Expression of LHFPL2 in hepatocellular carcinoma and its association with prognosis after transarterial chemoembolization

Abstract

BACKGROUND

Transarterial chemoembolization (TACE) is a standard treatment for recurrent hepatocellular carcinoma (HCC) after liver resection. However, resistance to TACE considerably limits its efficacy and worsens patient prognosis. Identifying molecular markers that predict TACE response is crucial for individualized treatment strategies.

AIM

To investigate lipoma HMGIC fusion partner-like 2 (LHFPL2) expression in patients with HCC and its association with TACE efficacy and overall survival (OS).

METHODS

A retrospective study was conducted including 60 patients with HCC who underwent ≥ 3 standardized TACE treatments for recurrence after curative hepatectomy between January 2014 and December 2020 at Jiangsu Provincial People’s Hospital and Wuxi People’s Hospital. The patients were divided into TACE-resistant (n = 30) and non-resistant (n = 30) groups. LHFPL2 protein expression in tumor tissues was assessed using immunohistochemistry, and its association with clinicopathological features, TACE response, and OS was analyzed. The Kaplan-Meier method, log-rank test, and Cox regression models were used to evaluate prognostic value.

RESULTS

The TACE-resistant group exhibited significantly higher LHFPL2 expression than the non-resistant group (P < 0.05). Significant differences were observed between the two groups in hepatitis B virus carrier status, alpha-fetoprotein (AFP) levels, tumor differentiation, and LHFPL2 expression (P < 0.05). The median OS in the LHFPL2 high-expression group was 2.28 years, which was significantly shorter than that in the low expression group (P < 0.05). Multivariate Cox regression analysis indicated that high LHFPL2 expression, AFP ≥ 400 ng/mL, and TACE resistance were independent risk factors for OS (P < 0.05). A combined prognostic model using LHFPL2 and AFP revealed that patients with high LHFPL2 and AFP ≥ 400 ng/mL had the poorest prognosis (median OS 1.44 years), whereas patients with low LHFPL2 and AFP < 400 ng/mL had the best prognosis (median OS not reached), with significant differences (P < 0.05).

CONCLUSION

High LHFPL2 expression was closely associated with TACE resistance and was an independent predictor of poor prognosis in patients with HCC. Combining LHFPL2 with AFP enhanced prognostic stratification, providing a novel molecular marker and clinical basis for personalized therapy and TACE response prediction.

Key Words: Hepatocellular carcinoma; Lipoma HMGIC fusion partner-like 2; Transarterial chemoembolization; Transarterial chemoembolization resistance; Prognosis; Alpha-fetoprotein

Core Tip: Lipoma HMGIC fusion partner-like 2 overexpression contributes to transarterial chemoembolization resistance in hepatocellular carcinoma patients and predicts unfavorable outcomes. The combination of lipoma HMGIC fusion partner-like and alpha-fetoprotein levels allows for improved prognostic assessment, offering a potential molecular marker for individualized treatment planning and early identification of transarterial chemoembolization non-responders (No. AF-SW-07-02.1).

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. The majority of patients are already at an intermediate or advanced stage at diagnosis and have lost the opportunity for curative resection[1]. Transarterial chemoembolization (TACE) is the standard treatment for intermediate-to-advanced HCC. However, marked inter-individual heterogeneity in responses has been observed in daily practice. Some patients experience continuous tumor progression despite repeated, guideline-conforming TACE sessions, a phenomenon termed “TACE refractoriness”[2]. TACE refractoriness not only delays effective therapy but also causes progressive deterioration of liver function owing to repeated embolization, seriously compromising the quality of life and prognosis[3].

