The Role Of Hepatic Inflammatory Status In Postoperative Recurrence/metastasis Of Hepatocellular Carcinoma And Its Predication And Intervention

Primary liver cancer is one of the most common malignant tumors,and is currently the third and second leading cause of tumor-related death worldwide and in China,respectively.Hepatocellular carcinoma(HCC) undoubtedly contributes to the majority primary liver cancer cases.Despite tremendous achievements being carried out in HCC clinically and basically during past decades,the overall prognosis of HCC remains dismal with a 5-year survival at about 5%.Even after curative resections,the 5-year recurrence/metastasis rates remain as high as 60%-70%.Therefore,recurrence or metastasis has emerged as the main obstacle to get better curative effects.The vast majority of previous studies focused solely on malignant cells themselves,regarding tumors just as masses of autonomous cells and aiming at identifying the molecular and genetic changes associated with this malignant transformation.However,the potential of a tumour cell to metastasize depends on its interactions with the homeostatic factors that promote tumour cell growth,survival, angiogenesis,invasion and arrest in distant organs.Given the "site-specific metastases" characteristic of tumor metastases,Stephen Paget had developed the theory of "seed and soil" in 1898,and recently,with the advent of high throughput analyses like genomic,protemics and tissue microarray,numerous growth factors, chemokines,guidance molecules,signaling pathways and,more importantly,new genes in host microenvironment are discovered and provide a new identity for organ-specific metastasis.Inflammatory/immune related elements contribute a great deal to the establishment of host microenvironment and modulate the progression of cancers.For instance,chronic inflammation is capable of generating a potentially "vicious self-sustaining loop(s)" which are resulted in the pro-cancer microenvironment favorable for survival of tumor cells and their growth.Therefore. the inflammatory responses of the target organs may be also of great significance regarding to the prognosis of cancer patients.However,inflammation can either promote or inhibit cancer metastasis/ recurrence.If we can modulate this double-edged sword of inflamrnation rigorously in order to impress the favorablc host inflammatory microenvironment or irritate unfavorable one for tumor metastasis, tumor recurrence may be under control.To achieve this goal,it is indispensable for us to get a panorama about the status of host inflammatory response,and to understand the accurate relation between one certain inflammation status and tumor recurrence/metastasis;otherwise,any intervention targeted at inflammation response will be aimless.The liver has its own unique immune system,acting as a key immune regulator locally and systemically.In addition,considering the background of hepatitis B and/ or C virus infection,the involvement of inflammation in HCC is of great significance. Among the abundant resident lymphocytes and inflammatory-related cells,hepatic stellate cell(HSC) is significantly involved in the progression of chronic hepatitis to hepatic fibrosis or even liver carcinogenesis.All these inflammatory or inflammatory-related cells are critical for the maintenance of immune homeostasis via their interaction with the circulating lymphocytes.However,in the context of infections,these cells will protract the inflammation reaction and inevitably result in fibrosis or carcinogenesis.For instance,our previous study has proved that the intratumoral inflammation/ immune status could influence the invasion and metastastic capability of hepatoma cells and was related to the prognosis of HCC. More importantly,the recurrence/metastasis of HCC after curative resections mostly occur within the residual liver tissues;hence liver itself is the major target organ for HCC recurrence/metastasis.It is reasonable for us to suggest that the inflammation response in the peritumoral liver tissue is likewise indispensable for HCC recurrence/ metastasis.Unfortunately,no systemic evaluations of the peritumoral inflammation