Evidences from animal models

Initial studies on animal models suggested Sm infection to be acting in synergism with other factors and advancing induction of HCC where this other carcinogenic factor, independently, might have initiated carcinoma later and in milder form.

One such study involved a mouse model where female mice were divided in three groups where the first group was orally given a carcinogen (N-2-fluorenylacetamide), the second group was exposed to twenty cercariae of Sm, and the third group received both. At forty weeks post infection, 6.3% of mice in the first group (carcinogen alone) developed liver tumours, whereas no mice in second group (exposed to cercariae) developed tumours. Interestingly, about 47% of mice in third group developed liver tumours at forty weeks, signifying early and distinct production of liver tumours (Kakizoe Reference Kakizoe1985).

In a similar but relatively recent study, 500 parasite-free mice were divided into four groups where group 1 was exposed to a known carcinogen (diethylnitrosamine (DEN)), group 2 was exposed to a carcinogen as well as infected with Sm, group 3 was only exposed to Sm infection, and group 4 was the control. Tumour markers (i.e., alpha fetoprotein (AFP) and ferritin) were examined by ELISA, and the mice were later sacrificed for histo-pathological examination of livers. Results showed that Sm enhanced and aggravated the carcinogenic effects of DEN. When compared to controls, the serum level for AFP and ferritin showed earlier and statistically significant differences in group 2 than in group 1. This study also concluded that Sm accelerates hepatic dysplastic changes, which, along with other risk factors for carcinogenesis, makes an early and more aggressive cancer appearance (El-Tonsy et al. Reference El-Tonsy, Hussein, Tel-S, Tawfik, Koriem and Hussein2013).

Apart from the mouse model, a study reported a chimpanzee with HCC who had a chronic Sm infection and was negative for HBV and HCV infection (Abe et al. Reference Abe, Kagei, Teramura and Ejima1993).

Evidences from humans

Studies linking Sm infection with liver carcinoma in humans began in late 1900s. One such study dates back to 1978 when K. E. Mott published an observation suggesting a possible relationship between infection with Schistosoma spp. (Sm and Schistosoma japonicum (Sj)) and liver carcinoma (Mott Reference Mott1978). However, a few years later, another study suggested that the association between Sm and HCC may be coincidental especially in regions where Sm is endemic (Pereira and Goncalves Reference Pereira and Goncalves1984).

A study that aimed at understanding the prevalence of hepatitis viruses and Sm infection among patients with chronic liver disease found that HCV infection along with Sm infection is a major risk factor for chronic liver disease (including liver cancer) in Egypt (Halim et al. Reference Halim, Garry, Dash and Gerber1999). In another study conducted in Brazil, an institutional database was screened from 2002 to 2015 to retrospectively identify patients with HCC who had Sm infection. A total of seven patients were identified in this analysis. All of these patients had negative HCV serology, and four were positive for HBV core antibodies but lacked any evidence of viral replication. The study concluded that Sm acts as an important co-factor for hepatic lesions and injury, but they could not conclude if Sm infection alone could induce HCC (Toda et al. Reference Toda, Kikuchi, Chagas, Tanigawa, Paranaguá-Vezozzo, Pfiffer, MdeS, Alves and Carrilho2015).

Another group from Egypt focused specifically on understanding the relationship between Sm infection and HCC induction. In this study, two groups were formed. The first group had 29 patients who were negative for schistosomiasis (tested by indirect hemagglutination assay (IHA)) with no history or evidence of current or previous Sm infection but were HCV positive. The second group had 46 patients who had positive IHA for schistosomiasis and were HCV positive. As all the patients in both the groups were positive for HCC, the study concluded that co-infection increased incidence of HCC in the second group. The aggression of HCC was also higher in the second group, which is in line with observations from previous studies (El-Tonsy et al. Reference El-Tonsy, Hussein, Tel-S, Tawfik, Korie and Hussein2016).

A clinical study carried out in Brazil analysed 6 HCC patients who were admitted for liver transplantation. Patients belonged to endemic regions of Sm infections and had Sm-associated fibrosis, but cirrhosis was not observed in the histo-pathological analysis of the resected livers. Further, all the patients had a negative serology for HBV and HCV, which suggested that Sm infection might have predisposed the patients towards HCC, but a causal relationship could not be established in this study as well (Filgueira et al. Reference Filgueira, Saraiva CM de, Jucá, Bezerra and Lacerda2018).