At present, the most commonly used indices to predict TACE efficacy are indirect and include alpha-fetoprotein (AFP), tumor size and number, vascular invasion, and hepatic function (Child-Pugh grade, Albumin-Bilirubin grade score, etc.); however, their sensitivity and specificity are limited[4,5]. Hence, novel biomarkers that directly reflect intrinsic tumor biology are urgently required. Lipoma HMGIC fusion partner-like 2 (LHFPL2) is a four-transmembrane-spanning protein belonging to the lipoma HMGIC fusion partner family that has recently attracted attention for its role in tumor biology. Emerging evidence suggests that LHFPL2 is involved in tumor progression, immune microenvironment remodeling, and hypoxia-related signaling pathways[6,7]. For example, LHFPL2 was associated with macrophage M2 polarization and adverse prognosis in renal cell carcinoma and is included in several tumor microenvironment-related prognostic signatures for HCC.

Hypoxia is the central mechanism underlying TACE resistance. A multi-omics study of hypoxia-associated molecular landscapes in HCC identified LHFPL2 as a key navigational molecule within hypoxia-enriched pathways, suggesting that it participates in tumor adaptation to ischemic stress induced by TACE[8]. However, the clinical significance of LHFPL2 expression in HCC, particularly its association with TACE refractoriness and patient prognosis, has not been systematically investigated.

Therefore, this study aimed to investigate the expression pattern of LHFPL2 in HCC and explore its association with TACE efficacy and overall survival (OS), thereby identifying a potential biomarker for predicting TACE resistance and optimizing individualized management strategies for patients with HCC.

MATERIALS AND METHODS

Patient cohort

We retrospectively reviewed patients with HCC treated at the Department of Hepatobiliary Surgery or Interventional Radiology of Jiangsu Provincial People’s Hospital and Wuxi People’s Hospital between January 1, 2014 and December 31, 2020.

Inclusion criteria: (1) Prior curative-intent hepatectomy for HCC; (2) Intrahepatic recurrence confirmed radiologically and/or histologically during follow-up; (3) Recurrence treated with ≥ 3 sessions of lipiodol-based, guideline-conforming TACE; (4) Complete clinicopathologic, imaging, and follow-up data available; and (5) Sufficient paraffin-embedded tumor tissue from the resected specimen for analysis.

Exclusion criteria: (1) Concomitant malignancies; (2) Additional anti-cancer therapies (targeted agents, immunotherapy, radiotherapy, etc.) during or after TACE; and (3) Death from non-tumor causes, such as liver failure or gastrointestinal bleeding.

Sixty patients fulfilled these criteria and were divided into TACE-resistant (n = 30) and non-resistant (n = 30) groups according to the definition of TACE refractoriness[9].

Clinicopathologic data collection

Using hospital electronic medical records and pathology databases, the following variables were extracted: Demographics, liver disease background, sex, age (years), hepatitis B virus infection status (hepatitis B surface antigen positive/negative), and presence of cirrhosis (determined by preoperative contrast-enhanced computed tomography/magnetic resonance imaging and/or histology). Serum tumor markers: Preoperative AFP (within 1 week before hepatectomy, ng/mL), categorized as < 400 ng/mL or ≥ 400 ng/mL[10]. Tumor characteristics: Maximum diameter of recurrent tumor on preoperative computed tomography/magnetic resonance imaging (≤ 3 cm vs > 3 cm), and histologic differentiation assessed independently by two senior pathologists using the Edmondson-Steiner grading system (well, moderately, or poorly differentiated)[11]. Outcome variables: LHFPL2 protein expression level determined by immunohistochemistry (high vs low), final survival status (0 = alive, 1 = dead), and OS calculated from the date of the first TACE session to the date of death or last follow-up (days). The final follow-up date was December 31, 2023.

Immunohistochemical staining and evaluation

Formalin-fixed (10% neutral-buffered), paraffin-embedded surgical specimens were cut into 4-μm sections and mounted on adhesive slides.

Immunostaining was performed using an EnVision Two-Step Kit[12]. Briefly: (1) Dewaxing and rehydration; (2) Antigen retrieval in ethylenediaminetetraacetic acid buffer (pH 9.0) under high pressure; (3) Blocking of endogenous peroxidase with 3% H₂O₂ for 10 minutes at room temperature; (4) Incubation with 5% bovine serum albumin for 30 minutes at 37 °C; (5) Overnight incubation at 4 °C with primary antibody (rabbit anti-human LHFPL2 polyclonal antibody, Proteintech, 24557-1-AP, 1:200); (6) 30-minute incubation at 37 °C with horseradish peroxidase-labeled secondary antibody; and (7) 3,3’-Diaminobenzidine chromogen, hematoxylin counterstain, dehydration, clearing and mounting.