response and its relevance to HCC recurrence have been carried out yet.As a result. there is no available information for an accurate monitoring and guiding purpose for postoperative therapies.Hepatic inflammation-oriented interventions should be helpful in suppressing HCC recurrence/metastasis.According to the "seed and soil" theory,therapies should be targeted not only against the cancer cells themselves,but also against the homeostatic inflammatory factors,lnterferon-α(IFN-α) can both ease the inflammation responses of HCV and/or HBV infection,and inhibit the proliferation, invasion and metastasis of tumor cells in vitro.Clinically.IFN-αhas been proved to be able to postpone the recurrence of HCC after curative resection.Therefore,IFN-αfits well with above-mentioned criteria.However,these contributions are based upon the long-term application with a high-dose of IFN-αwhich is near the toxic dose and related to visible by-effects.Moreover,the resistances to IFN-αin renal cancer, gladder carcinoma and so on have been detected.Cycloxygenase-2(COX-2) has been proved to be the key in IFN-αresistance,and agents such as curcumine and celecoxib can suppress COX-2 expression and improve the effect of IFN-α.With respect to HCC,there was also resistance to IFN-α.So,it is urgently demanded to find a way to decrease the dose of IFN-αmeanwhile improve its effect.Although chemosynthesis agents such as celecoxib are available candidates,their clinical utility is restricted by their side effects including cardiovascular complaints.The traditional Chinese medicine,however,is another choice,for its gentle effects with little side effects. Oxymatrine is now widely employed in the treatment for HBV with an effect similar to that of IFN-α.Besides,oxymatrine can modulate immune activity,and inhibit hepatic fibrosis and tumor progression.In vitro,oxymatrine were able to inhibit proliferation and induce apoptosis of liver cancer cells.Hence,oxymatrine,similar to IFN-α,can both modulate hepatic inflammation and repress tumor cells.We suggest that IFN-αin combination with oxymatrine may display better anticancer effects.COX-2 is heavily involved in IFN-αresistance.However,its role in HCC IFN-αtreatment is still unknown.In chronic liver diseases,COX-2 is unregulated to induce local infiltration of T lymphocytes and macrophages.Additionally,COX-2 is actively involved in HCC initiation via facilitating the integrating of HBx gene into host hepatocytes.Using COX-2 inhibitors can usually block HCC progression and improve the prognosis.However,the roles of COX-2 in liver inflammation,fibrosis and carcinogenesis are paradoxical.Therefore,a better understand of the role of COX-2 in HCC IFN-αtreatments should been based upon a thorough understanding of the relationship between COX-2 and HCC invasion,metastasis and prognosis.The aim of this study is(a) illuminate the correlation between hepatic inflammation and HCC metastasis;(b) estimate the effect of low-dose IFN-αin HCC treatments;(c) explore the role of COX-2 in IFN-αtreatments based upon identification the involvement of COX-2 in HCC metastasis and prognosis.The hepatic inflammation status is evaluated both with periphery parameters includingγ-glutamyltransferase(GGT),alanine aminotransferase(ALT) and their ratio (GGT/ALT),and with peritumoral inflammation(related) cells including activated hepatic stellate cell(aHSC) and mast cell(MC) by tissue microarray(TMA) and quantitive RT-PCR methods.With respect to the interventions,low-dose IFN-αis used both in vitro and in vivo to determine its influence over HCC.In addition. oxymatrine is utilized in combination with low-dose IFN-αto exam whether this combination can improve IFN-αeffect or not,and to evaluate its effect over tumor cells and hepatic inflammation.Partâ… .The relevance of peripheral GGT and ALT to HCC prognosisPurpose:To investigate the involvement of hepatic inflammation in HCC reucrrecne/metatstasis by evaluating peripheral GGT,ALT and GGT/ALT.Methods:In this study we enrolled one independent and random cohort of 219 HCC patients underwent curative resection in Zhongshan Hospital,Fudan University from Jan 2002 to Dec 2006.All the