Treatment strategy of adding transcatheter arterial chemoembolization to sorafenib for advanced stage hepatocellular carcinoma

Abstract Background Therapeutic effect and immunosuppressor cell alteration in adding transcatheter arterial chemoembolization (TACE) to sorafenib for advanced stage hepatocellular carcinoma (HCC) remain unclear. Aims To examine the therapeutic effect and immunosuppressor cell alteration in adding TACE to sorafenib. Methods Forty‐four advanced stage HCC patients were divided into group A (n = 17) treated by sorafenib (400‐600 mg/day) alone and group B patients (n = 27) treated by sorafenib and TACE. The frequency of regulatory T‐cells and myeloid‐derived suppressor cells (MDSC), and patients' outcomes were examined. Advanced HCC patients' survival was improved by adding TACE to sorafenib if N/L was reduced from ≥2.5 to <2.5 by TACE. Results The median (interquartile) follow‐up for all patients was 8.5 (3.5 to 15.5) with a range from 1 to 71 months. The median (interquartile) survival was 5.0 (2.3‐11.3) months for group A and 11.0 (5.0‐19.0) months for group B patients (P = .024). In group A, the patients (n = 8) with neutrophil‐to‐lymphocytes ratio (N/L) < 2.5 had better survival than the patients (n = 9) with N/L ≥ 2.5 (P = .006). In group B, 6 of 13 patients with N/L ≥ 2.5 had N/L reduction to <2.5 after combination therapy of sorafenib and TACE, and their 6‐month, 1‐year and 2‐year survival were improved (P = .013). For immune cell examination, the frequency of CD4+ and CD8+ T‐lymphocytes, regulatory T‐cell and MDSC were not altered by sorafenib treatment. However, actual number of lymphocytes had a tendency to increase (from 978.5 ± 319.4/mm3 prior to treatment to 1378.0 ± 403.3/mm3, P = .086) for the patients with N/L reduction. Conclusion Immunosuppressor cells were not altered by sorafeinb. Patients' survival was improved if N/L ≥ 2.5 was reduced to <2.5 by TACE.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the liver. HCC in early stage is silent and difficult to be found unless the patients with chronic liver diseases are regularly screened for liver tumors. Therefore, HCC may be already in advanced stage once upon the tumors are found. Even HCC is found in early stage and treated by liver resection or radiofrequency ablation, HCC is easy to recur and progress into advanced stage. 1,2 Therefore, HCC is the fifth most common malignancy in the world, but is the third common cause of cancer death. 3,4 How to treat advanced stage HCC and prolong patients' survival is more crucial than the treatments for early stage HCC.
When HCC is advanced with portal vein thrombus or extrahepatic spread and patients' Eastern Cooperative Oncology Group (ECOG) performance status is 1-2, systemic treatment is recommended in accordance with Barcelona Clinic Liver Cancer (BCLC) staging and treatment strategy. 5 In Hong Kong cancer staging, HCC with portal vein thrombus or extrahepatic spread is stage IVa and the therapy is the same as BCLC staging. 6 Sorafenib is the first molecular targeting drug approved to treat advanced stage HCC. In the Sharp and the Asia-Pacific randomized clinical trials, sorafenib was applied to treat advanced stage HCC and overall survival could be prolonged for 2.7-2.8 months. 7,8 However, the median overall survival time in these two studies was only 6.5-10.7 months. How to further prolong survival of advanced stage HCC patients is challenging.
Multimodality therapy may be one of the options for advanced stage HCC treatment. Based on intension-to-treat, advanced stage HCC patients may receive sorafenib or immunotherapy for systemic treatment and transcatheter arterial chemoembolization (TACE) for intra-liver tumor local treatment. [9][10][11] Radiotherapy may also be added for intravascular tumor-thrombus treatment. Whether local therapy such as TACE yields clinical benefit for advanced stage HCC remains to be determined.
Immunity is known to be important for cancer treatment. Alternation of immune system in the patients with large HCC tumor burden is already known. CD4 + CD25 + Foxp3 + regulatory T (Treg) cells are immunosuppressive cells and stay in cancer 12,13 .
HLA-DR − CD33 + myeloid-derived suppressor cells (MDSC) is another immunosuppressive cell of myeloid origin and characterized by production of reactive oxygen, nitrogen species and arginase I to suppress immunity. [14][15][16] In this study, we would examine the alteration of immune cells and clinical outcomes while advanced stage HCC was treated by sorafenib with or without TACE.

