Correlation of pathological examination with indocyanine green (ICG) intensity gradients: a prospective study in patients with liver tumor.

BACKGROUND: Intraoperative indocyanine green (ICG) fluorescence imaging has been shown to be a new and innovative way to illustrate the optimal resection margin in hepatectomy for hepatocellular carcinoma. This study investigated its accuracy in resection margin determination by looking into the correlation of ICG intensity gradients with pathological examination results of resected specimens. METHODS: This was a prospective, single-center, non-randomized controlled study. Patients who had liver tumors indicating liver resection were recruited. The hypothesis was that the use of intraoperative near-infrared/ICG fluorescence imaging would be a promising guiding tool for removing hepatocellular carcinoma with a better resection margin. Patients were given ICG (0.25 mg/kg) 1 day before operation. Resected specimens were inspected under a fluorescent imaging system. Biopsies were taken from tumors and normal tissue. Color signals obtained from ICG fluorescence imaging were compared with biopsies for analysis. RESULTS: Twenty-two patients were recruited for study. The median size of their tumors was 2.25 cm. One patient had resection margin involvement. Under ICG fluorescence, the tumors typically lighted up as yellow color, wrapped by a zone of green color. Tumors of 17 patients (77.3%) displayed yellow color and were confirmed malignancy, while tumors of 12 patients (54.5%) displayed green color and were confirmed malignancy. Receiver operating characteristic curve was used to measure the sensitivity and specificity of the green color to look for a clear resection margin. The area under the curve was 85.3% (p = 0.019, 95% confidence interval 0.696-1.000), with a sensitivity of 0.706 and specificity of 1.000. CONCLUSION: The use of ICG fluorescence can be helpful in determining resection margins. Resection of tumor should include complete resection of the green zone shown in the fluorescence image.

Stage-by-stage analysis of the effect of blood transfusion on survival after curative hepatectomy for hepatocellular carcinoma-a retrospective study.

OBJECTIVE: This study is to examine the impact of perioperative (intraoperative/postoperative) blood transfusion on the outcomes of curative hepatectomy for hepatocellular carcinoma. Hepatectomy is a well-established curative treatment for hepatocellular carcinoma, and blood transfusion cannot always be avoided in treating the disease. METHODS: A retrospective study of patients having curative hepatectomy for hepatocellular carcinoma from January 2010 to December 2019 at a single center was conducted. The patients were stratified by their disease stage. Patients with and without perioperative blood transfusion were matched by propensity-score matching and compared for each disease stage. Univariate and multivariate analyses were performed to identify prognostic factors for overall survival for each stage. RESULTS: A total of 846 patients were studied. Among them, 125 received perioperative blood transfusion and 720 did not. Patients with blood transfusion had worse disease-free and overall survival. After stratification and matching, the ratios of transfusion to non-transfusion were 33:165 (stage 1), 28:140 (stage 2), and 45:90 (stage 3). Perioperative blood transfusion was associated with a higher incidence of postoperative complications in all three disease stages (p = 0.004/0.006/0.017), and hence longer hospitalization (p < 0.001 in all stages), but had no significant impact on hospital mortality (p = 0.119/0.118/0.723), 90-day mortality (p = 0.259/0.118/0.723), disease-free survival (p = 0.128/0.826/0.511), or overall survival (p = 0.869/0.122/0.122) in any disease stage. Prognostic factors for overall survival included tumor size, tumor number, alpha-fetoprotein level, and postoperative complication of grade ≥ 3A. CONCLUSION: Perioperative blood transfusion was associated with a higher incidence of complications but had no significant impact on survival after curative hepatectomy for hepatocellular carcinoma.

Options and survival benefits of conversion therapy for unresectable hepatocellular carcinoma.

In the study by Wu et al, patients with unresectable hepatocellular carcinoma were subjected to transarterial chemoembolization (TACE) as a conversion therapy in order to render their tumors suitable for resection. A nomogram was devised and shown to be effective in predicting the survival of these patients. Generalization of the results, however, is questionable since the study subjects consisted of patients who had resection after TACE while excluding patients with the same disease but not suitable for TACE. Immunotherapy can be considered to be an option for conversion therapy. However, markers for determining responses to a conversion therapy and for guiding the decision between TACE and sequential immunotherapy have been lacking. The question of whether effective conversion therapy can truly enhance overall survival remains unanswered.

Recompensation in MASLD-related cirrhosis via metabolic bariatric surgery.

The prognosis of patients with decompensated cirrhosis is poor, with significantly increased liver-related mortality rates. With the rising tide of decompensated cirrhosis associated with metabolic dysfunction-associated steatotic liver disease (MASLD), the role of metabolic bariatric surgery (MBS) in achieving hepatic recompensation is garnering increasing attention. However, the complexity of preoperative assessment, the risk of postoperative disease recurrence, and the potential for patients to experience surgical complications of the MBS present challenges. In this opinion article we analyze the potential of MBS to induce recompensation in MASLD-related cirrhosis, discuss the mechanisms by which MBS may affect recompensation, and compare the characteristics of different MBS procedures; we highlight the therapeutic potential of MBS in MASLD-related cirrhosis recompensation and advocate for research in this complex area.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.

