| Hepatic progenitor cells (HPCs), which reside in canals of Hering, when activated cangive rise to ductular reactions (DRs). DRs correlate with the degree of inflammationand fibrosis in the course of many chronic human liver diseases. Since most of thesediseases are strong etiological factors for primary liver cancer, activation of the HPCcompartment has been strongly linked to hepatic carcinogenesis. Progenitor cell-likefeatures suggesting an HPC origin has been strongly associated with a poor prognosisin hepatocellular carcinoma (HCC).Combined hepatocellular-cholangiocarcinoma (CHC) is a malignant primary livertumor that contains elements of both hepatocellular carcinoma (HCC) and intrahepaticcholangiocarcinoma (ICC). Although relatively rare among liver cancers, CHC hasgarnered recent attention due to its distinct pathological features suggesting an HPCorigin, aggressive biological behavior, and related poor clinical outcome. While aprogenitor cell-like phenotype of CHC has been well established by morphologicaland immunohistochemical observations, and while certain clinical correlates ofsurvival have been described, pathological correlates of clinical outcome have notbeen previously identified.In the present study, we performed a clinicopathological study on80patients withCHC who underwent hepatectomy to explore the prognostic predictive utility ofintratumoral and non-tumor pathological findings, focusing on HPC-related features. The study of non-tumor transit-amplifying compartments is critical because suchfeatures may suggest a "field effect" that predicts the development of metachronoustumor recurrence. We found that proliferative ductular reactions in non-tumor tissuewere independent prognostic factors for both OS and DFS and independentlyassociated with multicentric occurrence. PATIENTS AND METHODSPatients and Clinical Data. From January1997to December2003, patients whounderwent hepatectomy in the Department of Hepatic Surgery and who werepostoperatively confirmed as Combined hepatocellular-cholangiocarcinoma wererecruited for prospective follow-up. Informed consent was obtained from each patientunder a protocol approved by the Hospital Research Ethics Committees. Neutrophil-to-lymphocyte ratio (NLR) was measured as an indicator of inflammatory status,previously established as a prognostic indicator in HCC. Tumor stage wasdetermined according to the2009UICC TNM classification system. We definedresection with curative intent as complete excision of the primary tumor with negativemicroscopic margins≥2cm from the tumor and with no residual tumors indicated byultrasonography and computed tomography (CT) scan within1month of initialsurgery.Follow-up and detection of recurrence. All patients were followed regularly every2–3months after surgery until study closure in July2008with serum AFP/CA19-9andabdominal ultrasonography. Progressive elevation of serum AFP/CA19-9levelsand/or ultrasonographic detection of a new hepatic lesion prompted hospitalization forconfirmation of diagnosis and appropriate management, including repeat resection,radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE) orsupportive therapy. Recurrence was confirmed by contrast-enhanced imaging studiesor cholangiography according to standard guidelines for HCC, ICC or radiologicfeatures of CHC described previously. Overall survival (OS) was defined as theinterval between the dates of surgery and death while disease-free survival (DFS) wasdefined as the interval between the dates of surgery and recurrence. If recurrence was not diagnosed, patients were censored on the date of death or the last follow-up.Clinical follow-up was not disclosed to laboratory personnel until statistical analysis.Histopathological diagnosis and analysis. Histological staining methods and criteriafor CHC diagnosis are presented in Supplemental Methods. The degree ofinflammation and fibrosis was graded and staged according to the method of Ishak etal. Since some cases showed marked geographical histological heterogeneity indifferentiation, the histopathological grade of each tumor component was defined bythe poorest degree of differentiation identified within the tumor. Histological grade oftumors was determined according to previously described schemas."Multicentricoccurrence"(MO) was also assessed in HCC components. Briefly, MO was definedby the presence of either (1) at least2nodules which included an early tumor within adysplastic nodule or without substantial destruction of the hepatic architecture (so-called "nodule in nodule" appearance) or (2) moderately-or poorly-differentiatedtumors within a margin of a well-differentiated tumor. Dysplastic foci or noduleswere diagnosed following recent consensus guidelines.Immunohistochemistry and evaluation. We performed immunohistochemistry onparaffin-embedded4μm sections of80CHCs from which