Role of zinc finger protein 71 in hepatocellular carcinoma: Methodological concerns, clinical relevance, and future directions

Abstract

A recent study by Qin et al emphasized the potential of zinc finger protein 71 (ZNF71) as a promising biomarker for hepatocellular carcinoma (HCC). The authors offered valuable insights into the relationship between ZNF71 and various clinical and pathological stages of HCC. However, several limitations are required to be addressed to improve the findings. These limitations include concerns regarding patient selection, the generalizability of the results, and the necessity for functional validation to establish ZNF71’s specific role in the progression of HCC. Furthermore, statistical issues related to multiple comparisons, confounding variables, and the inherent heterogeneity of high-throughput datasets warrant careful consideration. Future research should focus on multi-institutional cohorts, utilize in vivo models, and compare ZNF71 with established biomarkers to strengthen the clinical relevance of ZNF71.

Key Words: Zinc finger protein 71; Hepatocellular carcinoma; Biomarkers; Genomic data; Prognostic value; Functional validation; Clustered regularly interspaced short palindromic repeats; Tumor progression

Core Tip: A study by Qin et al showed significant upregulation of zinc finger protein 71 (ZNF71) in hepatocellular carcinoma and its association with disease progression. While their findings are valuable, limitations include the need for diverse patient cohorts, larger non-cancerous tissue samples, and functional validation through in vitro and in vivo studies. Statistical improvements, multivariate regression, and multiple comparison corrections are essential. Future research should integrate genomic, epigenomic, and tumor microenvironment data to compare ZNF71 with established biomarkers, such as alpha-fetoprotein. Addressing these gaps will strengthen ZNF71’s potential as a diagnostic and therapeutic target in hepatocellular carcinoma.

TO THE EDITOR

We read with great interest the study by Qin et al[1], which shows that zinc finger protein 71 (ZNF71) is significantly upregulated in hepatocellular carcinoma (HCC) and its association with the clinical and pathological stages of the disease. We commend the authors for their rigorous analysis and insightful findings; however, as a potential diagnostic and therapeutic target, several important limitations warrant further discussion. If addressed, these shortcomings could significantly improve our understanding of the disease and strengthen the findings and their clinical relevance.

PATIENT SELECTION AND GENERALIZABILITY

The current study used samples from a single institution, which may impact the broader applicability of its findings. HCC is a highly heterogeneous disease characterized by various underlying etiologies[2]. The study focused on a specific patient population by excluding individuals who had undergone preoperative treatments such as radiotherapy, chemotherapy, or immunotherapy. However, we want to point out that the study did not explicitly address other potential confounding factors. Among these, the presence of underlying liver diseases such as cirrhosis, including viral hepatitis, alcohol-related liver disease, and metabolic dysfunction-associated steatohepatitis, highlights the importance of stratifying patients in future research according to their specific underlying liver conditions, which may impact the expression of ZNF71[3]. Doing so will help determine whether the prognostic significance of ZNF71 remains consistent across various subtypes of HCC or if it is affected by particular etiological factors. In addition, the study analyzed 235 HCC specimens alongside 13 non-cancerous liver tissue samples. While the sample size for HCC tissues was considerable, the relatively small number of non-cancerous samples may introduce bias and limit the generalizability of the findings.

Furthermore, the non-cancerous group is heterogeneous, encompassing conditions such as cirrhosis and benign hepatic lesions, which could complicate the interpretation of results. The molecular characteristics of HCC can vary significantly among different populations and geographic regions[4]. It is essential to incorporate multi-institutional cohorts to enhance the representativeness of future research. This will not only provide a more comprehensive understanding of HCC but also facilitate the identification of potential differences in ZNF71 expression and function related to various etiological factors and genetic backgrounds. Studies should also stratify patients based on their liver health and implement more detailed exclusion criteria to enhance the rigor of future research[5]. This would help minimize selection bias and enhance the overall validity of the finding. Additionally, we recommend that the study team incorporate a larger and more homogeneous control group to improve the robustness of future research. Ideally, this group should be matched for factors such as age, sex, and underlying liver conditions to facilitate more accurate comparisons.

FUNCTIONAL VALIDATION OF ZNF71 IN HCC

The authors estimated the expression of ZNF71 using immunohistochemistry and high-throughput datasets. Although these techniques help identify potential biomarkers, they do not establish ZNF71’s direct functional role in the progression of HCC. For instance, the study lacks experiments demonstrating that the overexpression of ZNF71 contributes to tumor growth, invasion, or metastasis. In vitro and in vivo functional studies are crucial for substantiating ZNF71’s oncogenic role and elucidating its mechanistic pathways[6]. These studies may involve knockdown or overexpression experiments in HCC cell lines and animal models. Additionally, assays focusing on cell proliferation, migration, invasion, and apoptosis should be incorporated to provide substantial evidence of ZNF71’s functional influence on HCC cells.