Dual-tracer PET/CT in the management of hepatocellular carcinoma.

Background & Aims: Combined 18F-fluorodeoxyglucose (FDG) and 11C-acetate (dual-tracer) positron-emission tomography/computed tomography (PET/CT) is being increasingly performed for the management of hepatocellular carcinoma (HCC), although its role is not well defined. Therefore, we evaluated its effectiveness in (i) staging, (ii) characterization of indeterminate lesions on conventional imaging, and (iii) detection of HCC in patients with unexplained elevations in serum alpha-fetoprotein (AFP) levels. Methods: We retrospectively assessed 525 consecutive patients from three tertiary centers between 2014 and 2020. For staging, we recorded new lesion detection rates, changes in the Barcelona Clinic Liver Cancer (BCLC) classification, and treatment allocation due to dual-tracer PET/CT. To characterize indeterminate lesions and unexplained elevation of serum AFP levels, the sensitivity and specificity of dual-tracer PET/CT in diagnosing HCC were evaluated. A multidisciplinary external review and a cost-benefit analysis of patients for metastatic screening were also performed. Results: Dual-tracer PET/CT identified new lesions in 14.3% of 273 staging patients, resulting in BCLC upstaging in 11.7% and treatment modifications in 7.7%. It upstaged 8.1% of 260 patients undergoing metastatic screening, with estimated savings of US$495 per patient. It had a sensitivity and specificity of 80.7% (95% CI 71.2-88.6%) and 94.8% (95% CI 90.4-98.6%), respectively, for diagnosing HCC in 201 indeterminate lesions. It detected HCC in 45.1% of 51 patients with unexplained elevations in serum AFP concentrations. External review revealed substantial agreement between local and external image interpretation and patient assessment (n = 273, κ = 0.822; 95% CI 0.803-0.864). Conclusions: Dual-tracer PET/CT provides added value beyond conventional imaging in patients with HCC by improving staging, confirming HCC diagnosis with high accuracy in patients with indeterminate lesions, and detecting HCC in patients with unexplained elevation of serum AFP. Impact and implications: Compared to CT or MRI, dual-tracer positron-emission tomography/computed tomography (PET/CT) led to upstaging in 12% of patients with hepatocellular carcinoma (HCC) undergoing staging, resulting in treatment modification in 8% of cases and a cost saving of US$495 per patient. It also accurately detected HCC in high-risk cases where CT or MRI were equivocal or normal. Dual-tracer PET/CT provides added value beyond conventional imaging in patients with HCC by improving staging, confirming HCC diagnosis with high accuracy in patients with indeterminate lesions, and detecting HCC in patients with unexplained elevation of serum AFP.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Multimodal Integrative Genomics and Pathology Analyses in Neoadjuvant Nivolumab Treatment for Intermediate and Locally Advanced Hepatocellular Carcinoma.

Introduction: Immunotherapy has resulted in pathologic responses in hepatocellular carcinoma (HCC), but the benefits and molecular mechanisms of neoadjuvant immune checkpoint blockade are largely unknown. Methods: In this study, we evaluated the efficacy and safety of preoperative nivolumab (anti-PD-1) in patients with intermediate and locally advanced HCC and determined the molecular markers for predicting treatment response. Results: Between July 2020 and November 2021, 20 treatment-naive HCC patients with intermediate and locally advanced tumors received preoperative nivolumab at 3 mg/kg for 3 cycles prior to surgical resection. Nineteen patients underwent surgical resection on trial. Seven (36.8%) of the 19 patients had major pathologic tumor necrosis (≥60%) in the post-nivolumab resection specimens, with 3 having almost complete (>90%) tumor necrosis. The tumor necrosis was hemorrhagic and often accompanied by increased or dense immune cell infiltrate at the border of the tumors. None of the patients developed major adverse reactions contradicting hepatectomy. RNA-sequencing analysis on both pre-nivolumab tumor biopsies and post-nivolumab resected specimens showed that, in cases with major pathologic necrosis, the proportion of CD8 T cells in the HCC tissues predominantly increased after treatment. Moreover, to investigate noninvasive biomarker for nivolumab response, we evaluated the copy number variation (CNV) using target-panel sequencing on plasma cell-free DNA of the patients and derived a CNV-based anti-PD-1 score. The score correlated with the extent of tumor necrosis and was validated in a Korean patient cohort with anti-PD-1 treatment. Conclusion: Neoadjuvant nivolumab demonstrated promising clinical activity in intermediate and locally advanced HCC patients. We also identified useful noninvasive biomarker predicting responsiveness.