Histological Heterogeneity of Primary Liver Cancers: Clinical Relevance, Diagnostic Pitfalls and the Pathologist’s Role

Objective To investigate the correlation between the clonal origin of hepatocellular carcinoma (HCC) and the recurrence of pluri-nodular HCC after liver transplantation. Methods Sixty pluri-nodular HCC patients, conformed to the UCSF's standard, were selected and given liver transplantation. Then tumor nodules were separated from the normal tissue by microdissection. The state of 12 HCC high frequency microsatellite loss of heterozygosity (LOH) was detected by SSCP-PCR, and the tumor clone origin for intrahepatic metastasis (IM), multicentric occurrence (MO) and IM+ MO type was determined. The correlation between different types of clonal origin and the three-year tumor-free survival rate, tumor pathological features and the level of serum AFP was analyzed. Results Forty-five cases had two nodules of HCC, and the rest 15 cases had three nodules. After excluding two cases whom the clonal origin pattern was not identified, the remaining 58 cases were enrolled in the study. The IM group, MO group and IM+ MO group were set up, accounting for 34.48% (20/58), 56.90% (33/58) and 8.62% (5/58) respectively. In IM group, MO group and IM+ MO group, the three-year tumor-free survival rate was 50.00%, 78.79% and 40%; the incidence of MVI was 100%, 18.18% and 100%; the low differentiation rate of tumor was 80%, 51.52% and 80%; and the mean concentrations of AFP were 231.25 (2.78-9736.27), 24.59 (1.16-2674.50) and 122.58 μg/L (16.20-1608.03 μg/L) respectively. Conclusion The three-year tumor-free survival rate of MO was significantly higher than that of IM and IM+ MO types, which was closely related to the low incidence of tumor thrombi, the high degree of pathological differentiation and low preoperative AFP. Tumor clonal origin may serve as an important reference index for the evaluation of the disease stage of multiple nodules HCC and predict the risk of tumor recurrence after liver transplantation. Key words: pluri-nodular hepatocellular carcinoma; clonal origin; loss of heterozygosity; liver transplantation; pathology; tumor recurrence

Background & Aims: The molecular pathogenesis of primary liver cancer is extremely complex and heterogeneous, which was further complicated by multiple carcinogenesis. We aimed to elucidate the pathogenesis of multifocal primary liver cancer by genomic profiling. Methods: A patient with synchronous hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) who underwent resection and experienced post-operative intrahepatic metastases (IMs) was enrolled. Multiregion whole-exome sequencing was applied on the three primary tumors and two IMs to infer tumor clonality and evolution. The mutational and expression profiles, clinical relevance (n = 236) and functional implications of the identified potential driver gene FAT4 in HCC were investigated. Results: In total, we identified 12, 184, 126, and 54 of protein coding mutation in the cirrhotic liver, two HCCs (designated as HCC-A and HCC-B) and one ICC respectively. The mutational data of the two HCCs and one ICC showed almost no overlaps, suggesting that they developed through an accumulation of complete different sets of genetic alterations. For each tumor, multiregion sequencing data showed varied intratumoral heterogeneity (18.1% in HCC-A, 15.7% in HCC-B, 45.6% in ICC). The mutational profile of two IMs showed obvious similarity with HCC-A (87.6% and 87.7% shared with HCC-A respectively), rather than HCC-B and ICC tumors, indicating that they originated from HCC-A. Notably, FAT4 was the only gene that mutated in both HCCs and the two IMs, but in different locations (HCC-A and two IMs have the same G to A mutation at the coding position 2530, while HCC-B genomes have the A to C mutation at the coding position 14804). Prevalence screen revealed that FAT4 was mutated in over 15% of HCC patients and also several HCC cell lines. Significant down-regulation of FAT4 mRNA and protein expression was found in HCC, and loss of FAT4 independently correlated with early recurrence in HCC patients. In HCC cell lines, knockdown of FAT4 promoted and re-expression of FAT4 reduced cell growth and invasion. Conclusions: Spatial and temporal dissection of genomic alterations during the progression of liver cancer may help elucidate the basis for its dismal prognosis. FAT4 acts as a putative tumor suppressor that is frequently inactivated in human HCC. Citation Format: Qiang Gao, Zhi-Chao Wang, Rui-Bin Xi, Jia Fan. Inferring liver cancer evolution from spatial and temporal genomic heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2015-4814