STATISTICAL CONSIDERATIONS AND CONFOUNDING VARIABLES

The authors used multiple statistical tests but did not adequately address the issue of multiple comparisons, which raises the risk of type I errors (false positives). For instance, while correlation analyses between ZNF71 expression and immune cell infiltration include various immune cell types, a correction method for multiple testing, such as Bonferroni correction, is not applied[7]. This lack of adjustment may result in misleading associations being incorrectly reported as significant. Furthermore, the study used univariate analysis to evaluate the correlation between ZNF71 expression and clinicopathological features. This method does not account for confounding variables, such as age, sex, or pre-existing liver disease, that may affect the results. For example, older patients or those with advanced liver disease may exhibit elevated ZNF71 expression due to reasons unrelated to HCC[8]. Conducting multivariate regression analysis is advised to provide a more accurate assessment of ZNF71’s independent prognostic value. In addition, this approach would allow for the control of confounding factors, thereby clarifying whether ZNF71 expression is an independent predictor of HCC progression or is influenced by other variables. Future analyses should incorporate appropriate statistical corrections for multiple comparisons to enhance the reliability of the findings[9]. Furthermore, we recommend that the authors specify the statistical tests used in the methods section and whether any corrections were made.

HETEROGENEITY IN HIGH-THROUGHPUT DATASETS

The study combined various high-throughput datasets, including the Cancer Genome Atlas, Genotype-Tissue Expression, and Gene Expression Omnibus. While this increases the sample size, it also introduces heterogeneity due to variations in experimental protocols, platforms, and patient populations across the datasets. The authors note a significant degree of heterogeneity (I² = 88.9%) in their meta-analysis, which may compromise the reliability of the pooled results. It is recommended that subgroup analyses be conducted to determine whether their findings are consistent across different datasets and platforms[10]. Moreover, implementing more stringent inclusion criteria for selecting datasets could reduce heterogeneity and enhance the robustness of the results.

The study analyzed the link between ZNF71 expression and the infiltration of immune cells, yet it did not consider the variability present within the tumor immune microenvironment. Various factors, including previous treatments, tumor stage, and the patient’s immune status, can significantly impact immune cell infiltration and ZNF71 expression[11]. For instance, patients undergoing prior immunotherapy or chemotherapy might exhibit altered immune cell profiles, which could lead to confounding results. Future research should stratify patients based on their treatment history, tumor stage, and immune status to better understand the relationship between ZNF71 and immune infiltration[12]. The authors should also include markers indicative of immune activation or suppression, thereby providing a more comprehensive analysis of the tumor immune microenvironment. The study did not explore the potential genetic or epigenetic modifications that could regulate ZNF71 expression in HCC. Factors such as mutations in the ZNF71 gene, its regulatory regions, and epigenetic alterations, including DNA methylation, may impact its expression and function[13]. Integrative genomic and epigenomic analyses would be valuable in identifying the regulatory mechanisms of ZNF71 in HCC. Such analyses may include whole-genome sequencing, methylation profiling, and chromatin accessibility assays to identify genetic and epigenetic changes associated with ZNF71 overexpression.

TUMOR IMMUNE MICROENVIRONMENT AND ZNF71 EXPRESSION

The study underscored several correlations between ZNF71 expression and various clinical features, immune infiltration, and co-expressed genes. However, correlation does not equate to causation, meaning the precise mechanistic connections between ZNF71 and these associated factors remain unclear. For instance, although ZNF71 shows a positive correlation with immune cell infiltration, it has not been established whether ZNF71 directly influences immune cell recruitment or if this relationship is mediated by other variables[14]. The study’s discussion section stressed the correlative nature of the findings and cautioned against overinterpretation[14]. Further mechanistic research is necessary to establish causal relationships between ZNF71 expression and the observed associations with clinical features and immune infiltration.

The research positions ZNF71 as a promising biomarker for both diagnosis and prognosis of HCC. However, it did not compare ZNF71’s performance with established biomarkers, such as alpha-fetoprotein (AFP) or des-gamma-carboxy prothrombin (DCP), which limits the clinical significance of these findings. Without such comparisons, the question of whether ZNF71 provides additional benefits over already used biomarkers remains uncertain. ZNF71 has been correlated to various types of cancer, including laryngeal squamous cell carcinoma, where a decrease in its expression has been associated with poorer outcomes for patients[15]. By conducting comparative analyses across different tumor types, researchers may find the specific roles that ZNF71 plays in various tissues, which could improve its use as a biomarker.

Additionally, performing sensitivity and specificity analyses within multi-cancer cohorts would provide further evidence to support its potential as a reliable diagnostic tool. Future investigations should incorporate comparative analyses to assess the diagnostic and prognostic performance of ZNF71 concerning established biomarkers. This could involve using receiver operating characteristic curve analysis to evaluate the sensitivity and specificity of ZNF71 in conjunction with AFP, DCP, and other relevant biomarkers[16].

