HPHS Journal Watch: November/December 2022

American Journal of Clinical Pathology

Transcriptomic Analysis of Cirrhosis-Like Hepatocellular Carcinoma Reveals Distinct Molecular Characteristics and Pathologic Staging Implications

Van Treeck BJ, Moreira RK, Mounajjed T, et al. Am J Clin Pathol. 2022 Dec 1;158(6):750-758.


In this study, the authors evaluate the molecular biology of cirrhosis-like hepatocellular carcinoma (CL-HCC), characterized by numerous tumor nodules that clinically, radiologically, macroscopically, and microscopically mimic cirrhosis. 5 CL-HCC cases (each >50 nodules, all moderately differentiated) were compared to fibrolamellar HCCs (FLCs), steatohepatitic HCCs (SH-HCCs), and HCCs from The Cancer Genome Atlas (TCGA) using RNA sequencing. CL-HCC gene expression profiles showed discrete clustering away from FLCs, SH-HCCs, and HCCs from TCGA. Tumor nodules in the same patient were found to be clonally related (likely monoclonal, rather than representing multiple separate primaries), but no recurrent mutations, fusions, or aberrant regulation of tumorigenic pathways was identified. CL-HCC did, however, show aberrant expression of genes associated with matrix degradation and invasiveness, including ASPM, MMP11, and MMP16, indicative of extensive local invasion. The authors propose that based on these data, CL-HCC should be staged as a single tumor rather than multiple, separate tumors (pT2 unless there is involvement of a major branch of the portal vein, hepatic vein, or an adjacent organ other than gallbladder or perforation of the visceral peritoneum). Finally, the authors also show that tumor cells in CL-HCC recapitulate non-zone 1 or 3 lobule hepatocytes.

American Journal of Gastroenterology

Longitudinal Associations of Risk Factors and Hepatocellular Carcinoma in Patients With Cured Hepatitis C Virus Infection

Kramer JR, Cao Y, Li L, et al. Am J Gastroenterol. 2022 Nov 1;117(11):1834-1844.


The authors evaluated hepatocellular carcinoma (HCC) risk longitudinally after examining the risk factors (demographics, comorbidities, and liver/ HCV-related) associated with HCC at the time of sustained viral response (SVR) and 12 and 24 months after SVR. In addition, they examined associations of changes in these factors from baseline to different time points in subgroups defined based on the presence or absence of baseline cirrhosis. They conducted a retrospective cohort study of patients with HCV who achieved SVR with direct-acting antivirals from 130 Veterans Administration hospitals from 2014–2018, followed through 2021. Cox proportional hazards models were constructed at 3 landmark times (baseline and 12 and 24 months after sustained virological response) to examine associations between demographic, clinical, and behavioral factors and HCC risk, stratified by cirrhosis status. Among 92,567 patients (32% cirrhosis), 3,247 cases of HCC were diagnosed during a mean follow-up of 2.5 years. In patients with cirrhosis, male sex (hazard ratios [HR]: 1.89, 1.93, and 1.99), cirrhosis duration or =5 years (HR: 1.71, 1.79, and 1.34), varices (HR: 1.73, 1.60, and 1.56), baseline albumin (HR: 0.48, 0.47, and 0.49), and change in albumin (HR: 0.82 and 0.90) predicted HCC risk at each landmark time. HCV genotype 3, previous treatment, bilirubin, smoking, and race influenced HCC risk at baseline, but their effects attenuated over time. In patients without cirrhosis, diabetes (HR: 1.54, 1.42, and 1.47) and hypertension (HR: 1.59, 1.65, and 1.74) were associated with HCC risk at all landmark times. Changes in fibrosis-4 scores over time were associated with HCC risk both in patients with and without cirrhosis. Risk factors for HCC were different in patients with and without cirrhosis and some also evolved during follow-up. These factors can help with risk stratification and HCC surveillance decisions in patients with cured HCV.

American Journal of Surgical Pathology

PSMA Immunohistochemistry in Hepatic Neoplasms: A Promising Diagnostic Marker with Potential Theranostic Applications

Kmeid M, Park NY, Chung T, et al. Am J Surg Pathol. 2022 December;46(12):1688-1699.


