AFP — Α-fetoprotein
APC — Antigen-presenting cell
CD — Cluster of differentiation
CTA — Cancer testis antigen
CTL — Cytotoxic T lymphocyte
CTLA — Cytotoxic T lymphocyte-associated antigen
DC — Dendritic cell
FGF — Fibroblast growth factor
GM-CSF — Granulocyte-macrophage colony-stimulating factor
GPC3 — Glypican-3
HCC — Hepatocellular carcinoma
hTERT — Human telomerase reverse transcriptase
ICAM — Intercellular adhesion molecule
IDO — Indoleamine dioxygenase
IFN — Interferon
IL — Interleukin
LFA — Lymphocyte function-associated antigen
LSEC — Liver sinusoidal endothelial cells
MDSC — Myeloid-derived suppressor cells
MHC — Major histocompatibility complex
NK — Natural killer
PBMC — Peripheral blood mononuclear cells
PD-1 — Programmed death receptor 1
PD-L1 — Programmed death-1 ligand
PG — Prostaglandin
RFA — Radiofrequency ablation
TAA — Tumor-associated antigen
TACE — Transarterial chemoembolization
TGF — Transforming growth factor
TIL — Tumor-infiltrating lymphocytes
TLR — Toll-like receptor
TNF — Tumor necrosis factor
Treg — T regulatory cells
VEGF — Vascular endothelial growth factor
Гепатоцеллюлярная карцинома (HCC) становится все более важной причиной рак-ассоциированной смертности в Соединенных Штатах. В настоящее время это третья ведущая причина смерти от рака во всем мире, приближаясь к показателю в Соединенных Штатах, и остается одной из ведущих причин летальности пациентов с циррозом .
With rates of nonalcoholic fatty liver disease on the rise, hepatocellular carcinoma is also expected to increase. Effective therapy exists if hepatocellular carcinoma is caught in the early stages; however, when the disease progresses beyond the point at which surgical approaches or endovascular approaches can be utilized, very little options have been proven as effective forms of treatment. Current standard of care for advanced HCC with sorafenib has only been shown to provide a 2-month increase in life expectancy. Even with this marginal improvement, the estimated survival in patients with advanced HCC is only 1 year. It is for these reasons that many have gone back to the benchtop to understand the exact molecular and immunological pathophysiology so that it can be exploited and hopefully provide more a meaningful treatment for patients with HCC. In this chapter, we will discuss the immunological mechanisms underpinning the development of HCC and currently tested therapies using these mechanisms.
Современная терапия HCC
Предрасполагающие состояния, которые ведут к развитию HCC, включают все факторы, вызывающие фиброз и цирроз печени. Поэтому неудивительно, что были выявлены конкретные ассоциации между вирусным гепатитом В, вирусным гепатитом С, алкогольной болезнью печени, гемохроматозом, первичным билиарным циррозом, дефицитом α-1 антитрипсина и неалкогольным стеатогепатитом [2-4]. Индивидуальные показатели развития гепатоцеллюлярной карциномы варьируют от 0,5 до 2,4% в год. Кроме того, данные о 5-летнем кумулятивном инциденте варьируют от 8 до 30% для нескольких из этих состояний [2, 5]. Недавнее повышение инцидента HCC связано с глобальным увеличением распространенности HCV-инфекции . Ожидается, что с увеличением частоты метаболического синдрома неалкогольный стеатогепатит приведет к еще большему повышению заболеваемости HCC .
Liver transplantation can be offered to otherwise healthy individuals who have localized HCC based on the Milan criteria of either a solitary tumor <5 cm in diameter or up to three lesions, each <3 cm in diameter, without evidence of vascular invasion or extrahepatic spread and who are not otherwise resection candidates . Liver transplantation can eliminate HCC and is thought to represent the best chance for cure. Unfortunately, the availability of liver transplantation limits the widespread use of this approach.
Due to the limited availability and long wait times, the current treatment of choice remains hepatic resection. Strict selection criteria are employed in order to prevent postoperative complications and mortality . Unfortunately, even with resection, HCC recurs at an estimated 70% over the next 5 years . Additionally, only 20% of HCC is found at an early enough stage that curative procedures such as resection or total liver transplantation can be utilized.