Two pathologists who were blinded to the clinical data independently evaluated the slides. A semi-quantitative scoring system was used: Staining intensity (0, absent; 1, pale yellow; 2, brown-yellow; 3, dark brown) multiplied by percentage of positive tumor cells (0: < 5%; 1: 5%-25%; 2: 26%-50%; 3: 51%-75%; 4: > 75%) resulting in a total score of 0-12[13]. Scores ≥ 4 were defined as “LHFPL2 high expression” and < 4 as “LHFPL2 low expression”. Discrepancies were resolved by consensus or a third senior pathologist.

Statistical analysis

Data analysis was conducted using SPSS version 26.0 software. Continuous variables were tested for normality (Shapiro-Wilk) and are presented as mean ± SD; comparisons between groups were made with the independent-samples t test. Categorical data are given as n (%) and were compared with the χ² test or Fisher’s exact test. Univariate and multivariate logistic regression were performed to identify risk factors for TACE refractoriness; results were reported as odds ratios with 95% confidence intervals. Kaplan-Meier curves were constructed and compared using the log-rank test. Cox proportional hazards models were used for univariate and multivariate survival analyses; results were expressed as hazard ratios with 95% confidence intervals. All tests were two-tailed; P < 0.05 was considered statistically significant.

RESULTS

LHFPL2 expression in the two groups

Immunohistochemical analysis revealed that LHFPL2 was predominantly localized in the tumor cell membrane. In the TACE-resistant group, 22 of 30 patients (73.33%) exhibited high LHFPL2 expression, whereas only 6 of 30 patients (20.00%) in the non-resistant group showed high expression. The proportion of patients with high LHFPL2 expression was therefore significantly higher in the TACE-resistant group than in the non-resistant group (χ² = 17.140, P < 0.001; Table 1, Figure 1).

Figure 1 Representative immunohistochemical staining of lipoma HMGIC fusion partner-like 2 expression in transarterial chemoembolization-resistant and non-resistant groups. A: Transarterial chemoembolization-resistant group; B: Non-resistant group.

Table 1 Expression of lipoma HMGIC fusion partner-like 2 in transarterial chemoembolization-resistant and non-resistant groups (n = 60), n (%).

Group	Low LHFPL2	High LHFPL2	χ2	P value
TACE-resistant	8 (26.67)	22 (73.33)	17.140	< 0.001
Non-resistant	24 (80.00)	6 (20.00)	-	-

HFPL2: Lipoma HMGIC fusion partner-like 2; TACE: Transarterial chemoembolization.

Clinicopathologic characteristics of TACE-resistant and non-resistant patients

Sex, age, cirrhosis, and tumor size did not differ between the two groups (P > 0.05). In contrast, the hepatitis B virus (HBV) carrier status, AFP level, and histological differentiation did vary (P < 0.05; Table 2).

Table 2 Comparison of clinical and pathological features between transarterial chemoembolization-resistant and non-resistant groups, n (%).

Variable	TACE-resistant (n = 30)	Non-resistant (n = 30)	χ2/t/Z	P value
Sex			1.200	0.273
Male	18 (60.00)	22 (73.33)
Female	12 (40.00)	8 (26.67)
Age (years), mean ± SD	64.40 ± 10.53	64.27 ± 10.61	0.048	0.962
HBV carrier			6.787	0.010
Yes	22 (73.33)	12 (40.00)
No	8 (26.67)	18 (60.00)
Cirrhosis			3.360	0.067
Yes	16 (53.33)	9 (30.00)
No	14 (46.67)	21 (70.00)
AFP level			-	< 0.001
≥ 400 ng/mL	18 (60.00)	4 (13.33)
< 400 ng/mL	12 (40.00)	26 (86.67)
Tumor size			2.443	0.118
> 3 cm	20 (66.67)	14 (46.67)
≤ 3 cm	10 (33.33)	16 (53.33)
Histologic differentiation			8.131	0.017
Poor	15 (50.00)	5 (16.67)
Moderate	7 (23.33)	15 (50.00)
Well	8 (26.67)	10 (33.33)

TACE: Transarterial chemoembolization; HBV: Hepatitis B virus; AFP: Alpha-fetoprotein.