both tumor and non-tumortissues (>2cm away from tumor) were available. A standard two-stepimmunoperoxidase-labeled protocol with goat anti-mouse/rabbit HRP (EnVision,Dako, Glostrup, Denmark) was applied stringently on all slides.Evaluation of Reactive Lesions in Non-tumor Liver. Cell types and reactive lesionswere identified according to Roskams et al., with some terminology variations. In ourstudy, the term ductular reaction is specific for reactive ductules with biliary/HPCphenotype arranged in irregularly shaped structure residing along parenchymal- stromal boundaries. The description of terminology, morphology and evaluationschema for these reactive lesions is detailed. In addition, proliferation rate in ductularreaction was evaluated by calculating PCNA labeling index. Specifically, ten400×high powered fields including epithelial-stromal boundaries within each section wererandomly chosen and captured. The same fields were captured in sequential serialsections stained with K7for quantification of the number of K7+reactive ductularcells (RDCs) in reactive ductules. The proliferation index of DR (PI-DR) wascalculated as ratio between the number of PCNA immuno-reactive nuclei and the totalnumber of RDCs.Double-fluorescence immunostaining. For antigen co-localization studies, double-fluorescence immunostaining of formalin-fixed paraffin-embedded tissue wasperformed with a sequential fluorescent method as described previously. Alexa488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) and Alexa568-conjugatedgoat anti-rabbit IgG (Invitrogen) were used as secondary antibodies.To reduce autofluorescence, tissue sections were treated as previously described.Immunofluorescence was observed with the Olympus IX-71(Olympus Corp., Japan).Under these conditions, single labeling appears green (Alexa488) or red (Alexa568),whereas co-labeling appears to be yellow or orange.Statistical Methods. We applied normality tests on all numeric variables beforefurther analysis. Continuous normally distributed variables are summarized as mean±SD and represented graphically as mean with standard error of the mean (SEM) bar.Non-normally distributed variables are summarized by median and range. To comparethe means between groups, analysis of variance (ANOVA) or Student's t test wasperformed. To determine differences of non-normal variables between groups, medians were tested by Mann-Whitney U tests. The degree of association wasdetermined by Spearman or Pearson correlation as appropriate. Methods for survivaldata analysis were described in Supplemental Methods. Collinearity was diagnosedamong variables before all regression analyses. Reliability of the all models was thentested by residual or receiver operating characteristic (ROC) analysis. All analysiswas carried out using SPSS software version12.0(SPSS Inc., Chicago, IL) or Rsoftware (R Foundation, Vienna, Austria). P<0.05was considered significant.SUPPLEMENTAL METHODSTissue processing and histological staining. Dissection, fixation, embedding andstorage of resected surgical specimens were performed per routine hospital standardoperating procedures consistent with accepted standards. Specifically, specimens wereobtained from the tumor including the tumor margin, tumor with adjacent non-tumorliver, and distant non-tumor liver. To confirm histopathological diagnoses, all blockscontaining tumor including margin (5-8blocks), tumor with adjacent non-tumor liver(2-3blocks) and non-tumor liver (1-3blocks) were accessed. The hematoxylin/eosin(HE) and Alcian blue-Periodic acid-Schiff (AB-PAS) staining protocols wereperformed as previously described.Overview of immunohistochemistry evaluation. Computer-assisted analysis was usedto evaluate membrane and/or cytoplasmic antigen expression. Five200×high powerfield of each section were randomly chosen and captured by the HV-C20A CCDcamera (Hitachi Ltd., Japan) coupled to a Leica DM-RXA2microscope (LeicaMicrosystems, Germany). Identical settings were used for each photograph. Fields inareas with necrosis, with poor morphology, or at the margins of sections wereexcluded. Stromal staining of secreted AFP was also excluded from analysis. Image analysis software (ImagePro Plus6.0, Media Cybernetics, Bethesda, MD) was thenused to assess the mean immunoreactive density by measuring Integrated OpticalDensity (IOD) per case using the method described by Zhu et al. Box-Coxtransformation was then applied.Nuclear antigens, such as PCNA and p21Waf1/Cip1, were quantified by calculatinglabeling indices. Within non-tumor parenchyma, PCNA labeling indices werecalculated separately for periportal (Rappaport's hepatic acinus zone1), perivenular(Rappaport's hepatic acinus zone3) areas, or overall. A hepatocyte replicative arrestindex was calculated as the ratio between the number of strongly p21Waf1/Cip1-positivenuclei and the total number