Practice guidelines for the pathological diagnosis of primary liver cancer: 2015 update

In 2010, a panel of Chinese pathologists reported the first expert consensus for the pathological diagnosis of primary liver cancers to address the many contradictions and inconsistencies in the pathological characteristics and diagnostic criteria for PLC. Since then considerable clinicopathological studies have been conducted globally, prompting us to update the practice guidelines for the pathological diagnosis of PLC. In April 18, 2014, a Guideline Committee consisting of 40 specialists from seven Chinese Societies(including Chinese Society of Liver Cancer, Chinese Anti-Cancer Association; Liver Cancer Study Group, Chinese Society of Hepatology, Chinese Medical Association; Chinese Society of Pathology, Chinese Anti-Cancer Association; Digestive Disease Group, Chinese Society of Pathology, Chinese Medical Association; Chinese Society of Surgery, Chinese Medical Association; Chinese Society of Clinical Oncology, Chinese Anti-Cancer Association; Pathological Group of Hepatobiliary Tumor and Liver Transplantation, Chinese Society of Pathology, Chinese Medical Association) was created for the formulation of the first guidelines for the standardization of the pathological diagnosis of PLC, mainly focusing on the following topics: gross specimen sampling, concepts and diagnostic criteria of small hepatocellular carcinoma(SHCC), microvascular invasion(MVI), satellite nodules,and immunohistochemical and molecular diagnosis. The present updated guidelines are reflective of current clinicopathological studies, and include a novel 7-point baseline sampling protocol, which stipulate that at least four tissue specimens should be sampled at the junction of the tumor and adjacent liver tissues in a 1:1 ratio at the 12, 3, 6 and 9 o'clock reference positions. For the purposes of molecular pathological examination, at least one specimen should be sampled at the intratumoral zone, but more specimens should be sampled for tumors harboring different textures or colors. Specimens should be sampled at both adjacent and distant peritumoral liver tissues or the tumor margin in order to observe MVI, satellite nodules and dysplastic foci/nodules distributed throughout the background liver tissues. Complete sampling of whole SHCC ≤ 3 cm should be performed to assess its biological behavior, and in clinical practice, therapeutic borders should be also preserved, even in SHCC. The diagnostic criteria of MVI and satellite nodules, immunohistochemical panels, as well as molecular diagnostic principles, such as clonal typing, for recurrent HCC and multinodule HCC were also proposed and recommended. The standardized process of pathological examination is aimed at ensuring the accuracy of pathological PLC diagnoses as well as providing a valuable frame of reference for the clinical assessment of tumor invasive potential, the risk of postoperative recurrence, long-term survival, and the development of individualized treatment regimens. The updated guidelines could ensure the accuracy of pathological diagnoses of PLC, and provide a valuable frame of reference for its clinical assessment.

Surgicopathological classification of hepatic space-occupying lesions:A single-center experience with literature review

Accompanying rapid developments in hepatic surgery,the number of surgeries and identifications of histological types of primary hepatic space-occupying lesions (PHSOLs) have increased dramatically.This has led to many changes in the surgicopathological spectrum of PHSOLs,and has contributed to a theoretical basis for modern hepatic surgery and oncological pathology.Between 1982 and 2009 at the Eastern Hepatobiliary Surgery Hospital (EHBH) in Shanghai,31 901 patients underwent surgery and were diagnosed as having a PHSOL.In this paper,we present an analysis of the PHSOL cases at the EHBH for this time period,along with results from a systematic literature review.We describe a surgicopathological spectrum comprising more than 100 types of PHSOLs that can be stratified into three types:tumor-like,benign,and malignant.We also stratified the PHSOLs into six subtypes derived from hepatocytes;cholangiocytes;vascular,lymphoid and hemopoietic tissues;muscular,fibrous and adipose tissues;neural and neuroendocrine tissues;and miscellaneous tissues.The present study provides a new classification system that can be used as a current reference for clinicians and pathologists to make correct diagnoses and differential diagnoses among various PHSOLs.