patients with other diseases which can influence the level of GGT and ALT were excluded.The live reserve functions of all patients are in Child-Pugh A stage.The serum samples were taken two days before operations and the normal rang of GGT and ALT were 11-50 IU/L and＜75 IU/L,respectively. According to the documents,60 and 80 IU/L were used as the cutoff points for GGT and ALT,respectively.The overall survival(OS) and time to recurrence(TTR) were compared between high and low subgroups of GGT and ALT.Moreover,we calculated the ratio of GGT to ALT(GGT/ALT),and divided GGT/ALT into high and low subgroups by a optimal cutoff point which was created by X-tile software. The prognostic value of GGT/ALT was examined as well.Results:GGT and ALT were related to each other in a linear fashion(r=0.411,P＜0.001).However.as dichotomized variables,GGT and ALT were distributed unequally(P＜0.001).Most patients had high levels of GGT(n=110) while low levels of ALT(n=185).ALT displayed no relation to recurrence or survival,while GGT was independently associated with survival(P=0.002).The GGT/ALT ratio could predict survival precisely either in a continuous or dichotomized fashion(P＜0.001 and=0.001.respectively),and also related to recurrence when dichotomized(P =0.002).Additionally,high GGT/ALT ratio was associated with high early-recurrence rates,more recurrence-related death and various aggressive tumor characteristics such as lager tumor size,vascular invasion,poor encapsulation and advanced BCLC stage.In further stratified analyses,this ratio could discriminate the outcomes of patients with high- or low-α-fetoprotein level.Conclusion:The hepatic inflammation status which was evaluated by GGT/ALT, could predicate HCC prognosis perfectly.Additionally,GGT/ALT was also related to the tumor burden although it was an inflammatory indicator.Therefore,we suggested that the hepatic inflammatory status was correlated with HCC recurrence/metastasis, and GGT/ALT was a good parameter for us to select patients with high recurrence risks.Moreover,since GGT/ALT was conveniently available even for out-patients,it should be possible for us to judge the fluctuation of tumor burden and hepatic inflammation status,and further to modify the strategy of certain therapies.Partâ…¡.The relevance of peritumoral inflammatory(related) cells to HCC prognosisPurpose:To invest the involvement of peritumoral inflammatory(related) cells in HCC recurrence/metastasis,and their relation to hepatic inflammation status.Methods & Results:Two independent and random cohorts of HCC patients underwent curative resection in Zhongshan Hospital,Fudan University,were enrolled as study populations:cohort A,from Fed 2002 to Nov 2005,n=130;cohort B,from Jan 2002 to Dec 2006,n=207.The messenger RNA(mRNA) levels of the functional genes in aHSC(ie,FAP,SPARC,and TNC),quantitated by real-time quantitative polymerase chain reaction,and the density of peritumoral Foxp3+T-regulatory cells (Tregs) and CD68+ macrophages(Mφ),assessed immunohistochemically in tissue microarray sections,were positively correlated with the density of peritumoral aHSC. The density(P=0.007 for recurrence-free survival[RFS]and P=0.021 for overall survival[OS]) and functional genes(FAP,P=0.001 for RFS;SPARC,P=0.007 for RFS and P=0.021 for OS) of peritumoral aHSC independently contributed to high recurrence or death rates,as did peritumoral Tregs or Mφ.In addition,both density and FAP expression of aHSC were positively related to peripheral GGT/ALT level(r =0.176 and 0.204,respectively;P=0.04 and 0.02,respectively).Moreover. peritumoral aHSC were related to more early recurrences.It is important to note that the density of peritumoral aHSC,in combination with FAP and SPACR mRNA or density of Tregs and Mφ,might predict prognoses more effectively.In the context of cohort B,we found density of MC was positively associated with that of peritumoral Treg(r=0.353,P＜0.001) and peripheral GGT/ATL(r=0.146,P=0.036).MC can only predicate recurrence independently;however,when combined with Treg,MC was independently associated with both OS and recurrence.Similar to aHSC.high MC related to more early intrahepatic recurrence rather than later ones.Elevated peritumoral MC density was also related to larger tumor size and poor encapsulations. Conlusion:(a) Peritumoral aHSC was associated with HCC prognosis both in cellular and in gene level,aHSC can aggravate hepatic inflammation via its functional genes including FAP and SPARC,and through its role accumulating Treg and Mφ. Therefore,aHSC should be a promising therapeutic target to modulate hepatic inflammation response and prevent recurrence/metastasis.