HBV Genome-Enriched Single Cell Sequencing Revealed Heterogeneity in HBV-Driven HCC

Background: HBV-HCC is heterogeneous and frequently contains multifocal tumors. To interrogate heterogeneity of HBV-HCC, we developed a HBV genome enriched single cell sequencing (HGE-scSeq) procedure and a computational method to identify HBV integration sites and infer DNA copy number variations (CNVs). Results: We performed HGE-scSeq on 269 cells from 4 tumor sites and 2 tumor thrombi of a HBV-HCC patient. HBV integrations were identified in 142 out of 269 (53%) cells sequenced, and were enriched in two HBV integration hot spots chr1:34,397,059 (CSMD2) and chr8:118,557,327 (MED30/EXT1). There were also 162 rare integration sites. HBV integration sites were enriched in DNA fragile sites and sequences around HBV integration sites were enriched for microhomologous sequences between human and HBV genomes. Cells were grouped into 4 clonal groups based on CNVs. The HBV integration heterogeneity was associated with single cell’s CNVs. All of 269 cells carried chromosome 1q amplification, a recurrent feature of HCC tumors, suggesting that 1q amplification occurred before HBV integration events in this case study. Further, we performed simulation studies to demonstrate that the sequential events (HBV infecting transformed cells) could result in the observed phenotype with biologically reasonable parameters. Conclusion: Our HGE-scSeq data reveals high heterogeneity of HCC tumor cells in terms of both HBV integrations and CNVs.

Real-world outcome of immune checkpoint inhibitors for advanced hepatocellular carcinoma with macrovascular tumor thrombosis

Programmed cell death protein-1 (PD-1) inhibitors have shown promising results for treating advanced hepatocellular carcinoma (HCC). However, the clinical utility of such inhibitors in HCC patients with vascular tumor thrombosis remains unclear. This study investigated PD-1 inhibitor efficacy in advanced HCC with macrovascular invasion in a clinical setting. Among the 110 patients with unresectable HCC treated with PD-1 inhibitors, 34 patients with vascular metastases in the portal vein and inferior vena cava were retrospectively compared with 34 patients without tumor thrombi. The vascular response and its effect on survival were assessed. Predictors of survival were identified using multivariate analysis. Among patients achieving objective response, those with and without thrombi exhibited similar response to immunotherapy and comparable survival. Among the 34 patients with tumor thrombi, including 13 receiving PD-1 inhibitors alone and 21 receiving it in combination with tyrosine kinase inhibitors, the median overall survival was 8.9 months (95% confidence interval 3.2–12.6). The objective response rate of vascular metastasis was 52.9%, and vascular responders had a significantly longer survival than did non-responders (11.1 vs 3.9 months). Failure to obtain a vascular response correlated significantly with increased post-treatment Child–Pugh score or class. Multivariate analysis showed that vascular response was a significant positive factor for longer overall survival. Treatment-related grade 3/4 adverse events occurred in 3 (8.8%) of the patients with tumor thrombi. Immunotherapy with PD-1 inhibitors may be a feasible treatment option for HCC with tumor thrombi owing to the high response rate of tumor thrombi and favorable survival outcomes.

Evaluation of the Efficacy and Toxicity of Radiotherapy for Type III-IV Portal Vein Tumor Thrombi