Combined hepatocellular and cholangiocarcinoma originating from the same clone:a pathomolecular evidence-based study

Background:Combined hepatocellular and cholangiocarcinoma(CHC) is a unique subtype of liver cancer comprising both hepatocellular carcinoma(HCC) and intrahepatic cholangiocarcinoma(ICC);however,its cellular origin remains unclear.The purpose of this study was to investigate the clinicopathologic features and the clonal relationship between HCC and ICC in 34 patients with CHC.Methods:The clinicopathologic features and prognosis of the 34 CHC patients were compared with those of 29 patients with separated HCC and ICC(5HC).Loss of heterozygosity(LOH) at 10 highly polymorphic microsatellite markers was detected in 16 CHC and 10 SHC tissues for determination of the clonal origin of CHC.Expression of hepatocyte markers[hepatocyte paraffin 1(Hep Par 1) and glypican 3(GPC3)]and cholangiocyte markers[cytokeratin(CK)7 and 19]in tumor tissues was examined by immuno histochemical analysis.Results:In the 16 CHC specimens,the difference in LOH patterns between HCC and ICC was less than 30%,suggesting the same clonal origin of HCC and ICC.Consistent with this finding,immunohistochemical analysis revealed that hepatocyte markers(Hep Par 1 and GPC3) and cholangiocyte markers(CK7 and CK19) were simultaneously expressed in both the HCC and ICC components in 52.9%of CHC specimens,suggesting that the two components shared a similar phenotype with hepatic progenitor cells(HPCs).On the contrary,in all 10 SHC cases,the difference in LOH patterns between the HCC and ICC components was greater than 30%,suggesting different clonal origins of HCC and ICC.Overall survival and disease-free survival were shorter for patients with CHC than for patients with SHC(P < 0.05).Conclusions:Our results suggest that the HCC and ICC components of CHC may originate from the same clone,having the potential for dual-directional differentiation similar to HPCs.CHC tended to exhibit the biological behaviors of both HCC and ICC,which may enhance the infiltrative capacity of tumor cells,leading to poor clinical outcomes for patients with CHC.

Micrometastasis in surrounding liver and the minimal length of resection margin of primary liver cancer

AIM： To describe the distribution of micrometastases in the surrounding liver of patients with primary liver cancer （PLC）, and to describe the minimal length of resection margin （RM） for hepatectomy. METHODS： From November 2001 to March 2003, 120 histologically verfied PLC patients without macroscopic tumor thrombi or macrosatellites or extrahepatic metastases underwent curative hepatectomy. Six hundreds and twenty-nine routine pathological sections from these patients were re-examined retrospectively by light microscopy. In the prospective study, curative hepatectomy was performed from November 2001 to March 2003 for 76 histologically verfied PLC patients without definite macroscopic tumor thrombi or macrosatellites or extrahepatic metastases in preoperative imaging. Six hundreds and forty-five pathological sections from these patients were examined by light microscopy. The resected liver specimens were minutely examined to measure the resection margin and to detect the number of daughter tumor nodules, dominant lesions, and macroscopic tumor thrombi inside the lumens of the major venous system. The paraffin sections were microscopically examined to detect the microsatellites, microscopic tumor thrombi, fibrosis tumor capsules, as well as capsule invasion and the distance of histological spread of the micrometastases. RESULTS： In the retrospective study, 70 micrometastases were found in surrounding liver in 26 of the 120 cases （21.7%）. The farthest distance of histological micrometastasis was 3.5 mm, 5.3 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Macroscopic tumor thrornbi or rnacrosatellites were observed in 18 of 76 cases, and 149 rnicrometastases were found in the surrounding live in 25 （43.1%） of 58 cases with no macroscopic tumor thrombi. The farthest distance of histological micrometastasis was 4.5 mm, 5.5 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Two hundred and sixty-seven rnicrometastases were found in surrounding liver in 14 （77.8%） out of 18 cases with macroscopic tumor thrombi or macrosatellites. The farthest distance of histological micrometastasis was 18.5 mm, 18.5 mm and 19.0 mm in 95%, 99% and 100% cases, respectively. CONCLUSION： The required minimal length of RM is 5.5 mm and 6 mm respectively to achieve 99% and 100% rnicrometastasis clearance in surrounding liver of PLC patients without macroscopic tumor thrornbi or rnacrosatellites, and should be greater than 18.5 mm to obtain 99% rnicrometastasis clearance in surrounding liver of patients with macroscopic tumor thrornbi or rnacrosatellites.