of hepatocytic nuclei in non-tumor sections. Within tumor,PCNA was used to measure cellular proliferation using a PCNA labeling index (PI-T)defined as the ratio between the number of strongly PCNA-positive nuclei and thetotal number of tumor cell nuclei. In addition, hepatic progenitor cell (HPC)-liketumor cells were identified as small and oval-shaped tumor cells with hyperchromaticnuclei and scant cytoplasm. These cells, that were always intensively K7-positive,were counted and averaged per1000tumor cells on K7-stained tumor sections.Diagnosis of combined hepatocellular-cholangiocarcinoma. CHC diagnosis wasestablished by two highly experience pathologists identifying unequivocal histologicpresence of intimately mixed hepatocellular and cholangiocellular elements within anindividual tumor. Until the latest WHO classification (4th edition,2010) detailedsubtypes of CHC, detailed diagnostic criteria were not available. We applieddiagnostic criteria largely based upon previous WHO guidelines from1994and2000.We excluded separate HCC/ICC and "collision tumors" so that all included casesconsisted of Allen and Lisa type3tumors, which correspond to WHO4thedition classical type combined hepatocellular-cholangiocarcinoma or subtypes with stem-cell features.The typical hepatocellular characteristics of combined hepatocellular-cholangiocarcinoma were a trabecular pattern of growth and bile production.Confirmation of hepatocellular differentiation was obtained by immunostaining forHepPar1, K18or pCEA. Cholangiocellular elements were identified by smallglandular formation composed of small cuboidal cells with round nuclei withoutnucleoli resembling a biliary epithelium with abundant fibrous stroma. Though theproduction of PAS+mucin could be demonstrated in a majority of ICC components,the amount of mucin was variable. As proposed by Maeda et al., K7and K19in ICCareas were used to confirm biliary differentiation when mucin was not detected. Areasin which HCC elements and ICC elements were difficult to discriminate were labeled"intermediate areas". Cholangiolocarcinoma (CLC) component has small tumor cellswith high nuclear:cytoplasmic ratio and hyperchromatic, oval nuclei. The tumor cellsgrow in a monotonous tubular, cord-like, anastomosing pattern embedded in a fibrousstroma, which is so-called "antler-like" pattern. Tumors with predominantlycholangiolocarcinoma morphology that were diagnosed clinically ascholangiolocarcinoma were not recruited to the present study.Survival data analysis. Continuous variables were quantized for potential applicationin clinical decision-making. Univariate Kaplan–Meier estimation was used forevaluating the effect of variables on postoperative recurrence and overall survival.Acting on the principle that the p-value or hazard ratio should not influence the choiceof cutoff value, we pre-determined the cutoff values to reduce the risk of false-positive associations. For previously validated predictive variables, cutoff values weredetermined either based upon previous reports or upon clinically relevant threshold values. For instance, since the normal range for serum CA19-9was reported as0-37U/ml,37U/ml was used as the cutoff value. Values for positive staining for PCNA(clone PC10), a well-accepted marker of cellular proliferation, were categorized basedupon previously described criteria:1,0-25%of the cells positive;2,26-50%of cellspositive;3,51-75%of cells positive;4,76-100%of cells positive, respectively."Strong" proliferative activity within ductular reaction was defined as more than halfof reactive ductular cells being involved in cell cycle (PI-DR≥50%). For continuousvariables with no or less well established cutoffs, median values were used as cutoffvalues for Log-Rank tests. Time-dependent ROC methods were used to definealternative cutoff values with the highest Youden's J value to confirm lack ofassociation. An R software (R Foundation, Vienna, Austria) package "survivalROC"was used. Variables with univariate associations with p<0.10were selected for furtherCox proportional hazards models analysis. A backward elimination approach wasused to remove non-significant variables and determine the most parsimonious modelin multivariate analysis. Model assumptions were tested on the basis of deviance andSchoenfeld residuals. RESULTSClinical, histological and follow-up data.88Chinese patients were recruited. Fourpatients presenting with recurrent tumors and four patients whose non-tumor tissues(>2cm away from the primary tumor) were not available were excluded. Of theremaining80patients,70had undergone surgery with curative intent. None hadreceived any preoperative anti-tumor treatment. Samples from51patients wereassessed as "HCC-predominant",18"ICC-predominant", and8"intermediate area"predominant; the latter phenotype has, previously been described as "CHC withstem-cell