Objective To explore the differential diagnostic significance of clone analysis for multicentric occurrence (MO) and intrahepatic metastasis (IM) in hepatocellular carcinomas (HCCs).Methods Loss of heterozygosity (LOH) and microsatellite instability (MSI) were analyzed by microsatellite polymorphism test and the integration sites of HBV were assessed by Southern blot in each nodule of the HCCs. The clonalities were then compared between each nodule of the same patient and the diagnosis of MO or IM was concluded. Finally, the results based on clonality analysis were compared with those according to clinicopathological and imaging data. Results According to the results of LOH and MSI in 79 nodules and nontumorous tissue from 35 cases of mutiple HCCs, 5 (14.3%)and 29 cases (82.9 %) were divided into MO and IM, respectively. Both MO and IM presented simultaneously in 1 patient (2.9%). The integration sites of HBV could be analyzed in 77 nodules from 34 multiple HCCs. Among them, 6 (17. 6%) and 27 cases (79.4%) were divided into MO and IM, respectively. Both MO and IM presented simultaneously in 1 patient (2.9%). The classification results of microsatellite polymorphism test and HBV integration sites analysis demonstrated a significant positive correlation (rs = 0.909, P＜0.001). Nevertheless, neither the classification of microsatellite polymorphism test nor that of HBV integrate sites analysis was correlated with the discrimination according to clinicopathologic and imaging data (rs=0. 133, P=0. 468, rs =0. 262, P=0. 155, respectively). Recurrence in patients in the MO group occurred significantly later than that in IM cases who were diagnosed by clonality analyses (P=0. 001). Conclusion The clonality analysis based on the results of LOH and MSI or assessments of HBV integrate sites is useful for the differential diagnosis of MO and IM and their treatment and prognosis. Key words: Carcinoma, hepatocellular; Multicentric occurrence; Intrahepatic metastasis; Clonality analysis

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the outcomes for patients are still poor. It is important to determine the original type of synchronous multinodular HCC for preoperative assessment and the choice of treatment therapy as well as for the prediction of prognosis after treatment. To analyze clinicopathologic characteristics and prognoses in patients with multicentric occurrence (MO) and intrahepatic metastasis (IM) of synchronous multinodular hepatocellular carcinoma (HCC). The study group comprised 42 multinodular HCC patients with a total of 112 nodules. The control group comprised 20 HCC patients with 16 single nodular HCC cases and 4 HCC cases with a portal vein tumor emboli. The mitochondrial DNA (mtDNA) D-loop region was sequenced, and the patients of the study group were categorized as MO or IM based on the sequence variations. Univariate and multivariate analyses were used to determine the important clinicopathologic characteristics in the two groups. In the study group, 20 cases were categorized as MO, and 22 as IM, whereas all 20 cases in the control group were characterized as IM. Several factors significantly differed between the IM and MO patients, including hepatitis B e antigen (HBeAg), cumulative tumor size, tumor nodule location, cirrhosis, portal vein and/or microvascular tumor embolus and the histological grade of the primary nodule. Multivariate analysis further demonstrated that cirrhosis and portal vein and/or microvascular tumor thrombus were independent factors differentiating between IM and MO patients. The tumor-free survival time of the MO subjects was significantly longer than that of the IM subjects (25.7 ∓ 4.8 months vs. 8.9 ∓ 3.1 months, p=0.017). Similarly, the overall survival time of the MO subjects was longer (31.6 ∓ 5.3 months vs. 15.4 ∓ 3.4 months, p=0.024). The multivariate analysis further demonstrated that the original type (p=0.035) and Child-Pugh grade (p<0.001) were independent predictors of tumor-free survival time. Cirrhosis (p=0.011), original type (p=0.034) and Child-Pugh grade (p<0.001) were independent predictors of overall survival time. HBeAg, cumulative tumor size, tumor nodule location, cirrhosis, portal vein and/or microvascular tumor embolus and histological grade of the primary nodule are important factors for differentiating IM and MO. MO HCC patients might have a favorable outcome compared with IM patients.