The Food and Drug Administration (FDA) has approved prostate-specific membrane antigen (PSMA) radiopharmaceuticals for the imaging diagnosis and treatment of men with metastatic prostate cancer. Other non-prostatic malignancies have also been found to be PSMA PET avid, possibly because PSMA is associated with angiogenesis in malignancy. The authors of this study reviewed the expression of PSMA in cirrhotic liver as well as hepatocellular carcinoma (HCC) and adenoma (HA) to assess this marker for diagnosis and possible future imaging and treatment modalities. They found that PSMA was a more specific marker of neovascularization when compared to CD34 in HCC vs. HA. PSMA also stained the neovasculature of metastatic carcinoma to the liver. Of note, background PSMA staining was identified in a cytoplasmic or canalicular pattern in the majority of hepatocellular adenomas, but this was weak and distinct from the endothelial staining present in HCC. The authors conclude that a neovascular distribution of PSMA staining can be a useful marker in malignant versus benign hepatocellular neoplasms and that patients with this staining may benefit from future PSMA-based radiotherapies.

Archives of Pathology & Laboratory Medicine

Impact on Liver Biopsy Size on Histopathological Evaluation of Liver Allograft Rejection

Agarwal AN, Nania J, Qiu L, et al. Arch Pathol Lab Med. 2022. December;146:1530-34.


In general, the better the tissue sample, the more precise the pathologic diagnosis can be. In this study, the authors reviewed 68 consecutive allograft liver core biopsy specimens to determine if the diagnosis of acute cellular rejection (ACR) correlated with sample size. The pathologists blindly reviewed the same set of liver samples 5 times, each time at a different bracketed tissue length. Unsurprisingly, the number of portal tracts directly correlated with the length of the liver core biopsy available for review. In addition, smaller biopsy samples were found to sometimes result in an under calling of rejection. In fact, five biopsy samples were called negative for ACR when reviewed at 1 cm length but were considered positive for ACR at 3 cm. Similarly, zero samples had severe ACR at 1 or 1.5 cm, but 9 were considered severe ACR at the 3 cm length. The authors conclude that at least 2 cm of tissue (approximately 10 portal tracts) are necessary for adequate pathologic assessment of ACR.

Liver Histology in Septic Patients: Is It All About Ductular Cholestasis?

Bisirini C, Katerji R, Lee EJ, Gonzalez RS. Arch Pathol Lab Med. 2022. November;146:1329-37.


The authors of this retrospective study evaluated the utility of ductular cholestasis as a marker of sepsis. They also cataloged other histologic features in the liver biopsies of patients with a clinical diagnosis of sepsis. Overall, the histologic features between clinically diagnosed septic patients and those who ultimately were not diagnosed with sepsis were very similar. Ductular cholestasis was seen in both patient populations, although it was slightly more common in septic (68%) vs non-septic (55%) patients (p=0.17). Both groups often had portal mononuclear inflammation and ductular reaction, but portal neutrophilic inflammation, necrosis, and duct injury were uncommon histologic features. The authors conclude that clinical parameters are necessary to definitively diagnose sepsis in a patient.

Clinical Gastroenterology and Hepatology

A Purple Liver

Jiang G, Pu X, Cao L. Clin Gastroenterol Hepatol. 2022 Nov;20(11):A34.


This short article shows clinical images of a purple-colored liver, which was incidentally discovered during laparoscopic cholecystectomy due to Dubin-Johnson syndrome. Immunohistochemical staining was negative for MRP2 on biopsy of the liver.


Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies

Preston SP, Stutz MD, Allison CC, et al. Gastroenterology. 2022 Dec;163(6):1643-1657.e14.


