surgical
[8,9]
treatment appears suitable in 30%-35% of all diagnosed cases , therefore much effort is directed towards new
therapeutic agents. Encouraged by the good results obtained from treating metastatic renal cell carcinoma with
[10]
immunotherapy , we offered the same procedure, with palliative intent, to patients with advanced disease
who were not eligible either for hepatic resection or for percutaneous ablation based on BCLC classification
[12]
[11]
obtaining interesting preliminary results before approval of a new drug for treatment of HCC . Sorafenib®
[13]
is the only approved drug for patients with advanced HCC but has shown limited activity . It acts as a
multikinase inhibitor suppressing cell proliferation and angiogenesis. Recently it has been reported that other
oncogenic targets may contribute to the anti-proliferative activity of the drug [14,15] . Herein we report the results
of our pilot study in a cohort of patients with HCC in the pre-terminal stage who were not suitable for any
curative interventions, before Sorafenib® - period.

METHODS
From January 2003 to March 2009, 39 patients with advanced HCC were enrolled in our study. Among these,
26 underwent at least 3 cycles of immunotherapy, but only 16 who completed at least 6 cycles were able to
evaluate the efficacy of the treatment. In 13 patients the treatment was interrupted before the third cycle
because of local skin reaction (n = 1), early death (n = 2) and worsened clinical conditions (n = 10). An historical
control group is represented of 15 patients with similar characteristics of advanced HCC who underwent
standard therapy without immunotherapy. The protocol of the immunotherapy which was already reported
[11]
6
6
by our group consisted in monthly endolymphatic infusions of 1.5 × 10 -3.0 × 10 autologous activated
lymphocytes (LAK) and 250IU of IL-2. Lymphocytes were obtained through the centrifugation of 30 mL of
the patients’ peripheral blood on a Ficoll-Hypaque gradient. The lymphocytes were then suspended in Roswell
6
Park Memorial Institute-1640 (Sigma Aldrich, Germany) 2 × 10 /mL and incubated with 20 U/mL of IL-2
at 37 °C for 72 h. After the incubation the cells were washed with saline solution and suspended in 5-10 mL of
saline solution containing 250IU of IL-2.

Surgical procedure consisted of three steps. Firstly, the lymphatic vessels on the back of the foot was identified
using the standard lymphographic technique (subcutaneous injection of violet patent blue between two finger).
Then the main lymphatic was isolated and cannulated with a needle catheter (27G). A syringe containing the