feature". HCC and ICC components were equally dominant in3patients."Antler-like" features representing a cholangiolocarcinoma component was observedin12cases. Within ICC areas,4cases contained a component of squamous cellcarcinoma and1case also contained sarcomatous changes. Within non-tumor sections,51cases (63.8%) were confirmed cirrhotic, whereas only2patients showed nofibrosis. Adjacent to tumors, foci or nodules (>1mm) with dysplastic features werefrequently observed. Large cell change (LCC) was found in64(80%) cases and smallcell change (SCC) in55(68.8%). Coexisting dysplastic nodules (withoutdistinguishing low-grade or high-grade) were found in16(20.0%) cases. Among64patients undergoing non-curative resections (n=10), median survival was only88days.In the curative resection group (n=70), probability of overall survival (OS) at1-,3-and5-years was74.3%,38.6%and30.0%.64out of70(91.4%) patients developedintrahepatic recurrence (n=63) and/or distant metastases (n=4,3lung and1abdominalwall). The1-,3-and5-year probability of disease-free survival (DFS) was41.4%,18.6%and10.0%. After detection of intrahepatic recurrence,31patients receivedTACE and9patients received repeat resection (2of whom after initial TACE). Onepatient received RFA. After repeat resections, seven patient samples were available for pathological evaluation,3of which revealed HCC with biliary differentiation,1HCC,2ICC and1complete necrosis.Ductular reaction is observed in non-tumor tissue from CHC patients:relationship with background transit-amplifying compartments. In non-tumorK7staining sections of entire group (n=80), all patients showed increased intensity ofDR, with5patients in1+(≤10%),11patients in2+(10%to25%),22patients in3+(26%to50%),42patients in4+(≥50%). In the curative resection group,37of70(52.9%) showed4+(≥50%) in intensity of DR. In terms of PI-DR which measuredproliferation status of DR,21of80patients (26.3%) had PI-DR≥50%. In thecurative resection group, PI-DR≥50%was found in19/70patients (27.1%).Importantly, we observed some cases with intensive K7staining DR (4+) showinglow level of PCNA. The observation of this dissociation of K7expression andproliferation within DR was supported by statistical analyses across the cohort(r=0.074, P=0.512). Around portal areas of non-tumor sections stained with K7, HPCscan be observed with intensive staining in the Canals of Hering (CoH) location.Intermediate hepatobiliary cells (IHBCs) were found around HPCs or in continuitywith reactive ductules. EpCAM or OV-6positive hepatocytes were also observed.K7-DR was only found associated with number of HPCs (r=0.423, P<0.001). Bycontrast, cases with high PI-DR were found not only to have increased number ofHPCs (r=0.714, P<0.001), but also more IHBCs (r=0.506, P<0.001) as well as higherOV-6expression in parenchyma (r=0.514, P<0.001). A subgroup of DR expressingBmi-1was also found with higher PI-DR (p<0.001). To reduce potential impacts ofunspecific proliferation stimulators or confounding factors, correlations werecorrected for PCNA labeling index within hepatocytes, necroinflammation score, stage of fibrosis, gender, age at surgery, status of virus infection and chronicalcoholism. After correction, PI-DR was still significantly associated with all offactors. Cumulatively, these associations suggest that HPC activation is a dominantfeature in background liver.Consistent with prior studies, both PI-DR and K7-DR correlated with hepaticinflammation as measured by the Ishak grade score. Correlations were also foundbetween PI-DR/K7-DR and fibrosis stage. In addition, higher level of PI-DR wasfound associated with older age at surgery and impaired hepatocyte replicationmeasured by hepatocyte p21Waf1/Cip1labeling index (p21-LI). Multivariate analysisshowed that PI-DR was independently associated with fibrosis stage, hepatocytereplicative arrest and age. By contrast, K7-DR only correlated withnecroinflammation score.PI-DR, but not intratumoral HPC/biliary markers expression, is associated withdisease-free survival (DFS). We then asked if these HPC related futures can predictOS or DFS without or with correcting for demographic, clinical and pathologicparameters in the curative resection group (n=70). In univariate analysis, the OS wassignificantly associated with "multicentric occurrence"(MO), absence of AFPexpression by the tumor, PI-DR, K7-DR and PCNA-LI of preportal hepatocytes. DFSwas associated with MO, microvascular invasion (MVI), neutrophil-to-lymphocyteratio (NLR), necroinflammation score, and PI-DR, but not K7-DR. Thus, specificfeatures of the tumor (MO and MVI) as well as markers of non-tumor liver wereassociated with both OS and DFS on univariate analysis. As Model A shown, MO,K7-DR, PI-DR and absence of AFP remained significant predictors for OS.Histological grade of the ICC component showed a trend for association with