Angiogenic inflammation and formation of necrosis in the tumor microenvironment influence patient survival after radical surgery for de novo hepatocellular carcinoma in non-cirrhosis

Liver cirrhosis is an established risk factor for HCC. However, HCC also arises de novo in non-cirrhotic livers in approximately 20% of all cases, with host inflammatory responses having a key importance in hepatocarcinogenesis [1‐3]. There is a rising clinical interest in patients with de novo HCC, because this subgroup commonly presents at an advanced stage, as surveillance is usually not performed in patients without liver disease. Tumor growth in these non-cirrhotic patients is clinically silent in its early stages because of the lack of symptoms and compensated hepatic function. On the other hand, the importance of non-alcoholic steatohepatitis (NASH) in driving the process of hepatocarcinogenesis has been recently recognized and put into a causal context with de novo HCC [4]. Of note, NASH-driven hepatocarcinogenesis is mechanistically involved in the process of necrosis formation in the tumor microenvironment, and the latter has also been related to enhanced infiltration with immune-competent cells [5‐9]. Furthermore, experimental studies reported on novel angiogenic pathways playing a key role in de novo or NASH-driven hepatocarcinogenesis, implicating the complex immunologic mechanisms involved in cancer progression [10‐13].

The significance of complex angiogenic properties of the tumor microenvironment in HCC has come to the fore in recent years. Tumor angiogenesis has been validated as an attractive therapeutic target in the process of hepatocarcinogenesis, mainly in clinical trials targeting the vascular endothelial growth factor (VEGF) pathway [14]. However, a deeper insight into the biology of solid cancer reveals that the host cellular immune competence in the tumor microenvironment is mechanistically intertwined with angiogenesis and necrosis formation, and the blockade of only one functional pathway does not reach the desired long-term efficacy in cancer patients. Recently, the angiopoietin family of ligands, angiopoietin-1 and -2, has been demonstrated to selectively activate the endothelial cell membrane receptor tyrosine kinase TIE2 and to espouse tumor progression [15, 16]. In the scope of tumor angiogenesis and metastasis, this angiopoietin axis-TIE growth factor receptor pathway represents the key regulator of pathological vascular permeability and remodeling, and its pharmacological blockade is in clinical development in oncologic settings [14]. In this scenario, the role of novel angiomodulatory monocytes/macrophages subsets in hepatocarcinogenesis is vastly unknown. Angiogenic immune–competent cells represent a unique subpopulation of tumor-infiltrating bone marrow-derived myeloid cells, which differ from the classical tumor-associated macrophages (TAMs) [17]. These cellular effectors have immense angiogenic potential, express functionally active TIE2-expressing macrophages (TEMs) and directly respond to angiopoietin activity [18]. Interestingly, the tissue-infiltrating fraction of TEMs, which promotes angiogenesis and carcinogenesis, is localized only in the tumor microenvironment and not in healthy tissues [17, 18]. In line with this, our data demonstrated significant influence of TEMs on patient outcome in other cancer types [19‐21]. Thus, the study of TEMs in HCC may identify attractive targets for immunologic checkpoint inhibition.

In rapidly growing tumors, the formation of necrosis can be attributed to the relative hypoperfusion in the vicinity of the tumor. However, novel scientific results demonstrate that the nature of necrosis formation in the vicinity of the tumor is much more complex, as its occurrence is functionally mediated by infiltrating monocytes/macrophages and delineated its role as a prognosticator of aggressiveness in primary solid tumors [22‐24]. The presence of tumor necrosis in HCC may adequately characterize the tumor biology and provide additional beneficial prognostic information. Furthermore, we previously demonstrated the importance of histologic tumor necrosis and its relation to monocytes/macrophages in poorly vascularized human cholangiocarcinoma [25, 26]. However, HCC is in most cases a highly vascularized tumor [27]. Therefore, formation of necrosis might also be a typical feature of subsets of HCC. However, early stage and recurrence of HCC are difficult to detect by non-invasive imaging, and alpha-fetoprotein (AFP) as a surveillance biomarker has been removed from present guidelines because of its low sensitivity and specificity [28]. Novel biomarkers for the management and prognosis of HCC patients are needed, and thus, in the current work, we focused on the potential of tumor necrosis to be facilitated by immunological components of the tumor microenvironment (i.e., invading monocytes/macrophages).

Patients with de novo HCC are at enhanced risk for adverse disease outcome. Intriguingly, despite tumor necrosis induced by impaired tissue oxygen delivery, which is generally due to a compromised blood supply to cancerous tissue, recent studies also revealed that histologic tumor necrosis was associated with high levels of angiogenesis and increased inflammation in the tumor microenvironment [29, 30]. Interestingly, a strong association between liver steatosis and formation of tumor necrosis in de novo HCC in non-cirrhotic livers has been recently revealed [5]. Thus, possible immunologic coherence comprising tumor-infiltrating angiogenic monocytes/macrophages, associated molecular factors of angiogenesis and formation and extent of tumor necrosis could effectively characterize HCC patients. Recently, our group reported on the clinicopathologic characteristics of patients with de novo HCC and the clinical significance of tumor-infiltrating classical monocytes and lymphocytes in this hepatic pathology [31]. Insofar, the present work aimed to investigate the angiopoietin axis, related angiogenic TEMs, and tumor necrosis in de novo HCC in non-cirrhotic patients, and thus, deliver a deeper insight concerning the importance of novel immunologic biomarkers, which may help improve the personal risk and prognosis stratifications by defining subgroups of patients with beneficial or deleterious tumor characteristics.