A recent observational study with a much larger sample size, which ranged from years 2009 to 2019 (1,446 patients with HCC), divided the patients in two groups. Group 1 (688 patients) had patients with a history of Sm infection, whereas group 2 (758 patients) had no apparent history of Sm infection. It was concluded from all the studied parameters that Sm infection not only increases the risk of HCC but also interferes with HCC treatment response, as the response was found to be better in group 2 (Shousha et al. Reference Shousha, Abdelaziz, Nabeel, Omran, Abdelmaksoud, Elbaz, Salah, Harb, Hosny, Osman, Atef, Gaber, Zayed, Yosry and Leithy2022).

Pathogenesis

Sm pathogenesis develops with the initiation of a moderate Th1 response, which is marked by high levels of pro-inflammatory cytokines including interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ). After about 6 weeks of infection, Sm worms mature, and females start depositing eggs. These deposited eggs then release soluble egg antigens (SEAs), which interact with the immune system (Hiatt et al. Reference Hiatt, Sotomayor, Sanchez, Zambrana and Knight1979). SEAs lead to the release of pro-inflammatory cytokines through resident macrophages leading to recruitment of monocytes, lymphocytes, and neutrophils, which leads to initiation of granuloma formation and inflammation (Stadecker Reference Stadecker1999; Shaker et al. Reference Shaker, Samy and Ashour2014). With time, SEAs act as a key force behind the transition of a pro-inflammatory Th1 response to a relative anti-inflammatory Th2 response, which is characterised by increased levels of IL-4, IL-10, and IL-13 and recruitment of eosinophils (Pearce et al. Reference Pearce, Caspar, Grzych, Lewis and Sher1991; Zheng et al. Reference Zheng, Zhang, Chen, Nie, Miller, Gong and Liu2020).

The development of granulomas is supposed to be a beneficial response for the host as it sequesters the eggs, which, if left unchecked, can lead to a heightened and uncontrolled inflammatory response, leading to permanent tissue damage (Nono et al. Reference Nono, Ndlovu, Aziz, Mpotje, Hlaka and Brombacher2017). However, granulomas become detrimental for the liver as it leads to liver fibrosis (Morais et al. Reference Morais, Souza, Melo, Aroucha, Miranda, Domingues, Abath and and Montenegro2008; Culver et al. Reference Culver, Watkins and Westbrook2016). Egg granulomas are majorly composed of a huge number of eosinophils, lymphocytes, monocytes, macrophages, and neutrophils (Liu et al. Reference Liu, Zhang, Liang and Lu2022) and are known to contain T lymphocytes of both Th1 and Th2 responses (Bogen et al. Reference Bogen, Flores Villanueva, McCusker, Fogelman, Garifallou, el-Attar, Kwan and Stadecker1995). An SEA (i.e., IPSE/alpha-1 (interleukin-4-inducing factor from schistosome eggs)) is a major hepato-toxin (Abdulla et al. Reference Abdulla, Lim, McKerrow and Caffrey2011) that is linked with the enlargement of these granulomas (Fahel et al. Reference Fahel, Maced, Pinheiro, Caliari and Oliveira2010).

Interestingly, the cores of granulomas have different immune cells depending upon the infecting schistosomal species (Jain and Rana Reference Jain and Rana2024). Apart from Sm, Sj has also been implicated in HCC induction and has been classified as a possible carcinogen (group 2B of IARC classification) (IARC 1994). Both Sj and Sm are capable of granuloma formation, but the granulomas elicited by them show distinct cellular composition. The core of a granuloma elicited by Sj is mainly composed of neutrophils, whereas that of Sm is primarily composed of eosinophils. A chemokine binding protein that is secreted by Sm eggs is capable of binding to a specific chemokine and inhibiting its action. It binds to neutrophil chemo-attractant CXCL8 but does not bind to eosinophil chemo-attractant CCL11, which blocks the infiltration of neutrophils but not eosinophils (Chuah et al. Reference Chuah, Jones, Burke, McManus and Gobert2014; Jain and Rana Reference Jain and Rana2024).

