The patients were treated with 400 mg SOR b.i.d., if they had been classified with advanced or stage C disease according to the Barcelona Clinic Liver Cancer (BCLC-C) staging system (n = 89) [9] or if they had disease progression after surgical or locoregional therapies (BCLC-A, n = 1 or BCLC-B, n = 24). SOR treatment was continued until clinical progression, unacceptable toxicity level or wish of the patient.

The study included patients with HCC confirmed by appearance of a focal liver nodule in a cirrhotic liver with EASL criteria in dynamic techniques [9] (n = 75). Tumor histology was obtained in cases of diagnostic uncertainty (n = 39), including patients with normal liver, chronic hepatitis and atypical images on Computarized Tomography and/or Magnetic Resonance Imaging (CT/MRI).

We considered patients with an ECOG scale status ≤ 2, Child-Pugh A or B rating < 8, life expectancy of at least 12 weeks, adequate hematologic function (hemoglobin ≥ 8.5 g/dL) and normal prothrombin time (INR ≤ 2.3, prothrombin time ≤ 6 seconds), acceptable liver function (albumin ≥ 2.8 g/dL, total bilirubin ≤ 3 mg/dL; alanine aminotransferase and aspartate aminotransferase ≤ 5 times the normal), and adequate renal function (serum creatinine ≤ 1.5 times the normal). Patients with chronic hepatitis B were treated with protease inhibitors (tenofovir/entecavir). Hepatitis C treatment was not given simultaneously with sorafenib. Patients coinfected with HIV were included.

All patients with liver cirrhosis (n = 96) were compensated; if they presented ascites, it was controlled with diuretics and a salt-free diet. All patients had a gastroscopy before the treatment; in cases of risk of esophageal varices bleeding, preventive treatment was performed according to the care protocol.

The patient had not received previously systemic treatment with other targeted therapies or chemotherapy. Patients who received SOR within a clinical trial or combined with radioembolization (Y90) or stereotactic radiotherapy were excluded. Patients with AFP measurements outside the reference laboratory and patients who received SOR for HCC recurrence in the post-transplant setting were also excluded.

All patients signed an informed consent form before beginning of treatment. The Ethics Committee, according with the provisions of the Good Clinical Practice Guidelines, the Declaration of Helsinki and local laws, approved the analysis.

Serum AFP was prospectively collected at baseline and every 4 weeks during SOR treatment, using microparticle enzyme immunoassay (Abbott Laboratories, Chicago, I); the upper normal limit is 10 ng/mL.

Patients were evaluated at 2, 4, 8, 12 and every 8 weeks later on. In addition patients had free access to medical team as frequent as needed in case of adverse effects (AEs) or serious AEs (SAEs).

Each assessment included a detailed medical interview; physical examination; assessment of the patient’s toxicity record; AFP measurement; blood count; biochemical, hydroelectrolytic, liver, urine sediment and thyrotropin-stimulating hormone analyses.

Tumor radiological evaluation was performed at 12 weeks and every 16 weeks thereafter; the first assessment point was chosen to select a moment before the median time to radiological progression [2] and before the median time to clinical progression [2] . A multiphase computed tomography was used to assess baseline lesions and determine the tumor response according to RECIST criteria [23] .

The radiological response was classified by RECIST criteria: complete response (CR), partial response (PR), stable disease (SD), and progression (P). Patients were managed with clinical, analytical, and imaging assessments until to exit the study. Follow-up was continued until the patient’s death.

SOR treatment was continued until the disease progressed clinically, defined by Eastern Cooperative Oncology Group (ECOG PS 3-4) (n = 47), unacceptable toxicity was observed (n = 53), or patient’s desire (n = 3). All patients were followed until the end of the study. Only one patient received second-line treatment, and in this case survival was censored at the moment of starting it.

The aim of the study was to evaluate if AFP variation during SOR reflects the absence of HCC progression and its potential use as a prognostic factor.

The secondary objectives were:

a) Overall survival (OS), defined as the time between the start of SOR treatment and death from any cause, last follow-up or beginning of a second-line therapy.

b) Identification of prognostic factors associated with OS, considering clinical and analytical variables at baseline and during 90 days;

c) Analysis of toxicity during SOR, measured according to the common toxicity criteria of the U.S. National Institute of Cancer or Common Terminology Criteria for Adverse Events (CTCAE), version 3.0.

a) In patients with normal AFP (<10 ng/mL), TPA was defined as the time between the start of SOR and the point at which AFP increases higher than 20 ng/mL; that is, when it reaches twice the upper normal limit [17] - [20] . We assume that an increase lower than 20 ng/mL may result from variations due to underlying liver biology.

b) In patients with high AFP (>10 ng/mL), TPA was defined as the time between the start of SOR and the point at which AFP rises above the nadir. In clinical practice, we have observed that when AFP rises, this increase occurs exponentially.

The cutoff defined as TPA was calculated considering patients with high AFP, and it was applied to the entire cohort. After analyzing this time in tertiles, a TPA of 90 days was selected as the variable to be evaluated as a prognostic factor, because comprised early and late SOR progressions.

Patients were classified in: Group 1, poor prognosis, if they met the above criteria before day 90 and Group 2, good prognosis, if by day 90 this condition had not been met. Thus, after measuring the TPA, survival time begins at the time when the patient is classified as Groups 1 or 2 (Figure 1).