In our work, the tissue densities of angiopoietins and angiopoietin receptor-bearing monocytes/macrophages as well as the occurrence of histologic tumor necrosis in specimens of HCC patients were associated with clinicopathologic characteristics and patient survival. The main findings were as follows: (1) angiopoietins and infiltrating TEMs were associated with metastatic and recurrent disease; (2) the presence of tumor necrosis was associated with an advanced tumor stage and associated with TEM frequency; and (3) necrosis and TEMs exerted a pernicious influence on patient survival.

In this study, we demonstrated that high angiopoietin expression and the presence of TEMs was associated with recurrent and metastatic HCC. These results are in accordance with previously published results on HCC and other hepatobiliary tumors, which delineate the negative impact of infiltrating monocytes/macrophages on patient survival and outcome [34‐37]. Angiopoietins represent a potential prognostic biomarker of therapeutic responsiveness to anticancer treatments, including immunotherapy using immunomodulatory agents [38]. The activation of the angiopoietin-TIE2 axis could be a potent tumor escape mechanism from anti-angiogenic treatment. Novel data implicates that, in HCC, angiopoietins are co-expressed with VEGF and transiently decrease during the window of normalization and return to baseline levels after anti-VEGF therapy [38]. Tumor-derived VEGF can increase the tumor expression of angiopoietins and promote metastases, suggesting that interactions between these angiogenic pathways promote tumor progression [39]. Furthermore, several preclinical studies have shown that monocytes/macrophages, and especially angiopoietin receptors-bearing TEMs, contribute to tumor neoangiogenesis in mouse tumor models [40]. Therefore, these monocytes/macrophages may exert potent abilities to enhance the extent of tumor neoangiogenesis and progression to increased malignancy in patients with HCC. These findings suggest that angiopoietin signaling may foster the invasion of monocytes/macrophages, resulting in deleterious effects in HCC; however, further studies are needed to elucidate the possible functional mechanisms and help conceptualize novel checkpoint inhibitor targets for cancer immunotherapy.

HCC is a highly vascularized tumor and the recurrent disease remains a major clinical obstacle and defines the outcome of the patients receiving curative therapy. In the current work, we provide data that could be used to identify subgroups of patients with advantageous or deleterious HCC characteristics, respectively. Such a personalized approach may have a useful clinical implication in order to improve the individualized risk stratification after surgery and curative treatments. This translates ultimately into more intensified and purposeful aftercare of patients with HCC at high risk for adverse outcomes. De Palma et al. were the first to report on the significance of TEMs in regard to tumor neoangiogenesis [18]. In the clinical setting, Matsubara et al. reported first on the importance of TEMs in human blood and associated their presence with tumor neoangiogenesis, recurrence, and patient survival rates [41]. Consequently, our group reported on the prognostic value of TEMs and histologic tumor necrosis in other hepatobiliary tumors [19‐21, 25, 26]. Here, we showed that tumor necrosis and the presence of TEMs and angiopoietins were associated with an advanced HCC. In the current work, tumor necrosis was also associated with TEM density in HCC. These characteristics of the tumor microenvironment also affected overall survival and recurrence-free survival of the patients. Thus, we propose a coherent construct comprising tumor necrosis, neoangiogenesis, and associated TEMs as an attractive diagnostic modality prognosticating the outcome of HCC patients following radical surgery.

The clinical translation of novel immunologic aspects of the tumor biology represents an urgent unmet need. In addition, a major clinical problem is the lack of adjuvant treatment strategies in patients with advanced HCC [34]. Novel experimental techniques demonstrated high efficacy in visualization of important localized sites of the tumor microenvironment and related tumor necrosis. Moreover, these approaches offer the possibility for nanoparticles uptake and consequent clinical imaging of monocytes/macrophages in the vicinity of the tumor, as well. Aghighi et al. established a novel MRI-based modality for selective visualization of the tumor central area and the infiltrating margin that is adjacent to normal non-cancerous tissues [42]. In this setting, the utilization of ultra-small superparamagnetic iron oxide nanoparticles in ferumoxytol-enhanced MRI provides an opportunity to better characterize the extra- and intracellular compartments of solid tumors, corresponding TAMs, and formation of necrosis in the tumor microenvironment, which is a novel diagnostic tool with a high sensitivity and potential for immediate clinical translation [42, 43]. Moreover, a novel tumor necrosis therapy (TNT), which provides a new and promising therapeutic anticancer modality, has been recently described, as well [44]. Through radiolabeled necrosis avid compounds (i.e., small molecules with a high affinity for and long-lasting retention in necrotic tissues), tumor necrosis can be a carrier of therapeutic radionuclides [45‐47].

In conclusion, formation of histologic tumor necrosis is associated with the intratumoral density of invading angiogenic TEMs of HCC patients. Furthermore, the tumor necrosis–TEM–angiopoietin axis was associated with disease recurrence, advanced tumor stage, and reduced survival after radical surgery in these patients. Thus, our data may indicate the tumor necrosis–TEM–angiopoietin axis is a novel checkpoint target in tumor immunotherapy and, at the same time, provides potent anti-angiogenesis modalities in advanced HCC. However, some limitations of the current study should be taken into account. The small number of cases in the different subgroups remains the main drawback. On the other hand, our work incorporated no functional studies and remains a descriptive study. Therefore, further research encompassing larger patient populations with focus on functional assays and mechanistic links is urgently needed. This will deliver a deeper biological insight into possible mechanistic molecular pathways might help develop novel immunologic checkpoint inhibitor targets for hepatic malignancies.