Liver fibrosis, which initiates in response to the deposited Sm eggs and mainly occurs in the hepatic sinusoids and portal veins (Hoffmann et al. Reference Hoffmann, Cheever and Wynn2000; Wang et al. 2020), encompasses an excessive deposition of the extracellular matrix (ECM), including a down-regulation of ECM degradation and an up-regulation of ECM production. Hepatic stellate cells (HSCs) are a source of collagen and have the potential to abundantly secrete ECM proteins and tissue inhibitors of metalloproteinases, all of which are responsible for changing the liver architecture (Puche et al. Reference Puche, Saiman and Friedman2013). These accumulated proteins initiate the distortion of the hepatic architecture (Bataller and Brenner Reference Bataller and Brenner2005), which is followed by periportal hepatic fibrosis (PHF), the most serious manifestation of chronic schistosomal infection.

PHF impedes the blood flow and the nutrient reach to the liver, causing impairment of liver function. This can lead to an advanced stage of liver fibrosis leading to cirrhosis. Cirrhosis involves permanent replacement of healthy liver tissue with scar tissue. This interferes with the normal function of the liver, disposing it towards cancer induction (Pinter et al. Reference Pinter, Trauner, Peck-Radosavljevic and Sieghar2016), and ultimately leads to portal hypertension, hepatomegaly, and splenomegaly. It also predisposes the patient to liver failure if not provided with appropriate treatment (Wiest et al. Reference Wiest, Wu, Zhong, McGarvey, Tan, Yuan, Peters, Olveda and Olds1993; Coutinho et al. Reference Coutinho, McGarve, Acosta, Manalo, Langdon, Leenstra, Kanzaria, Solomon, Wu, Olveda, Kurtis and Friedman2005; Kamdem et al. Reference Kamdem, Moyou-Somo, Brombacher and Nono2018; von Bülow et al. Reference von Bülow, Lichtenberger, Grevelding, Falcone, Roeb and Roderfeld2021; Jain and Rana Reference Jain and Rana2024).

A number of studies have shown a very high proportion of people being infected with HBV and/or HCV viruses as well as Sm, especially in endemic regions (Michielsen et al. Reference Michielsen, Francque and van Dongen2005; Gasim et al. Reference Gasim, Bella and Adam2015). In the case of HBV, 300 million people are globally co-infected with HBV and Sm (Khatami et al. Reference Khatami, Bahadory, Ghorbani, Saadati, Zarei, Soleimani and Zibaei2021). Multiple studies show that the concomitant infection with hepatitis viruses and Sm causes advanced liver diseases and worsens the outcome especially if viral load is high, leading to an increased mortality rate. This increased mortality is a result of increased incidences of cirrhosis as well as advanced and more aggressive HCC progression (Omar Reference Omar2019; Allam et al. 2023).

Carcinogenesis

All the pathologic factors from initiation of inflammation to fibrosis, cirrhosis, and co-infection with hepatitis viruses lead to a cascade of events at the molecular and genetic level that destabilises the genetic makeup. Mutations in proto-oncogenes and tumour suppressor genes lead to the induction of cancer. Chronic inflammation can lead to the production of reactive oxygen species (ROS), which exert an oxidative stress and induce oxidative damage to both nuclear and mitochondrial DNA as well as affect signal transduction pathways (Wang et al. Reference Wang, Li, Ye, Xie and Li2016).

Similarly, an array of studies have linked liver fibrosis and cirrhosis with the shortening of telomere length in the liver cells (Donati and Valenti Reference Donati and Valenti2016; Barnard et al. Reference Barnard, Moch and Saab2018; Shin et al. Reference Shin, Park, Yu, Jin, Suh, Lee and Kim2021), and such telomere shortening in hepatocytes has been correlated with chromosomal instability, which has the potential to initiate hepatoma in humans (Plentz et al. Reference Plentz, Caselitz, Bleck, Gebel, Flemming, Kubicka, Manns and Rudolph2004). A number of other signalling molecules, transcription factors, and cellular receptors have been linked with Sm and HCC and are described below.

A study involved the examination of the relationship between Sm infection and ROS production and inflammosome activation (Chen et al. Reference Chen, Cheng, Chang, Cao, Feng, Chen, Lam and Peng2019). It found that, along with a heightened release of ROS and other super-oxides, expression of an important transcription factor linked with expression of pro-inflammatory genes as well as cell survival (i.e., NF-κB) (Liu et al. Reference Liu, Zhang, Joo and Sun2017) is also increased along with activation of NLRP3 and AIM2 inflammasomes (Chen et al. Reference Chen, Cheng, Chang, Cao, Feng, Chen, Lam and Peng2019). This implied the role of Sm infection in prolonging cell survival as well as the creation of inflammatory conditions that predispose hepatic tissue to cancer. A recent study has shown that oxidative stress induced by deposited eggs triggers proliferation of human hepatoma cells, thus supporting cancer progression (von Bülow et al. Reference von Bülow, Schneider, Dreizler, Russ, Baier, Buss, Lichtenberger, Härle, Müller, Tschuschner, Schramm, Pons-Kühnemann, Grevelding, Roeb and Roderfeld2024).