Necroptosis refers to a lytic form of programmed cell death in hepatocytes that has been implicated in nonalcoholic steatohepatitis (NASH) in animal models and humans. However, there has been conflicting evidence supporting these claims. The critical proteins involved in necroptosis include receptor interacting protein kinase 3 (RIPK3), an essential initiator protein that is expressed in most cell types, and mixed lineage kinase domain-like pseudokinase (MLKL), an effector protein. The mechanism of necroptosis involves the activation of RIPK3 by the loss of caspase-8 function; RIPK3 then phosphorylates MLKL leading to the disruption of the cell membrane and necroptotic death. The authors of this study aimed to define the role and relevance of necroptosis in liver pathology by using animal models (diet-induced steatohepatitis in male mice) and diverse infections in both male and female mice. Necroptotic stimuli were applied to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. The study revealed that RIPK3 mRNA is repressed or not overtly expressed in the human NASH specimens tested, both before and after dietary intervention. The authors speculate that since both RIPK3 and MLKL are present in Kupffer cells, the detection of these molecules in hemopoietic cells may have confounded the interpretation of results from other studies that used whole liver lysates. The authors concluded that RIPK3, a critical activator of necroptosis, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis.


COVID-19 and liver disease

Dufour JF, Marjot T, Becchetti C, Tilg H. Gut. 2022 Nov;71(11):2350-2362.


In the recent advances in clinical medicine section, the authors reviewed liver manifestations of SARS-CoV-2 (COVID-19) and pathophysiological aspects related to COVID-19 infection in patients without liver disease and with chronic liver disease (CLD), particularly cirrhosis and liver transplantation (LT). Liver injury during COVID-19 infection is probably multi-factorial, including mechanisms such as direct cytopathic effect of the virus, exaggerated systemic immune response, vascular damage and coagulopathy, and drug-induced liver injury. In patients without liver disease, elevated transaminase affects up to 50% of infected subjects and correlates with severity of disease. Severe acute liver injury is rare. A cholestatic pattern is less frequent in COVID-19 and often found later in the disease course.

Risk factors for severe COVID-19 and death include CLD, especially decompensated cirrhosis. Clinical features of COVID-19 in patients with cirrhosis include acute hepatic decompensation mostly with new or worsening ascites and hepatic encephalopathy, acute on chronic liver failure, and GI symptoms. The cause of death in patients with cirrhosis is predominantly respiratory failure followed by liver-related complications. Regarding specific aetiologies of liver disease, NAFLD alone probably does not significantly impact COVID-19 course and outcome, although these patients are likely to be more vulnerable due to the presence of at-risk comorbidity. Alcohol-related liver disease has been associated with COVID-19 mortality after controlling for relevant cofactors. Immunosuppressed patients with autoimmune liver disease do not seem to have a worse prognosis compared to other liver disease etiologies.

While LT does not represent a risk factor per se for worse outcome, LT patients remain a delicate population, particularly in the setting of vaccination. COVID-19 vaccine-induced immunity seems to be impaired in CLD and LT recipients, advocating for a revised schedule of vaccine administration in this population and particular caution in both populations. Following an initial decline in transplant activity, there was then an increase during the pandemic. These trends could be related to the relevant increase in the listing rate for LTs with alcoholic etiology as an indirect effect of the COVID-19 pandemic. Donors infected with COVID-19 could represent a life-saving opportunity to implement the donor pool; however, studies that include long-term follow-up are needed. In the early pandemic, LT recipient outcome was not necessarily worse than that of the general population. The natural immunologic response to COVID-19, both serologic and T cell-mediated, in LT recipients appears to be only modestly reduced compared with that of healthy controls. However, some delay in mounting this response and a more rapid decline over time were observed.

Hepatology Communications

The future risk of primary biliary cholangitis (PBC) is low among patients with incidental anti-mitochondrial antibodies but without baseline PBC

Duan W, Chen S, Li S, et al. Hepatol Commun. 2022 Nov;6(11):3112-3119.


This retrospective study evaluated the natural history of AMA-positive patients including patients with non-PBC liver disease at baseline. The authors identified 139 patients who were AMA positive but did not fulfill their criteria for PBC (elevated alkaline phosphatase, presence of AMA or AMA-M2, and diagnostic or compatible liver biopsy), including 51 patients with non-PBC liver diseases and 88 cases with non-liver disease. Notably, none of the 51 patients with non-PBC liver diseases developed PBC at the end of the follow-up, leading the authors to conclude that AMA-positive patients with non-PBC diseases had a low risk of developing PBC, especially for those with other liver diseases. Lower ALT and higher IgM were independent predictors for developing PBC.

Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma

Kim M, Hui KM, Shi M, et al. Hepatol Commun. 2022 Nov;6(11):3247-3259.


In this study, authors evaluated the differential expression of hepatic cancer stem cells and hypoxia in residual HCC after transarterial chemoembolization (TACE) treatment. The authors utilized microarray profile data from the Gene Expression Omnibus (GEO) database to compare TACE responders to TACE non-responders. The authors found that pretreatment tissue expression of both hepatic CSC markers and hypoxia markers transcripts correlate with response to TACE treatment in patients. Immunohistochemical staining of explant liver tissues exhibited more intense positive staining of hepatic cancer stem cell markers (CD24, EpCAM) and hypoxia marker carbonic anhydrase 9 (CA9) in residual tumor nodules from patients with HCC treated with TACE compared with transarterial radioembolization (TARE) treatment. The authors conclude that the expression of hepatic CSC markers may reflect more aggressive biology in residual tumor after TACE therapy.

Journal of Gastroenterology and Hepatology

Machine learning algorithms based on proteomic data mining accurately predicting the recurrence of hepatitis B-related hepatocellular carcinoma

Feng G, He N, Xia HH, et al. J Gastroenterol Hepatol. 2022 Nov;37(11):2145-2153.


The authors aimed to identify a molecular profile in HCC associated with increased recurrence risk. To do so, they assessed 127 HBV-positive patients with HCC (50 of which had tumor recurrence) with data in the Clinical Proteomics Tumor Analysis Consortium (CPTAC) database. The authors then employed machine learning to develop predictive algorithms for HCC recurrence. Seven proteins were identified as significant by Cox regression analysis (BAHCC1, ESF1, RAP1GAP, RUFY1, SCAMP3, STK3, and TMEM230) and four separate machine learning algorithms were trained. The random forest algorithm produced the highest performance with an AURCO of 0.991.

Journal of Hepatology

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma

Calderaro J, Di Tommaso L, Maillé P, et al. J Hepatol. 2022 Dec;77(6):1586-1597.


In this multi-institutional study, the authors compared 461 cases of combined hepatocellular-cholangiocarcinoma (cHCC-CCA) to 589 control cases (368 HCCs and 221 intrahepatic CCAs (iCCAs)) to assess the utility of nestin immunohistochemistry in the diagnosis of cHCC-CCA and as a prognostic marker. Nestin expression was detected (>1%) in 75% of cHCC-CCA, 7% of HCCs, and 59% of iCCA. Using this threshold of >1% tumor cell staining, nestin was able to distinguish cHCC-CCA from HCC and iCCA with an AUC of 0.75, sensitivity of 0.75, and specificity of 0.72. In the distinction from HCC, the AUC was 0.85 with specificity of 0.93; in distinction from iCCA, the AUC was 0.59 with specificity of 0.41. These results were comparable in resected specimens and biopsies. Compared to other markers (glypican-3, EPCAM, and CK19 immunohistochemistry), nestin AUC was lower than these markers in differentiating cHCC-CCA from iCCA but was comparable to CK19 in distinguishing cHCC-CCA from HCC. Nestin-high tumors (>30% nestin positivity by IHC) were associated with worse disease-free survival with significantly shorter overall survival in patients after resection and transplantation.

Prepared by:
Lindsey Westbrook, MD (Editor); University of Colorado
Dana Balitzer, MD; University of California San Francisco
Soo-Jin Cho, MD, PhD; University of California San Francisco
Ashim Das, MD; Post Graduate Institute of Medical Education and Research, India
Gillian Hale, MD, MPH; University of Utah
Meredith Pittman, MD; Maimonides Medical Center
Daniel Roberts, MD; Cleveland Clinic
Nafis Shafizadeh, MD; Southern California Permanente Medical Group
Xuefeng Zhang, MD; Cleveland Clinic

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