(b) Peritumoral MC can facilitate HCC recurrence/metastasis on a similar fashion to aHSC.(c) The positive relationships between GGT/ALT and aHSC and MC suggested that both peritumoral pathological parameters and peripheral biochemistry parameters can reflect the hepatic inflammation properly.These two methods were related to each other.Partâ…¢.The relevance of cyclooxygenase-2 to the invasion and metastasis of hepatoma cells and HCC prognosisPurpose:To estimate the involvement of COX-2 in HCC IFN-αtreatment,this study is carried out to evaluate the relationships between COX-2 and the invasion and prognosis of HCC.Method:Seven hepatoma cell lines with changed metastatic potency were used, in addition to 80 randomly selected HCC patients without later or extrahepatic metastasis.COX-2 expressions were assessed in cell lines with Western-blot,and in tumor samples with quantitive RT-PCR.Results:(a) Cellular COX-2 expressions were simultaneously decreased with the decrease in matastastic power;(b) The intratumoral COX-2 expression was independent of any clinicopathologic parameters;(c) The higher COX-2 expression was.the poorer prognosis would be.The hazard ratios were 1.916 for recurrence and 3.522 for death,respectively(P=0.025 and＜0.001,respectively).Conclusion:High COX-2 expression may promote the metastasis of hepatoma cells and facilitate early intrahepatic recurrence.If intervention can inhibit COX-2 expression,its therapeutic effect should be improved.Partâ…£.The influence over the metastatic power of MHCC97-H from low-dose IFN-αalone and in combination with oxymatrine in vitroPurpose:To evaluate the therapeutic role of low-dose IFN-αalone and in combination with oxymatrine,and the involvement of COX-2 in IFN-αeffect.Methods:According to documents.1×10³IU/ml was defined as low-dose IFN-α, and MHCC97-H cell line was selected for in vitro study.There were four interventions:control group with no intervention,low-dose IFN-αonly group, oxymatrine only group and low-dose IFN-α+oxymatrine group.We compared the proliferation,apoptosis and metastasis power of MHCC97-H cells under different intervention.In addition,COX-2 expression was evaluated in each group.Results:(a) Low-dose 1FN-αcould not inhibit the proliferation and metastasis of MHCC97-H,even the intervention was prolonged.Also,it was invalid in inducing cell apoptosis.(b) Oxymatrine could inhibit cell proliferation to some extent,with an IC₅₀=44.5mg/ml,and 10mg/ml was chosen for further intervention in order to not mask the effect of combination.(c) In a time-dependent fashion,the combination could inhibit cell proliferation and metastasis power significantly,and also increase cell apoptosis significantly.(d) Low-dose IFN-αcould increase the COX-2 expression in a time-dependent fashion.However,when oxymatrine was added,the COX-2 expression shrunk significantly.Conclusion:The in vitro anti-tumor capability of low-dose IFN-αis demolished for a COX-2 inducing ability.Oxymatrine can retrieve and improve the anti-tumor power of low-dose IFN-αvia its role in reversing COX-2 expression.We proposed that low-dose IFN-αin combination with oxymatrine could result in synergetic anti-tumor effects.Partâ…¤.In vivo impact of low-dose IFN-αalone and in combination with oxymatrine on the hepatic inflammation status and the growth and progression of orthotopic implantated MHCC97-H tumorsPurpose:To validate the anti-tumor effect and inflammation modulation power of low-dose lFN-αin combination with oxymatrine in vivo.Methods:IFN-αwas used at 1×10⁶IU/ml,0.1ml/d(s.c.) and oxymatrine at 60mg/kg·d(i.p.).Male BALB/c nu/nu mice(20g) were divided into four subgroups: control group(â… ,n=12),low-dose