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Lugaresi et al. Hepatoma Res 2018;4:67 I http://dx.doi.org/10.20517/2394-5079.2018.88 Page 3 of 9

cells suspended in 5 saline mL with 250U of IL-2 was connected to a pump for micro-injections (0.5 mL/min):
the infusion lasted 10-20 min. The patients were also i.m. administered with chlorphenamine maleate (GSK,
Brantford, UK) and ranitidine (GSK, Brantford, UK) 1 h before the treatment, in order to block H1 and H2
lymphocytes receptors and reduce possible side effects.

For evaluating the impact of the treatment on the tumour mass we adopted Response Evaluation Criteria in
Solid Tumours criteria, a well known simple and pragmatic methodology to evaluate the activity and efficacy
[16]
of therapies towards tumours . In addition every three months we evaluated 12 biochemical parameters on
the peripheral venous blood of the patients, i.e., alanine-amino-transferase (ALT), aspartate-amino-transferase,
gamma-glutamil-transferase (GGT), bilirubin (BIL), alkaline phosphatase (ALP), α-fetoprotein (AFP), platelets,
white blood cells, total plasmatic proteins, albumin, prothrombin time, creatinine (Cr). The minimal acceptable
response to the therapy was defined as an improvement of at least 7 of these biochemical parameters. Finally
we compared the survival rate of the treated patient to that of the non-treated patients (control group).

The present study was conducted in accordance with the ethical standards of the Helsinki Declaration (1964,
amended most recently in 2008) of the World Medical Association. The local Institutional Review Board
approved the use of the database for this retrospective review of the case files. Each patient provided written
consent, and all patient information, including illustrations, were anonymous.

Statistical analysis
2
Data are represented as mean (range) for continuous variables and as n (%) for categorical variables. The χ test
or Fisher’s test and the Student’s t test were used to analyse categorical and continuous variables.

Survival analysis was performed using the Kaplan-Meier method and the log-rank test. P-values < 0.05 were
considered significant. Data were analysed using SPSS (version 15.0) (SPSS Inc., Chicago, IL, USA).

RESULTS
In Table 1 are reported and compared the main clinical and pathological characteristics of treated patients
and control group. There were no statistically significant differences between the two groups. Among the
26 patients enrolled in our study, ten patients completed 12 therapy cycles, six received 6 infusions and ten
patients underwent only 3 or 4 procedures.

Twelve patients showed a partial response to the therapy that is amelioration of at least 7 out of 12 biochemical
parameters considered [Figure 1]. Moreover, all the patients who completed the 12 cycles showed an
improvement in 9 or more of the analysed parameters [Figure 1].

All parameters, but ALP and GGT, either improved or remained stable in more than 50% of the cases [Figure 2].

The regression of the neoplastic mass was not evident at the imaging studies in neither group, but in the treated
group we observed 34% of patients with stability after 12 cycles and 0% of stability in the other patients treated
with ≤ 6 cycles of immunotherapy.

The survival rate was measured from the beginning of the therapy, and analyzed with the Kaplan-Meier curve.
The difference between the treated group and the control group was calculated with log-rank test and found to
be statistically significant (P < 0.0001). The 1-year survival was 0% in the control group vs. 50% in the treated
group [Figure 3].

A striking difference (even though not statistically significant due to small numbers of the groups) can be
noted between the group of patients who completed the 12 cycles and those with < 6 cycles; 1-year survival was