Another study (Roderfeld et al. Reference Roderfeld, Padem, Lichtenberger, Quack, Weiskirchen, Longerich, Schramm, Churin, Irungbam, Tschuschner, Windhorst, Grevelding and Roeb2020) analysed the activation of critical hepatic cancer regulators, c-Jun and STAT3, by Sm infection in the liver of hamsters, primary hepatocytes, Huh7 cells, and human liver biopsies. A permanent activation of these hepatic cancer-associated regulators (i.e., as c-Jun and linked transcription factor STAT3) was observed by SEAs released from tissue-trapped eggs, which seem to be significant contributors in the development of liver cancer in Sm-infected patient. Interestingly, IPSE/alpha-1 is one of the major SEAs behind the activation of these regulators (Roderfeld et al. Reference Roderfeld, Padem, Lichtenberger, Quack, Weiskirchen, Longerich, Schramm, Churin, Irungbam, Tschuschner, Windhorst, Grevelding and Roeb2020).

IPSE/alpha-1 is also linked with the development of B-regulatory cells (Bregs), which are known to accelerate HCC by inducing growth and promoting the migratory potential of cancer cells. Bregs also lead to the production of IL-10, which induces T-regulatory cells (Tregs) differentiation. Tregs induce a tolerogenic microenvironment encouraging cancer growth (Shao et al. Reference Shao, Lo, Ling, Liu, Ng, Chu, Ma, Li, Fan and Man2014; Haeberlein et al. Reference Haeberlein, Obieglo, Ozir-Fazalalikhan, Chay, Veninga, van der Vlugt, Voskamp, Boon, den Haan, Westerhof, Wilbers, Schots, Schramm, Hokke and Smits2017; Lurje et al. Reference Lurje, Hammerich and Tacke2020). This tolerogenic environment, which, along with Tregs, is also composed of Th2 milieu and tolerogenic dendritic cells, suppresses tumour-specific immune responses, and establishes an immunosuppressive tumour microenvironment linking Sm with HCC-inducing potential (Chaudhary and Elkord Reference Chaudhary and Elkord2016).

SEAs from Sm eggs are also known to promote endothelial cell proliferation in vitro as well as up-regulate VEGF in human endothelial cells, hence inducing angiogenesis-related processes that can contribute to neo-vascularisation and help in development and progression of hepatic cancer (Freedman and Ottesen Reference Freedman and Ottesen1988; Loeffler et al. Reference Loeffler, Lundy, Singh, Gerard, Hudson and Boros2002).

Similarly, the expression level of a protein, high mobility group box 1 (HMGB1), has been reported to be heightened in serum levels of patients with schistosomiasis. It not only contributes to liver inflammation and tissue injury but also is responsible for enabling HSCs to change to a proliferative myofibroblast-like phenotype (Zhong et al. Reference Zhong, Gui, Hou, Lv and Jin2022). The initiation and progression of liver fibrosis involves activation and proliferation of HSCs. Quiescent HSCs (qHSCs) get activated in response to fibrotic stimuli, including persistent inflammatory responses induced by chronic infection, to become activated HSCs (aHSCs) (Wynn Reference Wynn2008; Li et al. Reference Li, He, Guo, Chen and Shan2015), which subsequently transform into myofibroblasts (Zhang et al. Reference Zhang, Yuan, He, Lei and Wang2016). This predisposes heptic tissue to advanced fibrosis and probable cirrhosis, ultimately leading to a potential HCC.

Tumour aggressiveness in hepatic tissue cancer has been linked to the expression of programmed death ligand 1 (PD-L1). The expression of PD-L1 is increased in macrophages in patients with Sm infection. An increase in its expression promotes tumour progression and probably its induction as it helps cancer cells to escape the host’s immune response, which is linked to T cell anergy (Smith et al. Reference Smith, Walsh, Mangan, Fallon, Sayers, McKenzie and Fallon2004; Calderaro et al. Reference Calderaro, Rousseau, Amaddeo, Mercey, Charpy, Costentin, Luciani, Zafrani, Laurent, Azoulay, Lafdil and Pawlotsky2016).