Abbreviated recovery periods, minimally invasive approach, and comparable results have led some to consider the typically well-tolerated approach of percutaneous ablation for treatment of early-stage HCC lesions. Many have not yet adopted this approach, but large randomized controlled trials are currently ongoing [11–15]. For now, these procedures are reserved for intermediate-stage disease or in patients with underlying cirrhosis and who are otherwise not surgical candidates [16–18]. The use of radiofrequency ablation, cryoablation, or transarterial embolization can often slow the growth of HCC and essentially debulk the tumor. Due to the unique pathophysiology of HCC and the unique anatomy of the liver vasculature, embolization has been increasingly effective at treating HCC. Although generally better tolerated than surgery, transarterial embolization is not without its share of side effects [18–21].
For those with an advanced disease, palliative therapy with sorafenib, an oral multikinase inhibitor, can be used. Approved by the FDA in 2007, sorafenib acts by inhibiting cell growth, causing induction of apoptosis, and downregulating anti-apoptotic protein Mcl-1 . Additionally, it was also found to reduce tumor angiogenesis, tumor cell signaling, and tumor growth in a dose-dependent manner in mouse xenograft models of HCC by blocking Raf/MEK/ERK pathway and other extracellular receptor tyrosine kinases [23–25]. Despite these promising molecular mechanisms, actual results of sorafenib therapy have unfortunately only been shown to increase average life span by 2 months beyond the median survival of around 12–24 months .
Иммуносупрессорные факторы для HCC
The liver’s ability to evade the immune system is inherent and necessary; however, by doing so, it has detrimental effects when it comes to being able to monitor the liver for cancer. The liver’s natural tolerogenicity allows liver transplant candidates the ability to be maintained on minimal doses of immunosuppressants. Despite this positive effect, the innate and adaptive immune tolerance also leads to increased risk for metastasis and makes carcinogenesis from hepatocellular carcinoma possible . It is postulated that due to the numerous antigens that are presented to the liver from the gut on a routine basis via the portal circulation, the liver has evolved various immune tolerance mechanisms in order to inhibit unnecessary immune responses. These mechanisms include recruitment of immunosuppressive regulatory cells as well as alteration of cytokine pathways and other immunomodulators. Numerous reports have shown that hepatocytes, normally not thought to contribute to antigen presentation, under the influence of viral or autoimmune hepatitis aberrantly express major histocompatibility complex (MHC) class II [27, 28]. Normally, the expression of MHC class II causes activation of naпve CD4+ T cells; however, in cases of hepatocellular carcinoma, diseased hepatocytes lack the appropriate costimulatory signals needed for this interaction and instead cause naпve CD4+ T cells to become inactive and thus evade endogenous immune responses (Table 10.1). In the paragraphs that follow, we will discuss these mechanisms and the relevant data, which indicate the integral role they have in allowing carcinogenesis in HCC (immunosuppressive mechanisms of immune cells are illustrated in Fig. 10.1).
The exposure to exogenous antigens, both blood-derived and microbial byproducts from intestinal flora, places the liver in a constant state of immune activation. Without mechanisms to quell the immune response, these foreign molecules and antigens would elicit states of severe inflammation and damage. The liver’s ability to avoid immune response is accomplished through several mechanisms via interactions of cytokines and between antigen-presenting cells and T cells.
Supplied by sinusoidal vessels, hepatic lobules are units comprised of both hepatocytes and non-parenchymal liver cells that interact with nutrient-rich portal venous blood and hepatic arterial blood before fl wing into the central vein and fi into the hepatic vein. In general, non-parenchymal cells interact fi with the nutrient-rich sinusoidal blood. By doing so, the hepatocytes avoid direct interaction with the bloodstream. Instead, non-parenchymal cells such as stellate cells, liver sinusoidal endothelial cells, dendritic cells, Kupffer cells, natural killer (NK) cells, and other lymphocytes have fi contact with sinusoidal blood.