E-selectin and its ligand-sLeX in the metastasis of hepatocellular carcinoma

Objective: To study the significance of E-selectin andits ligand-sLeX in the metastasis of hepatocellularcarcinoma (HCC).Methods: Flow cytometry and immunohistochemistrywere used to detect the expression of E-selectin and itsligand-sLeX in both HCC cell lines and human HCCtissues.Results: The positive rate of E-selectin in vascular en-dothelial cells adjacent to cancer was 67.9% (19/28). Theexpression of E-selectin in tumors accompanied withemboli or satellite foci was significantly higher than thatin tumors without emboli or satellite foci (P<0.05),and it was not related to tumor size, tumor capsule,AFP content, and the degree of differentiation. Thepositive expression of sLeX in SMMU-7721, PLF/PRF/5 and HepGⅡ cell lines was 7.03%, 63.35% and97.29% respectively. The positive cells of sLeX mainlydistributed in the margin of tumor tissues. The positiveexpression of sLeX in HCC cells in emboli or invasivetumor tissues was much higher than in primary foci.Conclusion: E-selectin and its ligand-sLeX are closelycorrelated with the metastasis of HCC.

Postoperative adjuvant transcatheter arterial chemoembolization improves the prognosis of patients with huge hepatocellular carcinoma

Background: Surgical resection of huge hepatocellular carcinoma(HCC, ≥ 10 cm) is potentially curative. More adjuvant treatments are needed to reduce relapses in these patients. We evaluated the influence of postoperative adjuvant transcatheter arterial chemoembolization(PA-TACE) on the prognosis of huge HCC. Methods: Data from consecutive patients who underwent curative resection for huge HCC in our center were retrospectively collected. Recurrence-free survival(RFS) and overall survival(OS) were compared between patients who did and did not undergo PA-TACE. Propensity score matching(PSM) was used. Results: Among the 255 enrolled patients, 93 underwent PA-TACE. The clinical outcomes were significantly better in the PA-TACE group than those in the non PA-TACE group(5-year RFS rate: 33.5% vs. 18.0%;5-year OS rate: 47.0% vs. 28.0%, all P<0.001). After PSM, similar results were obtained(5-year RFS rate: 28.8% vs. 17.6%, P<0.001;5-year OS rate: 42.5% vs. 25.0%, P=0.004). PA-TACE decreased the possibility of early recurrence(<2 years, crude cohort: P<0.001, PSM cohort: P<0.001) but not late recurrence( ≥ 2 years, crude cohort: P=0.692, PSM cohort: P=0.325). Multivariable Cox regression analysis suggested that PA-TACE was an independent protective factor prolonging early RFS, RFS and OS. Conclusions: PA-TACE is a safe intervention for huge HCC patients after liver resection and improves outcomes.

Copy Number Aberrations of Multiple Genes Identified as Prognostic Markers for Extrahepatic Metastasis-free Survival of Patients with Hepatocellular Carcinoma