| TACE
Under adequate local anesthesia, an angiographic sheath was inserted into right femoral artery as a working channel for angiographic catheter.
The angiographic catheter was threaded into celiac trunk and common hepatic artery to perform angiographic study of liver tumors. Then, the tumor-located segmental arteries were cannulated superselectively with 3 French coaxial micro-catheter and embolized by emulsion of lipiodol mixed with 10-20 mg adriamycin (20 mg epirubicin) or elution beads loading with adriamycin. TACE was performed every 2-3 months unless the liver function was deteriorated to Child-Pugh score >8 or the tumors were already well-embolized.

| Survival time
The patients were followed up at outpatient clinic every month after sorafenib was prescribed. Dynamic computed tomography (CT) of the liver was performed every 3 months to see the tumor status. Overall  Figure 1). This result showed that the patients treated by a combination of sorafenib and TACE had better survival rate than the patients treated by sorafenib alone.

| Survival rates in accordance with neutrophil to lymphocyte ratio
In this study, 2.5 of N/L was applied as the cutoff point to further divide group A and B patients into subgroups. 17 In

| Clinical significance of neutrophil to lymphocyte ratio reduction
To determine why the patients with N/L ≥ 2.5 in group B had a better survival than the patients with N/L ≥ 2.5 in group A, the alternation of respectively, which were much better than 57.1%, 28.6%, 14.3% and 0%, respectively, for the other 7 patients without N/L reduction (P = .013, Figure 3). Therefore, the patients with N/L ≥ 2.5 in group B having better survival than in group A was due to 46.2% of group B patients having N/L reduction after sorafenib combined with TACE treatment.   Nevertheless, TACE is an invasive procedure and patients may be suffered from the adverse effects of TACE. Because advanced stage HCC patients are already sick, it is better to choose patients who may get benefits from TACE to perform TACE. In accordance with the results of this study, there were no survival difference between group A and group B patients whose N/L was <2.5; that is, TACE in group B patients with N/L <2.5 did not showed clinical benefits. For the patients with N/L ≥2.5 prior to treatment, there was no patients in group A having N/L reduction after a-month of sorafenib treatment; in the other hand, almost half of the patients in group B had N/L reduction and their survival was improved. Undoubtedly, TACE can be applied to advanced stage HCC patients whose N/L was ≥2.5, and the survival will be improved if N/L can be reduced. The suggested treatment strategy is showed in Figure 4.
Neutrophil to lymphocyte ratio (N/L) could be applied as a predictor of prognosis and a guidance of performing TACE for advanced stage HCC patients. When 2.5 of N/L was employed as the cutoff point for HCC patients, 17 the patients in group A with N/L below 2.5 had significantly better survival rate than the patients with N/L ≥ more than 2.5. In group B patients, 6 (46.2%) patients with high levels of N/L had N/L reduction after combination therapy of sorafenib and TACE, and their survival was improved. N/L reflects the systemic inflammation status. Chronic inflammation is a risk factor of cancers and also a significant component of tumor progresssion. 21,22 The development of HCC are frequently related to hepatitis B, hepatitis C or alcohol-associated inflammation or cirrhosis. As anti-cancer immunity is mainly mediated by T-lymphocytes, N/L may be used as a surrogate of hosts' immunity. In the literature, N/L was reported as a predictor of prognosis of hepatectomy for HCC, liver transplantation for HCC or even other treatments for HCC. [23][24][25] This study confirmed that reduction of N/L from high levels could be employed as a well sign to indicate the treatment efficacy of sorafenib combined with TACE.

CONFLICT OF INTEREST
There is no conflict of interest among authors.

ETHICS STATEMENT
All the patients signed informed consent of providing blood samples for immune cell analysis and clinical research. This study was confirmed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by institutional review board of Chang-Gung Memorial Hospital (IRB No. 101-3552B).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author, W.-C. Lee, upon reasonable request.