Clinicopathologic features between multicentric occurence and intrahepatic metastasis of multiple hepatocellular carcinomas related to HBV

Clonality Analysis for Multicentric Origin and Intrahepatic Metastasis in Recurrent and Primary Hepatocellular Carcinoma

PURPOSE: The indication for liver transplantation in malignant liver tumors has been controversial due to disappointing results and shortage of donor organs. The authors evaluated the experience and results of a single center in order to define present indications and selection criteria in hepatobiliary malignancy. PATIENTS AND METHODS: Retrospective analysis of 212 patients who underwent liver transplantation for malignant tumors between 1972 and 1995: Primary hepatobiliary tumors: hepatocellular carcinoma, n = 124 (with underlying cirrhosis, n = 86; fibrolamellar subtype, n = 8); intrahepatic bile duct (cholangiocellular) carcinoma, n = 24; proximal bile duct carcinoma, n = 29; other uncommon entities (n = 15); secondary liver tumors: neuroendocrine, n = 11, and nonendocrine, n = 9. RESULTS: Survival rates in primary liver cancer were correlated to International Union Against Cancer (UICC) tumor stage. For hepatocellular and proximal bile duct carcinoma significantly better outcome was found in UICC-tumor stage I and II versus III and IV. No long-term survival was found after transplantation for intrahepatic bile duct carcinoma, hemangiosarcoma and nonendocrine liver metastases. Comparison of transplant and resected patients with hepatocellular carcinoma stage I and II with underlying cirrhosis showed better survival after transplantation: 1-, 3-, 5-year survival rate of 83.3% versus 76.9%, 75.8% versus 44.0%, 60.6% versus 44.0%, and median survival 96.5 versus 23.2 months. Although this difference was not significant, no patient died from tumor recurrence in the transplant group versus three in the resection group. DISCUSSION AND CONCLUSIONS: Patients with malignant tumors can be selected for transplantation with predictable likelihood for long-term survival. According to the present data, liver transplantation can be considered in unresectable UICC-stage II hepatocellular and proximal bile duct carcinoma, the uncommon entities fibrolamellar carcinoma, epitheliod hemangioendothelioma and hepatoblastoma as well as liver metastases from neuroendocrine tumors. UICC-stage II and IV hepatocellular carcinoma as well as intrahepatic bile duct carcinoma, hemangiosarcoma and metastases from nonendocrine tumors should be excluded from transplantation alone. For hepatocellular carcinoma, multimodality treatment protocols have had a proven impact on the prevention of early recurrence and prolongation of survival. There is evidence that liver transplantation in still resectable hepatocellular carcinoma with underlying cirrhosis might be more appropriate in order to cure the cancer-bearing disease.

Multinodular hepatocellular carcinoma(HCC) might originate from multicentric occurrence (MO) or intrahepatic metastasis(IM). This study was to find out proteins which play important roles in clonal origin of multinodular hepatocellular carcinoma bt screening the differentially expressed proteins between the MO and IM tissues using comparative proteomic analysis. Total protein extracted was separated by two-dimensional gel electrophoresis. Comparative analyses of the 2-DE protein patterns between the two groups were carried out using computerized imaging techniques. Proteins exhibiting significant alternations were subsequently isolated and identified by mass spectrometry. A total 1025+/-52 and 900+/-98 spots were detected in the protein profile in IM and MO, respectively. Twenty-five protein spots were statistically different at expression levels between the two groups. Twenty of them were identified by MALDI-TOF-MS and bioinformatics. The protein profile of MO HCC tissues is different from that in IM HCC tissues. The twenty differentially expressed proteins might play a key role in the carcinogenesis and progression of multinodular HCC. These newly identified proteins might be potential and valuable biomarkers for identifying the multinodular HCC of clonal origin.

How should patients with hepatocellular carcinoma recurrence after liver transplantation be treated?