Extrahepatic metastasis confers unfavorable patient prognosis in patients with hepatocellular carcinoma(HCC),however,reliable markers allowing prediction of extrahepatic metastasis at the time of initial diagnosis are still lacking.This study was to identify gene-level copy number aberrations(CNAs)related to extrahepatic metastasis-free survival of HCC patients,and further examine the associations between CNAs and gene expression.Array comparative genomic hybridization(aCGH)and expression array were used to analyze gene CNAs and expression levels,respectively.The associations between CNAs of a panel of 20 genes and extrahepatic metastasis-free survival were analyzed in 66 patients with follow-up period of 1.6-90.5 months.The gene expression levels between HCCs with and without gene CNA were compared in 109 patients with HCC.We observed that gains at MDM4 and BCL2L1,and losses at APC and FBXW7 were independent prognostic markers for extrahepatic metastasis-free survival of HCC patients.Integration analysis of aCGH and expression data showed that MDM4 and BCL2L1 were significantly upregulated in HCCs with gene gain,while APC and FBXW7 were significantly downregulated in HCCs with gene loss.We concluded that gene gains at MDM4 and BCL2L1,and losses at APC and FBXW7,with concordant expression changes,were associated with extrahepatic metastasis-free survival of HCC patients and have potential to act as novel prognostic markers.

A Panel of Genes Identified as Targets for 8q24.13-24.3 Gain Contributing to Unfavorable Overall Survival in Patients with Hepatocellular Carcinoma

Copy number aberrations （CNAs） in chromosome arm 8q have been associated with unfavorable clinical outcomes of several cancers and progressive tumor characteristics of hepatocellular carcinoma （HCC）. This study was to identify correlation of CNAs in 8q with clinical outcomes of HCC patients, and further screen for differentially expressed genes in outcome-related CNAs. Array comparative genomic hybridization and expression arrays were performed to detect CNAs and expression levels, respectively. The correlations between CNAs in 8q and outcomes were analyzed in 66 patients, with a median follow-up time of 45.0 months （range, 2.6-108.6 months）. One hundred and nine cases were further evaluated to identify differentially expressed genes in the potential outcome-related CNAs. Copy number gain in 8q was observed in 22 （33.3%） of the 66 HCC cases. The most recurrent gains （with frequencies 〉20%） were 8q 13.3-21.3, 8q21.3-23.3, 8q23.3-24.13, 8q24.13-24.3, and 8q24.3. Survival analysis showed that 8q24.13-24.3 gain was significantly associated with reduced overall survival （P=0.010）. Multivariate Cox analysis identified 8q24.13- 24.3 gain as an independent prognostic factor for poor overall survival （HR=2.47; 95% CI=1.16-5.26; P=0.019）. A panel of 17 genes within the 8q24.13-24.3 region, including ATAD2, SQLE, PVT1, ASAP1, and NDRG1 were significantly upregulated in HCCs with 8q24.13-24.3 gain compared to those without. These results suggest that copy number gain at 8q24.13-24.3 is an unfavorable prognostic marker for HCC patients, and the potential oncogenes ATAD2, SQLE, PVT1, ASAP1, and NDRG1 within the regional gain, may contribute coordinately to the 8q24.13-24.3 gain-related poor prognosis.