OS(p=0.057). By contrast, DFS was only associated with MVI and PI-DR. Background "progenitor dominant" regeneration pattern is associated withworsened DFS. In parallel to OV-6, AFP is an immature hepatocyte markerrepresenting hepatocyte dedifferentiation that was expressed mostly in hepatocytesnear parenchymal-stromal boundaries. Scattered AFP positive hepatocytes also couldbe observed in centrilobular and intermediate zones in some cases. However, doubleimmunostaining showed that co-expression of AFP and OV-6or EpCAM inhepatocytes was extremely rare (2/80,2.5%). The observation were supported byfinding that OV-6and AFP expression (IOD) in hepatocytes were inversely correlated(r=-0.343, p=0.002). Additionally, lower hepatocyte OV-6expression is significantlyassociated with higher hepatocyte P21-LI (r=-0.343, P=0.002). This finding supportsthe concept that progenitor activation and hepatocyte dedifferentiation are the twoprocesses contributing to DR origin and related regeneration. We therefore classifiedthe predominant liver regeneration pattern in each case based on the expressionpatterns of AFP and OV-6in parenchymal as follows:(1) OV-6highAFPlowwereregarded as "progenitor dominant" regeneration;(2) OV-6lowAFPhighwere regarded as"dedifferentiation dominant" regeneration;(3) OV-6lowAFPlowwere regarded as "mild"regeneration;(4) OV-6highAFPhighwere regarded as "progenitor/dedifferentiationsynergistic" regeneration. In survival analyses within curative resection group, the"progenitor dominant" regeneration pattern was associated with the poorest OS,highest recurrence rate, and was characterized by the greatest level of PI-DR. InModel B, OV-6or AFP expression in hepatocytes was independent predictor for OS,and OV-6could independently predict DFS. Thus, an OV-6highAFPlowbackground"progenitor dominant" regeneration pattern may help define a "field effect" that isstrongly associated with DFS. HPCs features in tumor are associated with higher PI-DR: proliferative ductularreactions as a "field effect". Intratumoral cells with a HPC/DR-like phenotype, co-expressing of HPC/biliary markers (e.g. K7, K19, OV-6or EpCAM) and immaturehepatocyte markers (e.g. AFP), were common by double immunostaining (53/80,66.3%).We then found an interesting "field effect" that PI-DR in non-tumor liver wasassociated with expression of several intratumoral HPC/biliary markers including K7(P=0.010), K19(P=0.037), OV-6(P=0.001), and c-Kit (P=0.005), also number ofK7+HPC-like tumor cells (r=0.232, P=0.039). Increased PI-DR was also associatedwith a greater intratumoral proliferation index (PI-T)(P=0.003)."Multicentric occurrence"(MO) of CHC is associated with proliferative activitywithin DR."Multicentric occurrence" feature was observed in32patients, including6patients showing "nodule in nodule" appearance. Univariate analysis showedpresence of MO was associated with both higher PI-DR and OV-6expression in non-tumor sections. Patients with MO had smaller tumor size, consistent with MOdescribed originally in early HCC. By contrast, we found no association between MOand MVI. Multivariate analysis with the presence of MO as dependent variableshowed independent associations with PI-DR, tumor size, stage of fibrosis andconfluent multinodularity.ConclusionAll of results revealed that:1. Combined Hepatocellular-cholangiocarcinoma is a primary liver carcinomawith great intratumoral heterogeneity highly consistent with previoushypothesis indicating its hepatic progenitor cell origin.2. Diverse ductular reaction phenotypes being bound up with altered hepaticcellular repair coexist within the background disease entity, shaped bycomplexity of the disease evolution. Proliferative ductular reaction can partlydistinguish the heterogeneity of ductular reaction phenotypes representinghepatic progenitor cells transit-amplifying. 3. Proliferative ductular reaction is a predictor for recurrence of CombinedHepatocellular-cholangiocarcinoma after resection with curative intent.Activation of HPCs with the purpose of parenchymal regeneration may be oneof the critical drivers of CHC recurrence.4. Proliferative ductular reaction is associated with intratumoral heterogeneity ofCHC, defining a "field effect". The lack of association of recurrence andintratumoral HPC/biliary markers expression suggests that the poor prognosisof tumors with HPC features results not only due to aggressive tumor biology,but also due to the transit-amplifying HPCs related "field effect" in non-tumorliver associated with subsequent tumor development.5. Proliferative ductular reaction is independently associated with "multicentricoccurrence", a feature of CHC representing metachronous multifocaltumorigenesis suggesting HPC activation may contribute to CHC recurrenceby inducing metachronous multifocal tumorigenesis. |
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