Association between LHFPL2 expression and prognosis

Kaplan-Meier analysis showed that the median OS was 2.28 years in the high-LHFPL2 group, whereas the low-LHFPL2 group did not reach median OS (survival > 50% at last follow-up). The cumulative OS rate was significantly higher in the low-LHFPL2 group (log-rank χ² = 14.79, P < 0.001; Figure 2).

Figure 2 Correlation curve between lipoma HMGIC fusion partner-like 2 expression and patient survival. LHFPL2: Lipoma HMGIC fusion partner-like 2.

Univariate and multivariate Cox regression for OS

Variables were coded as follows: Age (continuous), sex (male = 1, female = 0), HBV (positive = 1, negative = 0), cirrhosis (yes = 1, no = 0), AFP ≥ 400 ng/mL (= 1), tumor size > 3 cm (= 1), poor differentiation (= 1), high LHFPL2 level (= 1), and refractory TACE (= 1). Factors that were significant in univariate analysis were entered into a multivariate model. AFP ≥ 400 ng/mL, high LHFPL2 expression, and TACE refractoriness were independent predictors of mortality (P < 0.05; Table 3).

Table 3 Univariate and multivariate Cox analyses of prognostic survival in patients with hepatocellular carcinoma.

Index	Univariate analysis			Multiple factor
	HR	95%CI	P value	HR	95%CI	P value
Age	1.040	1.000-1.084	0.067	-	-	-
Sex	0.688	0.318-2.920	0.342	-	-	-
HBV carrier	1.931	0.877-4.253	0.102	-	-	-
Cirrhosis	1.977	0.925-4.226	0.079	-	-	-
AFP ≥ 400 ng/mL	13.483	4.634-39.226	< 0.001	38.632	5.274-282.970	< 0.001
Tumor size > 3 cm	1.695	0.758-3.787	0.198	-	-	-
Poor differentiation	0.584	0.341-1.001	0.050	-	-	-
High LHFPL2	4.490	1.950-10.341	< 0.001	5.408	1.569-18.642	0.008
TACE refractoriness	12.444	4.131-37.489	< 0.001	6.955	1.458-33.170	0.015

CI: Confidence interval; HR: Hazard ratio; HBV: Hepatitis B virus; AFP: Alpha-fetoprotein; HFPL2: Lipoma HMGIC fusion partner-like 2; TACE: Transarterial chemoembolization.

Combined LHFPL2 and AFP for prognostic stratification

Because both high LHFPL2 and AFP ≥ 400 ng/mL were independent risk factors, we constructed a combined model: Group 1: Low LHFPL2 + AFP < 400 ng/mL (n = 25); group 2: Low LHFPL2 + AFP ≥ 400 ng/mL (n = 7); group 3: High LHFPL2 + AFP < 400 ng/mL (n = 13); and group 4: High LHFPL2 + AFP ≥ 400 ng/mL (n = 15) (Figure 3).

Figure 3 Forest plot of the multivariate Cox regression analysis for factors influencing patient survival prognosis. CI: Confidence interval; HR: Hazard ratio; TACE: Transarterial chemoembolization; LHFPL2: Lipoma HMGIC fusion partner-like 2; AFP: Alpha-fetoprotein.

Kaplan–Meier analysis revealed significant differences in OS among the four groups (log-rank χ² = 59.750, P < 0.001; Figure 4). Group 1 had the best outcome (median OS, not reached), whereas group 4 had the worst outcome (median OS 1.44 years). Median OS for groups 2 and 3 was 2.40 years and 3.91 years, respectively, with no significant difference between them (log-rank χ² = 1.960, P = 0.162).