Twist2 is a valuable prognostic biomarker for colorectal cancer

AIM: To investigate the significance of Twist2 for colorectal cancer (CRC). METHODS: In this study, 93 CRC patients were included who received curative surgery in Eastern Hepatobiliary Surgery Hospital from January 1999 to December 2010. Records of patients' clinicopathological characteristics and follow up data were reviewed. Formalin-fixed, paraffin-embedded tissue blocks were used to observe the protein expression of Twist2 and E-cadherin by immunohistochemistry. Two independent pathologists who were blinded to the clinical information performed semiquantitative scoring of immunostaining. A total score of 3-6 (sum of extent + intensity) was considered as Twist2-positive expression. The expression of E-cadherin was divided into two levels (preserved and reduced). An exploratory statistical analysis was conducted to determine the association between Twist2 expression and clinicopathological parameters, as well as E-cadherin expression. Furthermore, the variables associated with prognosis were analyzed by Cox's proportional hazards model. Kaplan-Meier analysis was used to plot survival curves according to different expression levels of Twist2. RESULTS: Twist2-positive expression was observed in 66 (71.0%) samples and mainly located in the cytoplasm. Forty-three (46.2%) samples showed reduced expression of E-cadherin. There were no significant correlations between Twist2 expression and any of the clinicopathological parameters. However, Twist2-positive expression was significantly associated with reduced expression of E-cadherin (P=0.040). Multivariate analysis revealed that bad M-stage [hazard ratio (HR)=7.694, 95%CI: 2.927-20.224,P < 0.001] and Twist2-positive (HR=5.744, 95%CI: 1.347-24.298,P=0.018) were the independent risk factors for poor overall survival (OS), while Twist2-positive (HR=3.264, 95%CI: 1.455-7.375, P=0.004), bad N-stage (HR=2.149, 95%CI: 1.226-3.767, P=0.008) and bad M-stage (HR=10.907, 95%CI: 4.937-24.096, P < 0.001) were independently associated with poor disease-free survival (DFS). Survival curves showed a definite trend for Twist2-negative patients to have longer OS and DFS than Twist2-negative patients, not only overall, but also for patients in different stages, especially for DFS of patients in stage Ⅲ (P=0.033) and Ⅳ (P=0.026). CONCLUSION: Our data suggests, for the first time, that Twist2 is a valuable prognostic biomarker for CRC, particularly for patients in stage Ⅲ and Ⅳ.

Diagnosis and Treatment of Cholangiocarcinoma: A Consensus from Surgical Specialists of China

Cholangiocarcinoma refers to malignant tumors that develop in epithelial lining of biliary system, and it is divided into two categories according to tumor location, intrahepatic cholangiocarcinoma （ICC） and extrahepatic cholangiocarcinoma （ECC）. ICC occurs from the epithelial cells of the intrahepatic bile duct, its branches and interlobular biliary tree; and ECC is divided into hilar cholangiocarcinoma and distal cholangiocarcinoma by the circumscription at the confluence of cystic duct and the common hepatic duct.

Pathologic assessment of hepatocellular carcinoma in the era of immunotherapy:a narrative review

Background and Objective:Immune checkpoint inhibitor(ICI)-based therapy has achieved impressive success in various cancer types.Several ICIs have been unprecedentedly approved as the treatment regimens for advanced hepatocellular carcinoma(HCC)in recent decade.Meanwhile,numerous clinical trials are being performed to exploit more ICIs into initially unresectable HCC and postoperative HCC to expectantly induce adequate tumor downstaging for further resection or implement adjuvant treatment for relapse-free survival,respectively.In this review,we aim to summarize some pragmatic histomorphologic,immunohistochemical,and molecular pathologic parameters which promisingly indicate the response of neoadjuvant/conversion ICI-related therapy and predict the efficacy of adjuvant/therapeutic ICI-related therapy for HCC.Methods:We searched PubMed using the terms hepatocellular carcinoma,immunotherapy,immune checkpoint inhibitor,immune checkpoint blockade,conversion therapy,neoadjuvant therapy,adjuvant therapy,biomarker,pathologic evaluation,pathologic assessment till February 2023.Key Content and Findings:Although there is no consensus regarding the pathologic evaluation of relevant HCC specimens,it is encouraging that a few of studies have concentrated on this field,and moreover,the methods and parameters noted on other cancer types are also worthy of reference.For the pathologic assessment of HCC specimens underwent immunotherapy,a suitable sampling scheme,identifying immunotherapy-related pathologic response,and quantification of pathologic response rate should be emphasized.For the patients of HCC who are scheduled to receive immunotherapy,tumor-infiltrating lymphocyte,intratumoral tertiary lymphoid structure,programmed cell death ligand 1,Wnt/β-catenin,microsatellite instability and mismatch repair,tumor mutational burden and tumor neoantigen,as well as some other signaling pathways are the potential predictive biomarkers of treatment response of ICI.Conclusions:The management of HCC in the era of immunotherapy arises a brand-new pathological challenge that is to provide an immunotherapy-related diagnostic report.Albeit many related researches are preclinical or insufficient,they may tremendously alter the immunotherapy strategy of HCC in future.