Table 10.1. Immune subsets involved in tumor-related immune suppression
|Immune cell subset||Known effects on immune function|
|Liver sinusoidal endothelial cell||Produces IL-10 and thus forms Tregs|
|Apoptosis of T cells in interaction of PD-L1 on LSEC with PD-1 on T cells|
|Kupffer cells||Downregulate immune response via TNF-α, TGF-β, IL-10, PGE2, CD95 ligand, galectin-1, and indoleamine dioxygenase (IDO)|
|Primary cells for apoptosis of activated T cells in the liver|
|Dendritic cells||Produce IL-10 and thus CD4+ T-cell polarization into Tregs|
|Myeloid-derived suppressor cell||Induces IL-10 and Foxp3 expression in CD4+ T cells|
|NK cell||Functional impairment of NK cells and decreased IFN-γ production|
|T regulatory cell||Major role in inhibition of tumor-specific T-cell response|
Фиг. 10.1. Иммуносупрессорные механизмы HCC: The various immune cells like LSEC, Kupffer cells, MDSC, and Treg cells together with the immune checkpoint pathway (PD1/PD-L1) play a major role in the immune escape mechanism of HCC. DC dendritic cell, Tregs T regulatory cells, LSEC liver sinusoidal endothelial cells, MDSC myeloid-derived suppressor cells, HCC hepatocellular carcinoma cell, PD-L1 programmed death-1 ligand
Liver sinusoidal endothelial cells (LSEC) are commonly the first to interact with exogenous antigens and proteins from the bloodstream because, as their name implies, they line the hepatic sinusoids. Once exposed, LSEC process these particles and present them via MHC class I and II molecules to CD8+ and CD4+ T cells. Upon interaction with LSEC, naпve CD4+ T cells differentiate preferentially into the T regulatory phenotype. This is thought to be from LSEC-produced IL-10 . With interaction between LSEC and naпve CD8+ T cells, only partial activation occurs. This interaction is thought to be secondary to PD-1 on the T cells and B7-H1/PD-L1 on the LSEC [30–32]. These partially activated CD8+ T cells later go on to passive apoptosis and eventual phagocytosis by Kupffer cells. Interleukin (IL)-2 has been postulated to reverse the liver sinusoidal-mediated CD8+ T-cell partial activation.
Both indirectly and directly, liver-specific macrophages known as Kupffer cells play an important role in immune regulation of HCC. Kupffer cells are known to produce anti-inflammatory cytokines and pro-apoptotic signals. Of these, tumor necrosis factor-α (TNF-α), TGF-beta, IL-10, PGE2, CD95 ligand, galectin-1, and indoleamine dioxygenase (IDO) have been implicated in downregulating the immune response and thus increasing tolerogenicity . Direct relationships between other cytokines also play a part. For instance, interferon γ (IFNγ), produced by activated CD8+ T cells, is known to increase the concentration of CD95 ligand and TNF-α produced by Kupffer cells, thereby leading to more apoptosis [33, 34]. Directly, Kupffer cells appear to be the primary cells implicated in the apoptosis of activated CD8+ T cells that enter the liver . After the presentation of antigen via MHC class I molecules by Kupffer cells, the increased affinity of ICAM-1 on Kupffer cells and LFA-1 binding on CD8+ T cell results in apoptosis. Given this, some postulate inhibition of antigen presentation to be a possible mechanism for allowing expansion of effector CD8+ T cells and thus increasing immune surveillance.
Stellate и MDSC
Both hepatocytes and non-parenchymal cells serve a regulatory immune role. Activated hepatic stellate cells, which reside in the subendothelial space of Disse , are thought to play an integral role in the development of hepatocellular carcinoma . Exact mechanisms remain unclear; however, it is thought that activated stellate cells cause a preferential increase in myeloid-derived suppressor cells (MDSC) . These cells go on to develop into neutrophils, monocytes, and macrophages. Induction of MDSC in the liver leads to suppression of the antitumor immune responses and therefore creates a favorable environment for the spread and development of HCC. MDSC induction occurs not only in HCC but also in acute and chronic hepatitis.
The majority of dendritic cells (DC) within the liver reside in the areas adjacent to the portal triad and in healthy conditions are primarily in their immature form . Once matured from their myeloid state after toll-like receptor 4 (TLR4) ligation, these DC have been found to produce large amounts of IL-10 and similar to LSEC cause CD4+ T-cell polarization into T regulatory induction and poor antigen recall response. Decrease in the number of circulating DCs and reduction in their cytokine production have been demonstrated by Ormandy et al. This may indicate impaired TAA presentation by DCs . Therefore, they shift the state of inflammation to one of immunosuppression and thus allowing a pro-tumorigenic environment for the spread and development of hepatocellular carcinoma. Marked dendritic cell infiltration in HCC nodules correlated well with better prognosis of HCC after resection .