IFN-αgroup(â…¡,n=8),oxymatrine group(â…¢,n= 8) and combination group(â…£,n=6).All interventions were given for 62 days unless the mice were dead before being sacrificed.There were 3,2 and 2 mice dead before day 62 in control,low-dose IFN-αand oxymatrine group,respectively.Another four subgroups(n=12 in each subgroup) were simultaneously established to compare the life span.So.there were respectively 15,14,14 and 12 mice in control,low-dose IFN-α,oxymatrine and the combination group for life span comparison.Results:(a) In groupâ…¡.the mice weights were lowest and the tumor volumes were largest;while the thorough opposite occurred in groupâ…£.(b) Among four groups,the lung metastasis incidence of groupâ…£was the lowest(P=0.011). Low-dose IFN-αcan increase the number of lung metastasis significantly(P=0.018). (c) Compared to groupâ… ,the life span of groupâ…£was significantly improved;the life span of groupsâ…¡andâ…¢were similar to each other.(d) The GGT or GGT/ALT,which was positively related to the volume of tumors in each group,was significantly elevated in groupâ…¡,while suppressed in groupâ…£.(e) Intratumoral and peritumoral aHSC density was increased in groupâ…¡,and decreased in groupâ…£.(f) Intratumoral COX-2 expressions in groupsâ… andâ…¡were much higher than those in groupâ…¢andâ…£.Conclusion:Low-dose IFN-αcould not inhibit the orthotopic growth, progression and lung metastasis of implanted tumors in vivo.In addition,the life span of this group was not prolonged significantly.Contrarily,groupâ…£displayed profound orthotopic tumor inhibition,lung metastasis depression and life span expansion effects.We suggested the combination could modulate the hepatic inflammation and directly inhibit tumor cells effectively.The former was displayed by a marked decrease in aHSC density and GGT/ALT level which was positively associated with tumor volumes.The latter was displayed by a profound decrease in lung metastasis incidence and numbers.Importantly,we proposed that the suppression of COX-2 was the key for oxymatrine to incorporate with low-dose IFN-α.Conclusions1.The hepatic inflammation status is of prognostic significance for HCC.Evaluations of either peripheral GGT/ALT or peritumoral inflammatory(related) cells can reflect the hepatic inflammation status and predicate prognosis perfectly. The former is convenient to practice and can be used in therapeutic effect monitoring.The latter provides us with mechanical explanations and promising therapeutic targets.2.Low-dose IFN-αis insufficient in inhibiting tumor cells and modulating hepatitis.When combined with oxymatrine,the invasion and metastatsis of tumor cells can be well handled.COX-2 decrease is the key for the cooperation of low-dose IFN-αand oxymatrine.3.This combination can inhibit orthotopic implanted tumors progression via modulating the hepatic inflammation status.The novelty of this study1.For the first time,we demonstrated and reported that peripheral GGT/ALT was tightly associated with HCC prognosis.This ratio was a linkage between hepatic inflammation and tumor burden.More importantly,it was applicable in screening patients with high recurrence risks and monitoring the therapeutic effect.2.For the first time,we demonstrated and reported that peritumoral aHSC and MC were related to the HCC prognosis.These parameters provided us with promising therapeutic targets and were also helpful in screening patients with high recurrence risks.3.We found that low-dose lFN-αwas insufficient in inhibiting the invasion and metastasis of tumor cells,and in modulating hepatic inflammation.On the contrary,the combination of low-dose IFN-αand oxymatrine were effective in tumor cell inhibition and hepatic inflammation modulation.COX-2 decrease was the key for the synergetic effect of this combination.The potential application of this project1.Both peripheral GGT/ALT level and peritumoral aHSC or MC can well reflect the hepatic inflammation status.These parameters with prognostic power are of clinical utility in screening high recurrence risk patients and monitoring therapies.2.The combination of low-dose IFN-αand oxymatrine is a novel adjuvant therapy for HCC patients after resection.