Postoperative adjuvant transarterial chemoembolization for multinodular hepatocellular carcinoma within the Barcelona Clinic Liver Cancer early stage and microvascular invasion

Background:The survival benefit of postoperative adjuvant transcatheter arterial chemoembolization(PA-TACE)remained controversial.We aimed to investigate the prognosis effect of PA-TACE on the Barcelona Clinic Liver Cancer(BCLC)early stage multinodular hepatocellular carcinoma(MHCC)patients with/without microvascular invasion(MVI).Methods:Two hundred and seventy-one patients from January 2010 to December 2014 undergoing curative hepatectomy were included in this study.Disease-free survival(DFS)rates and overall survival(OS)rates as well as prognostic factors were analyzed by the Kaplan-Meier method,the log-rank test and the Cox proportional hazard model.Results:Thirty-four point four percent(44/128)MVI positive and 55.2%(79/143)MVI negative patients underwent PA-TACE.Multivariate analysis revealed that HBV DNA load>103 copy/Ml,>three tumors,MVI,and without PA-TACE were independent risk factors for poor DFS.Higher alkaline phosphatase(ALP),three tumors,MVI,and without PA-TACE were independent risk factors for poor OS.Both DFS and OS were significantly improved in patients with MVI who received PA-TACE as compared to those who underwent hepatic resection alone(5-year DFS,26.3%vs.20.7%,P=0.038;5-year OS,73.6%vs.47.7%,P=0.005).No differences were noted in DFS and OS among MVI negative patients with or without PA-TACE(5-year DFS,33.7%vs.33.0%,P=0.471;5-year OS,84.1%vs.80.3%,P=0.523).Early recurrence was more likely to occur in patients without PA-TACE(P=0.001).Conclusions:PA-TACE was a safe intervention and could effectively prevent tumor recurrence and improve the survival of the BCLC early stage MHCC patients with MVI.

Expert Consensus on Pathological Diagnosis of Intrahepatic Cholangiocarcinoma(2022 version)

Intrahepatic cholangiocarcinoma(iCCA)can originate from the large bile duct group(segment bile ducts and area bile ducts),small bile duct group(septal bile ducts and interlobular bile ducts),and terminal bile duct group(bile ductules and canals of Hering)of the intrahepatic biliary tree,which can be histopathological corresponding to large duct type iCCA,small duct type iCCA and iCCA with ductal plate malformation pattern,and cholangiolocarcinoma,respectively.The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies,tissue structures,growth patterns,invasive behaviors,immunophenotypes,molecular mutations,and surgical prognoses.For these reasons,this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA,mainly based on the 5th edition of the World Health Organization Classification of Tumours of the Digestive System.

New strategy to distinguish clonal origin of RHCC/MHCC between intrahepatic metastasis and multicentric occurrence

Hepatocellular carcinoma (HCC) is a kind of malignancy with high potential of metastasis and multicentric occurrence. The treatment of recurrent hepatocellular carcinoma (RHCC) and multinodular hepatocellular carcinoma (MHCC) is always a nodus because of the diverse clonal origin of RHCC/MHCC. Theoretically, the RHCC/MHCC can originate from intrahepatic metastasis (IM type) or multicentric occurrence (MO type). Our previous study proposed that there are at least 6 subtypes of clonal origin patterns in RHCC. RHCC and MHCC with different clonal origins have variant biological behaviors, clinical prognosis as well as treatment strategy. Generally speaking, patients with IM type HCC have a poorer prognosis compared with those with MO type HCC. Therefore, it is essential to emphasize the distribution of the clonal origin in HCC in order to determine the choice of clinical treatment. Undoubtedly, the detection of clonal origin pattern will become a promising breakthrough in the molecular pathological diagnosis of HCC. We should attach more attention to the establishment of a standardized molecular pathological clonal origin detection method and a new stratification of clinical treatment choice for RHCC/MHCC in future.