Background: Type Ⅲ and Ⅳ portal vein tumor thrombi (PVTT) cannot be removed through surgery, and no effective therapeutic procedure is available. Type Ⅲ/Ⅳ PVTT can be downstage to type I/II PVTT by using Radiotherapy, and can further be can be removed surgically. Thus, radiotherapy may be an effective treatment for type Ⅲ/Ⅳ PVTT. This study aims to evaluate the efficacy and toxicity of radiotherapy for type III-IV PVTT. Methods: This prospective study was conducted from August 1, 2017, to September 30, 2019, for patients with type Ⅲ and Ⅳ PVTT. Patients received radiotherapy with a target dose of 50Gy/25f or 59.5Gy/17 f. Advanced radiological technique such as image fusion technique for CT image and MRI image were utilized to produce more precise lesion localization, and limit the dose to organs at risk in order to get a better downstage rate and less adverse complications. Results: Nine (9) patients with type Ⅲ PVTT and 5 patients with type Ⅳ PVTT were included in this study. 12 patients received a radiotherapy dose of 50Gy/25f, 2 patients received 59.50Gy/17 f. After radiotherapy, 92.9% of patients with PVTT were successfully downstage to type II/I. In patients with primary hepatocellular carcinoma, 8 patients (accounting 88.9%) achieved down-stage. 5 patients with other types of tumors achieved downstage which accounts 100%. In addition, none of the 14 patients observed radiation hepatitis and radiation liver failure. And none of the patients developed gastrointestinal ulcers and thrombocytopenia. Conclusion: Radiotherapy is a suitable treatment measure for type Ⅲ and Ⅳ PVTT to get downstage and make the opportunity for surgery. Image fusion technology for precise lesion location such as CT-MRI image fusion, and strict dose limitation of organ at risk, contributed to the improvement of radiotherapy efficiency and the significant decrease in adverse complications.

Use of Contrast-Enhanced Ultrasonography for the Characterization of Tumor Thrombi in Seven Dogs

Tumors of adrenal and thyroid glands have been associated with vascular invasions—so-called tumor thrombi, both in humans and dogs. The detection and characterization of venous thrombi is an important diagnostic step in patients with primary tumors for both surgical planning and prognosis. The aim of this study was to describe the use of contrast-enhanced ultrasonography (CEUS) for the characterization of tumor thrombi. Dogs with tumor thrombus who underwent bi-dimensional ultrasound (B-mode US) and CEUS were included. Seven dogs were enrolled in this retrospective case series. On B-mode US, all thrombi were visualized, and vascular distension and thrombus-tumor continuity were seen in three and two cases, respectively. On color Doppler examination, all thrombi were identified, seemed non-occlusive and only two presented vascularity. On CEUS, arterial-phase enhancement and washout in the venous phase were observed in all cases. Non-enhancing areas were identified in the tumor thrombi most likely representing non-vascularized tissue that could potentially be embolized in the lungs after fragmentation of the tumor thrombi. On the basis of these preliminary study, CEUS appeared to be useful for the characterization of malignant intravascular invasion.

ADRENAL METASTASES OF RENAL CELL CARCINOMA WITH INTRAVENOUS TUMOR THROMBI OF THE INFERIOR VENA CAVA (two case repor ts with literature review)

Intrvenous extension is one of the features of neoplastic process and is very characteristic for tumors of the kidneys and adrenal glands. However, reports about the penetration of adrenal metastases into the venous system are extremely rare. We have presented two case reports on intravenous extension of renal cell carcinoma adrenal metastases. One of them for the first time in worldwide literature presents intravenous extension of synchronous bilateral adrenal metastases RCC into inferior vena cava and left renal vein. The patient underwent bilateral adrenalectomy with the use of hormone replacement therapy and targeted therapy with pazopanib. MDCT after 2 and 6 months did not show tumor progression. Lungs metastases are reduced in size or remained unchanged. The patient does not complain. Blood pressure 110/70 mm Hg. Symptoms of Addison's disease are absent. In the second case, there was a metachronic contralateral metastasis of RCC, which penetrated through the right adrenal vein into the subhepatic section of the inferior vena cava. The patient underwent a right-sided adrenalectomy with thrombectomy. After 6 months multiple pulmonary metastases were identified. Pazopanib therapy has initiated. Three months later, tumor progression was revealed. The patient died after 5 months (14 months after surgery) from tumor intoxication. Although these cases are extremely rare (7 cases in the world literature), it is necessary to take into account possibility of presence of venous tumor thrombi with adrenal metastases. The incidence of this phenomenon of RCC is in the second place after hepatocellular carcinoma (15 cases). Organ-sparing surgical technique in these patients is not feasible. Targeted therapy along with hormone replacement therapy was not accompanied by increased side effects or exacerbation of adrenal insufficiency after removal of both adrenal glands.