In addition to the decreased NK cell function that is induced by the HCV envelope protein E2, expanded population of MDSC also wreaks havoc [42, 43]. MDSC have been found to cause inhibition of NK cell function thus leading to hyporesponsive NK cells. Aside from functional impairment in patients with HCC, decreased NK cell frequency as well as diminished production of IFN-γ was noted . The decreased IFN-γ is known to be associated with increased CD4+ T regulatory cells. NK cells are also being used as one of the adoptive cell therapies (other cells that are also being studied include DCs, chimeric antigen receptor T cells, cytokineinduced killer cells) in HCC . A study (NCT01147380) to evaluate feasibility and safety of the adoptive transfer of IL-2-activated NK cells (extracted from cadaveric donor liver graft perfusate) for liver transplant recipients with hepatocellular carcinoma (HCC) has been completed in December 2014 .
For patients with underlying hepatitis B or C virus, both innate and adaptive immune system alterations are known to occur as a result of viral-mediated effects. HBV, in particular, has been observed to increase the circulating levels of IL-10 . The increased levels of regulatory T cells (Tregs) have been correlated with viral load and ultimately impair CD8+ T-cell-mediated clearance of the virus .
HCV also leads to adaptive immune cell dysfunction. This T effector cell dysfunction that occurs is thought to be secondary to uptitration of PD-1 and Tim-3 receptors [49–51]. In a recent study, IFN-α, previously known to be a component of standard of care for treatment of HCV infection , leads to decreased memory T cells and telomere loss in naпve CD8+ T cells . Innate immunity is also impaired by HCV. As a consequence of viral proteins, monocyte-derived dendritic cells remain in their immature forms despite maturation stimuli, and NK cells lose their effector function [43, 54].
CD4+ CD25+ T regulatory cells (Tregs) play a major role in inhibiting tumorspecific T-cell response in HCC. Increased frequency of CD4+ CD25+ Tregs has been reported in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) . The increased Tregs then express Foxp3 and inhibit CD3/CD28-stimulated CD8+ T-cell proliferation . Depletion or inhibition of Tregs using anti-CD25 mAbs or cyclophosphamide has shown enhanced antitumor effects in preclinical models .
HCC-специфические антигены/опухоль-ассоциированные антигены (TAA)
An understanding of TAAs is prime in the development of tumor-specific immune therapy (summarized in Table 10.2).
Α-fetoprotein is an oncofetal antigen which is expressed during fetal development. It is the most abundant protein in the serum of a fetus . It is produced in large amounts by the yolk sac and fetal liver and is transcriptionally repressed shortly after birth. AFP is reexpressed in 40–80% of HCC, and germ cell tumors and serum assays of AFP help in diagnosing and monitoring response to therapy . AFP is currently the most well-studied target antigen for hepatocellular carcinoma immunotherapy.
AFP is a self-protein, and therefore it was thought that AFP-specifi T-cell responses are suppressed and thus diffi to activate. But now studies have shown that more AFP-specifi epitopes exist, which can mount AFP-specifi CD-8+ T-cell responses. In one such study , HLA-A2-restricted AFP-specifi CD-8+ T-cell epitope was identifi and its ability to mount CD-8+ T-cell responses in human lymphocyte cultures as well as HLA-A2 transgenic mice was demonstrated. In the same study, four dominant and ten subdominant AFP-specifi epitopes were identifi which generated low to moderate CD-8+ T-cell responses in peripheral blood mononuclear cells (PBMCs) of HCC patients [58, 59]. AFP-specifi T cells were found in patients with HCC as well as in patients with chronic HCV infection, some other liver diseases, and less commonly in healthy subjects . Depletion of T regulatory cells (CD4CD25Foxp regulatory T cells) has resulted in the unmasking of AFP-specifi T-cell responses in HCC patients and could be used with other immunotherapeutic approaches for HCC . AFP is also one of the candidates for peptide-based vaccines against HCC (tumor vaccines for HCC will be discussed separately).
