Carolina Armengol

p27Kip1 is an independent predictor of recurrence after surgical resection in patients with small hepatocellular carcinoma

Abstract Background/Aims : Alterations in p27 Kip1 (p27) and cyclin E (cycE) expression are found in tumors and are related to poor prognosis. This study assesses the role of these cell cycle regulators in the development of recurrence after surgical resection in 46 cirrhotic patients (age: 61.3±7 years, 30 males, 44 Child–Pughs A, 30 HCV-positive) with small hepatocellular carcinoma (HCC, size: 3.1±1.5cm, 40 solitary at pathological examination). Methods : p27 and cycE expression in tumoral and non-tumoral liver were analyzed by Western blot (WB). p27 was also assessed by immunohistochemistry (IHC). Results : Tumor p27 underexpression (50% decreased vs. non-tumoral liver) occurred in 12 cases. Throughout follow-up, 26 patients developed recurrence, which was significantly higher in patients with p27 underexpression than in those without (3-year recurrence: 80 vs. 44%, respectively, P =0.026). IHC showed concordant inverse findings: 13 tumors showed high p27 staining that was related to lower recurrence rate ( P =0.019). Multivariate analysis identified p27 measured by WB as an improved predictor of recurrence (OR: 3.09, 95% CI: 1.26–7.08, P =0.016). By contrast, cycE, increased in 66% of the tumors, had no impact on recurrence but was associated to poor differentiation ( P =0.015) and microvascular invasion ( P =0.016). Conclusions : p27 underexpression is frequent in relatively early stages of HCC and constitutes an independent predictor of recurrence after surgical resection.

Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.

Purpose: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage. Experimental Design: Tumor pieces (2 × 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21Cip1, p27Kip1, p16INK4a, pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression. Results: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27kip1 overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete α-fetoprotein, and has TP53 missense mutations in codons 192 and 242. Conclusions: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.

HEPACONTROL. A program that reduces early readmissions, mortality at 60 days, and healthcare costs in decompensated cirrhosis

BACKGROUND & AIMS Decompensated cirrhosis patients have an elevated incidence of early readmission, mortality and economic burden. The aims of HEPACONTROL were to reduce early readmission and to evaluate its impact on mortality and emergency department visits. PATIENTS AND METHODS Quasi-experimental study with control group which compared two cohorts of patients discharged after being admitted for cirrhosis-related complications. A prospective cohort (n=80), who followed the HEPACONTROL program, which began with a follow-up examination seven days after discharge at the Hepatology Unit Day Hospital and a retrospective cohort of patients (n=112), who had been given a standard follow-up. Outcome variables that were compared between both groups were early readmission rates, the number of emergency department visits post-discharge, financial costs and mortality. RESULTS The rate of early readmission was lower in the group with HEPACONTROL (11.3% vs 29.5%; P=.003). Also, the mean number of visits to the emergency department post-discharge (1.10±1.64 vs 1.71±2.36; P=.035), mortality at 60days (3.8% vs 14.3%; P=.016), and the cost of early readmission were all lower compared with the group with standard follow-up (P=.029). CONCLUSIONS HEPACONTROL decreases the incidence of early readmission the rate of emergency department visits and mortality at 60days in patients with decompensated cirrhosis, and it is cost-effective.

CD5L is upregulated in hepatocellular carcinoma and promotes liver cancer cell proliferation and antiapoptotic responses by binding to HSPA5 (GRP78)

CD5‐like (CD5L) is a soluble scavenger cysteine‐rich protein that modulates inflammatory responses. We studied the involvement of CD5L in liver cancer. Immunohistochemistry (IHC) of CD5L in 60 hepatocellular carcinomas and 34 adjacent nontumor livers, showed that CD5L staining was higher in tumor than in nontumor tissue (Mann‐Whitney test; P = 0.0039). High CD5L correlated with elevated proliferation (Ki67, linear regression; P < 0.0001) and lower patient event‐free survival (log‐rank; P = 0.0185). Accordingly, CD5L expression was detected in the liver cancer cell lines Huh7, HepG2, and SNU‐398. In vitro technologies using these cell lines, including small interfering RNA (siRNA) and cDNA transfection, showed that CD5L promoted colony formation and cell proliferation and protected against cisplatin‐induced apoptosis. To find a molecular explanation for these roles, novel CD5L‐interacting protein ligands in liver cancer cells were identified by immunoprecipitation followed by mass spectrometry. Among these, the molecular chaperone of the unfolded protein response (UPR), heat shock protein (HSP)‐A5, was selected for validation. The interaction was confirmed by confocal microscopy in the Huh7 and HepG2 cell lines. Furthermore, functional experiments revealed that CD5L activates the UPR and autophagy mechanisms in Huh7 cells, thereby providing a novel molecular link between the UPR and autophagy in liver cancer.—Aran, G., Sanjurjo, L., Barcena, C., Simon‐Coma, M., Tellez, E., Vazquez‐Vitali, M., Garrido, M., Guerra, L., Díaz, E., Ojanguren, I., Elortza, F., Planas, R., Sala, M., Armengol, C., Sarrias, M.‐R. CD5L is upregulated in hepatocellular carcinoma and promotes liver cancer cell proliferation and antiapoptotic responses by binding to HSPA5 (GRP78). FASEB J. 32, 3878–3891 (2018). www.fasebj.org