Figure 4 Survival curves of patients with hepatocellular carcinoma in the lipoma HMGIC fusion partner-like 2-alpha-fetoprotein combination group. LHFPL2: Lipoma HMGIC fusion partner-like 2; AFP: Alpha-fetoprotein.

DISCUSSION

To our knowledge, the present study is the first to demonstrate that high LHFPL2 expression is significantly associated with both TACE refractoriness and shorter OS in patients with HCC who underwent curative resection and subsequently underwent TACE for intrahepatic recurrence. By integrating LHFPL2 with AFP, we constructed a refined prognostic model that might facilitate the early recognition of TACE failure and guide individualized treatment strategies.

High LHFPL2 expression was observed in 73.33% of TACE-resistant cases vs only 20.00% of non-resistant cases. Multivariate Cox analysis identified high LHFPL2 as an independent risk factor for refractoriness. These clinical data align with previous mechanistic work showing that LHFPL2 acts as a “hypoxia-pathway navigator”[14]. TACE induces tumor ischemia, and the resulting hypoxic microenvironment is a cardinal trigger for therapeutic resistance[15,16]. Hypoxia-inducible factors (HIFs) enhance glycolysis, angiogenesis, and epithelial-mesenchymal transition, thereby increasing tumor cell fitness and invasion[17,18]. Elevated HIF-1α and HIF-2α after TACE correlate positively with refractoriness[15]. We speculate that LHFPL2 is embedded in a “hypoxia-HIF-LHFPL2–signaling axis”: HBV infection can integrate HBV X protein and activate HIF, indirectly up-regulating LHFPL2[19], while LHFPL2 itself may heighten tumor tolerance to ischemic injury, neutralizing the cytotoxic intent of TACE[6].

AFP, the most widely used serum biomarker in HCC, reflects tumor proliferative activity, but has a about 30% false-negative rate, especially in early or well-differentiated tumors[20]. Multivariate analysis here showed that both AFP ≥ 400 ng/mL^-1 and high LHFPL2 were independent predictors of OS, suggesting complementary biology. Stratification of patients into four groups revealed distinctly separated survival curves (log-rank χ² = 59.750, P < 0.001): The LHFPL2-low/AFP-low subgroup had the best outcome (median OS not reached), whereas the LHFPL2-high/AFP-high subgroup had the poorest (median OS 1.44 years). This mirrors the observation by Guo et al[21] that the addition of tetraspanin transmembrane 4 L six family member 1 to AFP improved the area under the curve for TACE prognosis from 0.68 to 0.83. AFP supplies information on the global tumor burden, whereas LHFPL2 specifically captures the hypoxia-driven resistance potential; therefore, their combination provides a more comprehensive appraisal of therapeutic sensitivity and survival risk.

Several limitations of this study should be acknowledged. First, the retrospective design and modest sample size (n = 60) from only two centers may have introduced selection bias, therefore prospective multicenter validation with larger cohorts is warranted. Second, only protein expression was evaluated. Consequently, mechanistic studies using LHFPL2 knockdown/overexpression in vitro (hypoxia models) and in vivo (embolization-based mouse models) are required to delineate the signaling circuitry underlying TACE resistance[22]. Third, time-to-progression and objective response rates were not recorded, precluding the assessment of the predictive value of LHFPL2 for short-term TACE efficacy[23]. Future integration of translational and clinical endpoints is essential to fully exploit LHFPL2 as a therapeutic target.

CONCLUSION

High LHFPL2 expression was independently associated with TACE refractoriness and shortened OS in patients with HCC. Combining LHFPL2 with AFP significantly refined prognostic stratification. These findings expand the molecular landscape of TACE resistance, offer a practical tool for precise TACE, and provide a rationale for developing LHFPL2-based diagnostic assays and targeted interventions to improve outcomes in patients with HCC.