Таблица 10.2. Таргетируемые опухоль-ассоциированные антигены
|Опухоль-ассоциированный антиген (TAA)||Тип TAA||Частота экспрессии в HCC||Potential for immunotherapy|
|Онкофетальный протеин||40–80%||AFP peptide vaccination|
|DC pulsed with AFP peptides|
|Гепаринсульфат и онкофетальный протеин клеточной поверхности||84%||In development for HLA-A2 individuals|
|Anti-GPC3 chimeric antigen receptor (CAR) T-cell therapy (NCT02395250)|
|NY-ESO-1||Канцер-тестикулярный антиген (CTA)||13–51%||A DC205-NY-ESO-1 vaccine with or without sirolimus in solid tumors expressing NY-ESO-1 is ongoing (NCT01522820)|
|TERT||Фермент для удлинения теломер||80–90%||hTERT-derived peptide (hTERT461) vaccine for HCC|
|HCA519/TPX2||Микротрубочка — ассоциированный протеин||100%||Potential for immunotherapy as it is highly expressed in HCC tissue|
GPC3 is a cell surface heparan sulfate proteoglycan which is also a fetal oncoprotein. It can bind growth factors like Wnt, Hedgehog (Hh) signaling protein, VEGF, and FGF (fibroblast growth factor) 1/2 and help in growth of the tumor [40, 62]. It is expressed by 84% of HCC and was found to be immunogenic in murine models and human cell culture . On one hand, GPC3 overexpression indicates a poor prognosis in HCC patients—with significant association with high tumor grade, late TNM stage, and vascular invasion [63, 64]. On the other hand, it is not expressed in normal adult tissue and benign liver lesions, thus making it an ideal tumor antigen for HCC Immunotherapy .
Previous studies had shown that GPC3 (144–152) and GPC (298–306) peptides induced specifi CD8+ CTLs in HCC patients with HLA-A2 and HLA-A24 restriction, respectively . Inoculation of these CTLs reduced the mass of the human HCC tumor implanted into nonobese diabetic/severe combined immunodefi y mice. In a recent study published by Dargel et al., peptide GPC3367 was identifi as a predominant peptide on HLA-A2. To overcome the problem of immune tolerance due to fetal expression of GPC3, dendritic cells from HLA-A2-negative donors were co-transfected with GPC3 and HLA-A2 RNA to stimulate the GPC3-specifi T cells. Expression of GPC3367-specifi T-cell receptor by T cells allowed them to eliminate GPC3-positive xenograft tumor grown from human HCC cells in mice .
GPC3 cDNA vaccine has also been studied for cellular antitumor activity of specific CTLs for treatment of HCC in a C57BL/6 mouse model .
NY-ESO-1 is one of the antigens of the cancer testis antigen (CTA) family (includes NY-ESO-1, members of SSX family, MAGE family, SCP-1, CTP11) which is expressed in multiple cancers including HCC (in 13–51% of HCC) but not in a normal tissue except for the testis [56, 68]. Therefore, it is a potential target for immunotherapy in HCC . Combined expression of CTA (MAGE-A3, MAGE-A4, and NY-ESO-1) mRNA enhances the prediction of recurrence of HCC, therefore acting as a potential prognostic marker.
In an initial study, spontaneous NY-ESO-1-specifi antibody response and functional CD4+ and CD8+ T-cell responses were seen in NY-ESO-1-expressing HCC . Another study demonstrated increased frequency of specifi CD8+ T-cell responses to HLA-A2-restricted NY-ESO-1b (p 157–165) in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients . As opposed to stimulating T-cell response to NY-ESO-1 antigen, it was seen that we could signifi enhance the antitumor cytotoxic T lymphocyte response to HCC by depleting Treg cells followed by stimulation by NY-ESO-1b peptide .
Теломеразная обратная транскриптаза (TERT)
Human TERT (hTERT) is a catalytic enzyme required for telomere elongation. Tumors have to maintain the telomeric ends of their linear chromosomes, and this is accomplished with the help of TERT. Most tumor cells express TERT, but most adult human cells do not express it. Eighty to ninety percent of HCC express hTERT, thus making it a possible target for immunotherapy in HCC . In a recent study by Huang et al., mutations of the TERT promoter region (which correlated with telomerase activity) were frequently seen in many tumors including HCC (31.4% of HCC) . This emphasizes the importance of telomere maintenance in the development of a tumor.