Ambiguous understanding of tumors and tumor microenvironments (TMEs) hinders accurate diagnosis and available treatment for multifocal hepatocellular carcinoma (HCC) covering intrahepatic metastasis (IM) and multicentric occurrence (MO). Here, we characterized the diverse TMEs of IM and MO identified by whole-exome sequencing at single-cell resolution. We performed parallel whole-exome sequencing and scRNA-seq on 23 samples from 7 patients to profile their TMEs when major results were validated by immunohistochemistry in the additional cohort. Integrative analysis of whole-exome sequencing and single-cell RNA sequencing found that malignant cells in IM showed higher intratumor heterogeneity, stemness, and more activated metabolism than those in MO. Tumors from IM shared similar TMEs while distinct TMEs were noticed in those from MO. Furthermore, CD20+ B cells, plasma cells, and conventional type II dendritic cells (cDC2s) were decreased in IM relative to MO while T cells in IM exhibited a more terminally exhausted capacity with a higher proportion of proliferative/exhausted T cells than that in MO. Both CD20 and CD1C correlated with better prognosis in multifocal HCC. Additionally, MMP9+ tumor-associated macrophages were enriched across IM and MO, which formed cellular niches with regulatory T cells and proliferative/exhausted T cells. Our findings deeply decipher the heterogeneous TMEs between IM and MO, which provide a comprehensive landscape of multifocal HCC.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma

Treatment for Hepatocellular Carcinoma in South Asia

Characteristics of multicentric hepatocellular carcinomas (HCCs) remain obscure. We therefore aimed to clarify them and compare them with HCC with intrahepatic metastases. A series of 118 patients who had definite hepatitis C viral status and multinodular HCC were divided into two groups: a multicentric occurrence (MO) group (n = 38), with multicentric HCCs; and an intrahepatic metastasis (IM) group (n = 80), with HCC having intrahepatic metastases. Clinicopathologic variables, including the patient's survival and disease-free survival rates, were compared between the MO and IM groups. Univariate analysis revealed the presence of esophageal varices, the presence of hepatitis C virus infection, a platelet count of less than 10 x 10(4)/microliter, hepaplastin test, gamma-globulin, the histologically active hepatitis, tumor size, des-gamma-carboxy prothrombin > 0.1 AU/ml, positive portal vein invasion, and histologic grade as discriminating factors. The MO score to differentiate multicentric HCCs from intrahepatic metastatic HCCs was determined using the following four independent factors selected by a stepwise regression analysis: the presence of hepatitis C virus infection, a platelet count of less than 10 x 10(4)/microliter, tumor size, and histologic grade. The sensitivity and specificity of the MO scores using those factors were 84% and 70%, respectively, when the cutoff value was 0.4. The disease-free survival rate in the MO group was similar to that in the IM group, whereas the survival rate in the MO group was significantly better than that in the IM group. The multivariate analysis revealed the multicentric occurrence of HCC as one of the independent prognostic factors. Clinicopathologic factors differentiating multicentric HCCs from intrahepatic metastatic HCCs were the presence of hepatitis C virus infection, a platelet count of less than 10 x 10(4)/microliter, small tumor size, and low histologic grade.

Recurrence after successful surgical or nonsurgical treatment of hepatocellular carcinoma (HCC) is caused either by intrahepatic metastasis or by metachronously multicentric occurrence. Intrahepatic metastasis is a major cause of recurrence of advanced HCCs with varying degrees of vascular invasion, and multicentric occurrence is a frequent cause of recurrence in small HCCs with no obvious vascular invasion. It is estimated that at least 20% of small HCCs have a high probability of recurrence due to multicentric occurrence, based on the finding that adenomatous hyperplasia (AH) and/or atypical adenomatous hyperplasia (AAH), which are considered premalignant lesions, are found in the vicinity of resected small HCCs with liver cirrhosis. However, because neither AH nor AAH occur in HCC cases without liver cirrhosis, most recurrence of HCC in noncirrhotic liver is considered to be due to intrahepatic metastasis or to de novo hepatocarcinogenesis. In a survey of autopsy cases of liver cirrhosis with small HCC, smaller HCC nodules were found in other liver slices in 50% of cases, and it is estimated that approximately 50% of HCC is already multicentric in the early stage.

An unusual risk factor for intrahepatic cholangiocarcinoma