The Kaplan-Meier method was used to calculate the overall survival, time to progression, TPA, and cumulative survival rate. Differences were determined using the log-rank test. The clinical variables and treatment responses of the two groups (TPA < 90 or TPA > 90 days) were compared using X², Fisher’s exact test, or independent t- tests. Statistically significant factors in the univariate analysis were estimated using the multivariate Cox proportional hazard regression models. Statistical significance was defined as a P value < 0.05. Data were analyzed using SPSS v 14.0 software (SPSS, Chicago, IL, USA) and R (http://www.r-project.org/index.html; from Bell Laboratories, formerly AT & T, now Lucent Technologies).

In addition to developing the TPA concept, we evaluated the Shao [18] and Personeni [20] model in our cohort to determine its applicability and predictive capacity in our patients. To calculate the time to radiographic progression (RECIST criteria) and the time to clinical progression, the combined event progression/death was considered, and Kaplan-Meier analysis was applied.

Between July 2007-October 2012, 114 patients were included (mean age: 64 years, 97 males, 96 with cirrhosis).

The etiologies of liver disease were as follows: alcohol, 47 (41%); hepatitis C virus (HCV), 31 (27%); mixed (HCV plus alcohol), 13 (11%); hepatitis B virus, 1 (0.9%). The liver cancer stages were classified according to the BCLC: BCLC-A, 1; BCLC-B, 24; BCLC-C, 89. The Child-Pugh classification was Class A in 89 cases. The general condition according to ECOG was 0 in 73 cases. The median baseline AFP was 29.3 ng/mL (interquartile range P₂₅ - P₇₅: 7.7 - 217.5). The details of population’s characteristics are shown in Table 1.

Treatment with SOR was maintained for a median of 5 months (95% CI 3.47 - 6.53; mean 9.6 months). The daily maintenance dose was 800 mg in 26 patients (22.8%), 600 mg in 12 patients (10.5%), 400 mg in 47 patients (41.2%), and 200 mg in 5 patients (4.4%). In 24 patients (21%), SOR was suspended before a maintenance dose was reached due to a serious AE.

SOR was temporarily reduced in response to grade 2 AE in 78 patients (68%), and was temporarily discontinued in 52 patients (45%) because of a grade 3-4 AE or decompensation of liver cirrhosis. The main cause of permanent interruption of SOR was tumor progression (47 patients, 41%).

Ninety-four serious AE (grade 3-4) were observed in 78 patients (67%). In addition, 24 patients showed a second serious AE, and 5 had a third serious AE. The most common toxicities were fatigue (80%) and diarrhea (78%). There were 3 deaths potentially related to SOR: 1 death at home without autopsy, 1 intracranial hemorrhage, and there was one death preceded by a hypertensive crisis. The details of 3-4 grade toxicities recorded during follow-up are presented in Table 2.

AFP was elevated during SOR treatment in 83 patients. AFP values did not increase in the first 3 months in 24 patients. Follow-up was less than 3 months in 7 patients, so they were excluded from the analysis. Mean time to progression of AFP was 150 days (95% CI 102 - 199 days). We grouped the TPA in tertiles and arbitrarily decided to split the study population (n = 107) into two groups according to the following characteristic: those who had elevated AFP before the 90^th day after SOR (Group 1, TPA < 90, n = 53) versus those who did not have elevated AFP plus those that had elevated AFP after the 90^th day after SOR (Group 2, TPA ≥ 90, n = 54). There were no differences in survival between those who did not have elevated AFP and those with late AFP elevation (P = 0.964). The applicability of the proposed primary endpoint was 93.9% (107/114).

We analyzed the differences between baseline characteristics of patients of Groups 1 and 2 (Table 1). The former were younger (P = 0.008) with higher baseline AFP levels (P < 0.001), impaired hepatocellular function (Child-Pugh stage, P < 0.001; MELD, P = 0.039), worse baseline ECOG (P = 0.004), advanced BCLC stages (P = 0.036), increased presence of vascular invasion (P = 0.02), and fewer prior treatments (P = 0.018). The median time to radiographic progression combined with overall survival was 4 months (IC 95% 3.86 - 6.55) in Group 1 and 8 months (IC 95% 6.81 - 12.82) in Group 2 (P = 0.006). The Group 1 had a shorter time to radiographic progression and decreased survival.

At the end of the study 101 patients died and 13 patients were alive; 5 of them continued with SOR. The median time to clinical progression was 5 months (P₂₅ - P₇₅: 3 - 15 months), and the median time to radiographic progression applying RECIST criteria was 4 months (P₂₅ - P₇₅: 3 - 9.25 months). Median OS was 9.3 months (7.31 - 11.28, 95% CI; Figure 2). Statistically significant predictors of OS in the Cox proportional hazard model were: age, Child-Pugh stage, ECOG PS, BCLC stage, and the vascular invasion at time of starting with SOR (Table 3(a)). Additionally, belonging to TPA Groups 1 or 2 was an independent prognostic factor. Body mass index, metastasis, sex, baseline AFP level, grade 2-4 hypertension during the first 3 months, and grade 2-4 skin toxicity during the first 3 months were not predictors of OS. After multivariate analysis with stepwise inclusion (forward likelihood), only the variables ECOG and TPA entered the model (Table 3(b)).

The median OS values according to TPA < 90 days (Group 1) and >90 days (Group 2) were 8.10 months and 18.85 months (P < 0.001; Figure 3). These medians were 5, 4 and 15 months from classification in Groups 1 and 2, respectively. As Table 4 shows, the TPA criterion is a survival prognostic factor that retains its value independent of the adjustment for baseline covariates, such as age, sex, baseline ECOG, baseline Child-Pugh stage, and baseline BCLC, with an HR of 2.422 (1457 - 4029, 95% CI), P = 0.001.

Model 1: + Age + Sex; Model 2: + Model 1 + ECOG + Child-Pugh + BCLC.