Mizukoshi et al. identifi hTERT-derived, HLA-A*2402-restricted cytotoxic T-cell (CTL) epitopes and found hTERT-specifi CTL responses in the peripheral blood of HCC patients . This was also observed in a patient with early stages of HCC.
HCA519 is a microtubule-associated protein which plays an integral role in the HCC replication cycle. It is also known as the targeting protein for Xklp-2 (TPX2). Significantly higher expression of HCA519/TPX2 is seen within HCC tissue (expressed in 100% of HCC as per Greten et al.) compared to a normal liver tissue [56, 76, 77]. Increased TPX2 production has also been seen in lung and pancreatic cancers . Peptides HCA519464–472 and HCA519351–359 derived from HCA519/ TPX2 were effective in generating HLA-A*0201-restricted CTLs. Therefore, HCA519/TPX2 can be a promising target for immunotherapy in HCC.
Другие CTA: SSX-2 и Melanoma Antigen Gene-A (MAGE-A) семейство
SSX-2 and MAGE-A are other cancer testis antigens which are overexpressed in <50% and <80% of HCC patients, respectively . In addition to melanoma, cytotoxic T cells response specifically to MAGE-A10, and SSX-2 was also demonstrated in vivo in HCC patients . Similar CD8+ T-cell response was also seen against MAGE-A1 and MAGE-A3 epitopes in tumor-infiltrating lymphocytes (TILs) but not peripheral blood lymphocytes of HCC patients . These studies point toward a potential use of these TAAs for immunotherapy in HCC.
Блокада иммунных чекпоинтов
Immune checkpoint inhibitors are one of the mechanisms to enhance antitumor immunity. The most studied immune checkpoint receptors are CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, and OX40 . Three checkpoint inhibitors have been approved by US FDA for the treatment of melanoma (ipilimumab, antiCTLA-4; pembrolizumab, antiPD1; and nivolumab, anti-PD-1) . Ipilimumab was the first to be approved in 2011. The roles of immune checkpoint inhibitors are being studied in the treatment of various solid tumors including HCC.
PD-1/PD-L1 иммунный чекпоинт
Programmed death-ligand-1 (PD-L1 also known as B7-H1 or CD274) is expressed by many immune cells as well as cancer cells. The PD-L1 can bind to T-cell receptors— programmed death-1 (PD-1) and B7.1 (CD80)—which suppress T-cell function (T-cell migration, proliferation, secretion of cytotoxic mediators) and therefore block the cancer-immunity cycle . Blockade of the PD-1/PD-L1 pathway has shown signifi effi y in patients with advanced non-small cell lung cancer, melanoma, renal cell cancer, and Hodgkin’s lymphoma including upon failure to several lines of therapy . A study of 46 metastatic melanoma patients by Tumeh P. C. and colleagues demonstrated that regression of tumor after PD-1 blockade therapy (pembrolizumab) required preexisting CD8+ T cells which are suppressed by PD-1/PD-L1 antitumor effect . Similar results were shown by Herbst et al. where engineered humanized anti-PD-L1 antibody therapy was used in multiple cancer types . HCC patients were found to have increased PD-1 expression in circulating and intratumor CD8+ T cells . Kupffer cells and cells with MDSC phenotype upregulate PD-1expressing CD8+ T cells in HCC patients [85, 86]. Anti-PD-1 antibody treatment had additional antitumor effect when combined with AMD3100 (a CXCR-4 inhibitor which targets sorafenib induced hypoxic and immunosuppressive microenvironment) in sorafenib-treated HCC in mice .
PD-1 antibodies that are being developed include nivolumab (BMS-936558, Bristol-Myers Squibb, USA), CT-011 (CureTech, Israel), lambrolizumab (MK-3475, Merck, USA), and AMP-224 (Amplimmune, USA). The PD-L1 antibodies currently being developed include MPDL3280A/RG7446 (Genentech, USA, and Roche, Switzerland) and MEDI4376 (MedImmune, USA, and AstraZeneca, UK) .
Анти-PD-1 антитела для HCC в клинических исследованиях
The phase I/II clinical trial (NCT00966251) of CT-011 (pidilizumab) in HCC patients not eligible for surgery, TACE, or other systemic therapies was started in 2009 but was terminated due to slow accrual.
Another phase I/II clinical trial (NCT01658878) of nivolumab (fully human IgG4 monoclonal antibody PD-1 inhibitor) in advanced HCC patients by El-Khoueiry et al. with a primary endpoint of safety and a secondary endpoint of antitumor activity  is ongoing. The results were presented at the American Society of Clinical Oncology (ASCO) in 2015. The study population included 3 cohorts of patients (total of 41 patients) stratified based on viral infection with HBV (11 patients), HCV (12 patients), or no viral infection. All patients had a Child-Pugh Class B scores of 5 or 6 (indicating relatively good liver function) and Eastern Cooperative Oncology Group (ECOG) performance scores of 0 or 1. Most had metastasis beyond the liver or portal vein tumor invasion. Approximately 75% had previously been treated for HCC, including prior treatment with sorafenib in 68% patients. All patients were treated with intravenous infusions of nivolumab every other week for up to 2 years. The results revealed that this therapy had a manageable safety profile and durable response in all the three patient cohorts. Five percent had complete response, 14% had partial response, and the overall survival rate was 62% at 12 months. These results compared favorably with a complete response rate of around 2% and a 1-year overall survival rate of about 30% with sorafenib.
Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) blockade
CTLA-4 (also known as CD152) is an inhibitory co-receptor expressed on T cells and Tregs and can bind CD80 and CD86 on APCs with a much higher affinity than CD28 (CD28 is a costimulatory molecule which also binds CD80 and CD86) and therefore inhibits T-cell activation . Blockade of CTLA-4 suppresses antitumor immune response mediated by T cells.
From the studies of CTLA-4 blockade in other malignancies (breast, colon, lung, prostate, and brain cancers, melanoma, lymphoma, and sarcomas), it has been seen that the effi y of CTLA-4 correlates with immunogenicity of the tumor and immunotherapy may have better results in smaller tumors . Ipilimumab (MDX-010, Bristol-Myers Squibb, USA) and tremelimumab (formerly referred to as ticilimumab, CP-675,206, MedImmune, USA, and Pfi , USA) are the two CTLA-4 antibodies which are currently in advanced stages of development.
Anti-CTLA-4 Antibody in Clinical Trials for HCC
Tremelimumab was studied in a phase I clinical trial of 21 patients with advanced HCC not amenable to percutaneous ablation or TACE, and all patients had chronic hepatitis C genotype 1b. Partial response was found in 17.6% of patients, and disease control rate was 76.4%. The time to progression was
6.48 months (95% CI 3.95–9.14) . Another phase I clinical trial of tremelimumab combined with RFA or TACE is ongoing (NCT01853618) .
Стратегии противоопухолевой вакцинации
The main goal of all the vaccination strategies that are being currently studied is to induce a tumor-specific immune response and overcome the inherent immune tolerance of HCC. This is being tried in the following three broad categories.
It involves the administration of recombinant full-length TAAs or its peptide to cancer patients to stimulate immune response against the tumor. AFP and GPC3 are two frequently used TAAs for this purpose. One of the initial studies was performed by Butterfield et al. in which six AFP-positive HCC patients were vaccinated (intradermal injection) with four immunodominant HLA-A*0201-restricted peptides. This generated measurable AFP-specific T-cell response . GPC3 has also been used for cancer immunotherapy and has been described under Sect. 10.10.2. Recently, an hTERT-derived peptide (hTERT461) was studied as a vaccine in 14 HCC patients. It was administered subcutaneously three times biweekly. This vaccination generated hTERT-specific immunity in 71.4% of patients, and 57.1% of patients who were administered hTERT461 peptide-specific T cells could prevent HCC recurrence after vaccination .
Дендритно-клеточная (DC) вакцина
Dendritic cells are the most potent APC with the capability to process and present tumor antigens to T cells and stimulate an antitumor immune response. DC-based vaccines are developed by collecting monocytes in peripheral circulation from cancer patients. In the presence of a source of TAAs (autologous tumor tissue or peptides of TAAs or cell line lysate) and maturation stimuli (like interleukins, GM-CSF), these cells are expanded ex vivo and reinfused into the patient to mount a tumorspecific immune response.
A phase I clinical trial was done in which tumor lysate-pulsed autologous DCs were generated ex vivo after stimulation with GM-CSF and IL-4. This was found to be feasible and without any toxicity in patients with unresectable HCC . Intratumoral injection of DC in HCC nodules was also found to be safe in another study . A phase II clinical trial showed that intravenous administration of autologous dendritic cells (DCs) pulsed ex vivo with a liver tumor cell line lysate (HepG2) in patients with advanced HCC was found to be safe, and the radiologically determined disease control rate was 28% . DC infusion performed during TACE was shown to enhance tumor-specific immune response but was not sufficient to prevent HCC recurrence .
DNA encoding one or multiple TAAs can be delivered to patients as naked plasmids or within vectors (Vaccinia virus , adenovirus , Listeria monocytogenes  have been used in preclinical studies). The DNA is expected to undergo transcription and translation to express the TAA peptide and induce immune response. In a study by Butterfi et al., two HCC patients who had received prior locoregional therapy were administered full-length AFP in a plasmid DNA
- Chakraborty et al.
construct together with an AFP-expressing replication-defi adenovirus (AdV) in a prime-boost vaccine strategy. This strategy generated AFP-specifi T-cell response, but both patient had recurrence of HCC (within 9 and 18 months, respectively) .
10.12.4 Oncolytic Virus Therapy
Oncolytic viruses are made to selectively replicate within cancer cells and subsequently lyse them. Pexa-Vec (pexastimogene devacirepvec, JX-594) is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus which expresses GM-CSF and lac-Z transgenes that causes replication-dependent cell lysis and stimulation of antitumoral immunity . Intratumoral injection of this virus in advanced HCC patients was shown to be safe and feasible . In a pilot study, Hoe et al. studied sequential JX-594 therapy followed by sorafenib in three HCC patients and found this to be well tolerated, having a signifi decreased tumor perfusion and associated with objective tumor response (Choi criteria; up to 100% necrosis) .
A multicenter, randomized phase III clinical trial (NCT02562755) to determine whether treatment with Pexa-Vec followed by sorafenib increases survival compared to treatment with sorafenib alone in patients with advanced HCC (mentioned in Table 10.3) who have not received prior systemic therapy is expected to open recruitment in October 2015 .
Таблица 10.3. Продолжающиеся и будущие клинические исследования иммунотерапевтических подходов к гепатоцеллюлярной карциноме
|Intervention||Registration no.||Фаза исследования||Start date||Primary outcome||Status|
|NCT01658878||I фаза||September 2012||Safety||Recruiting|
|Tremelimumab with TACE or RFA||NCT01853618||I фаза||April 2013||Safety and feasibility||Recruiting|
|Pexa-Vec (JX-594) followed by sorafenib versus sorafenib (PHOCUS)||NCT02562755||III фаза||Planned for October 2015||Overall survival||Not yet recruiting|
(allogenic DC) vaccine
|NCT01974661||I фаза||October 2013||Adverse events||Recruiting|
|MG4101(ex vivo expanded allogeneic NK cell) after curative liver resection||NCT02008929||II фаза||August 2014||Disease- free survival for 1 year||Recruiting|
We have now looked at the various ways in which immune regulation can affect HCC. The main goal in the future would be to translate all these preclinical and clinical research into a strategy which would be a successful immune therapy for HCC patients.
There have been reports of spontaneous regression of HCC, and in fact, HCC was found to be one of the most common types of cancer with spontaneous regression . The most common causes were thought to be immunologic and decrease blood flow to the tumor. This reemphasizes the potential of immunotherapy in HCC.
One important approach would be to individualize immune therapy for each patient based on the immune arm which is overactive in that patient. This would include using either activation of TAA-specific T cells via vaccination methods or inhibition of the immune evasion mechanisms (immune checkpoint blockade, inhibition of immunoregulatory cell like MDSC/Treg) or a combination of both. Studies have demonstrated increased AFP-specific CD4+ T cells and increased circulating NK cells following TACE and RFA, respectively [103, 104]. This immune response following ablative therapies points toward another therapeutic strategy combining conventional HCC therapy (TACE/RFA, chemotherapy) with immunotherapy.
Though significant development has been made in understanding the immune mechanisms involved in HCC, further well-designed, randomized, controlled clinical trials with appropriate patient population and thorough immunomonitoring are required to develop efficient immunotherapies for HCC.
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