CORRELATION VS CAUSATION IN ZNF71-ASSOCIATED FINDINGS

The study suggested several correlations between ZNF71 expression and various clinical features, immune infiltration, and co-expressed genes. However, it is essential to note that correlation does not imply causation, leaving the mechanistic relationships between ZNF71 and these factors largely speculative. For instance, while there is a positive correlation between ZNF71 and immune cell infiltration, it remains uncertain whether ZNF71 directly influences immune cell recruitment or if other mediating factors influence this relationship.

COMPARATIVE ANALYSIS WITH ESTABLISHED HCC BIOMARKERS

The study presented ZNF71 as a potential diagnostic and prognostic biomarker for HCC. However, it did not provide a comparative analysis against established biomarkers, such as AFP or DCP, which omits the clinical relevance of the findings, as it is unclear whether ZNF71 offers any advantages over currently used biomarkers[17]. The discussion should emphasize the correlative nature of the findings and avoid overinterpretation. Mechanistic studies are needed to establish causal relationships between ZNF71 expression and its observed associations with clinical features and immune infiltration. Future studies should include comparative analyses to evaluate the diagnostic and prognostic performance of ZNF71 relative to established biomarkers. This could involve receiver operating characteristic curve analysis to compare the sensitivity and specificity of ZNF71 with AFP, DCP, and other biomarkers.

The assessment of publication bias in this study utilized Begg and Egger tests; however, the reliance on publicly available datasets may introduce selection bias, as studies with negative or null findings were often underreported, which could limit results in an overestimation of the effect size and significance associated with ZNF71 expression in HCC[18]. Moreover, the study utilized tissue microarrays and samples obtained from a single institution, raising concerns about the representativeness of the findings regarding the diverse spectrum of HCC cases. The exclusion of patients who underwent preoperative treatments further restricts the generalizability of the results to a broader patient population. While two independent pathologists conducted the immunohistochemistry evaluation, it is important to note that inter-observer variability was not quantified, which could impact the reliability of the findings. We recommend that the authors acknowledge these potential biases and consider validating their results through independent cohorts or prospective studies to strengthen the conclusions drawn from this study[19]. Exploring unpublished datasets or conducting original experiments to verify the outcomes would be beneficial. For future research, including multi-institutional cohorts and recruiting patients with varied treatment backgrounds would enhance the representativeness and applicability of the findings across a wider array of HCC patients. Implementing automated quantification techniques, such as digital pathology with AI-based scoring systems, could also improve the reproducibility of the results.

FUTURE DIRECTIONS

Future research should explore several key areas in greater depth to expand upon the conclusions drawn from this study. First, ZNF71 facilitates the progression of HCC and should be thoroughly examined. Research influences critical processes, such as cell cycle regulation and metabolic pathways[20]. Understanding these mechanisms could unveil new insights into the role of ZNF71 in tumor biology. Additionally, the potential for ZNF71 to serve as a therapeutic target warrants further exploration, particularly through innovative strategies such as small-molecule inhibitors or cutting-edge gene-editing technologies, like clustered regularly interspaced short palindromic repeats-Cas9. This could pave the way for new treatment modalities to combat HCC[21].

Furthermore, conducting well-designed prospective clinical trials is crucial to assessing the diagnostic and prognostic significance of ZNF71 in patients with HCC. By correlating ZNF71 expression levels with patient outcomes, researchers can better determine its utility in clinical settings. As liquid biopsies continue to play an increasingly important role in detecting and monitoring HCC, future research should investigate the expression of ZNF71 in circulating tumor DNA and exosomes[22]. By correlating ZNF71 Levels found in tissue samples with results from liquid biopsies, we may significantly improve its effectiveness as a non-invasive biomarker for HCC. An integrative approach, combining transcriptomic, proteomic, and epigenomic data, should be adopted to construct a comprehensive picture of ZNF71’s involvement in the pathogenesis of HCC[23]. This multi-omic perspective could reveal the nuanced relationships between ZNF71 and various biological phenomena within the context of cancer.

While the study establishes a noteworthy correlation between elevated ZNF71 expression and the progression of HCC, it stops short of providing causal evidence. To address this gap, in vivo studies utilizing clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout models are necessary to confirm whether ZNF71 has a direct oncogenic role in the development and progression of HCC. Moreover, despite the suggestion that ZNF71 may be a viable therapeutic target, there remains a lack of experimental data substantiating its role in tumor growth inhibition. To further investigate this hypothesis, preclinical models should rigorously evaluate the efficacy of targeting ZNF71 in enhancing the therapeutic effects of current HCC treatments, such as the multikinase inhibitor sorafenib or various immunotherapy approaches[24]. This line of investigation could significantly influence the future landscape of HCC treatment strategies.

CONCLUSION

The present study by Qin et al[1] offered valuable insights into the function of ZNF71 in HCC, particularly regarding its relationship with disease progression and immune infiltration. Nevertheless, several methodological and analytical limitations require further investigation. Expanding patient cohorts, utilizing more sophisticated statistical methods, and conducting functional validation studies can improve the robustness of the findings. In addition, handling the outlined limitations and following the suggested future research directions could significantly strengthen the study’s impact, ultimately aiding the development of innovative diagnostic and therapeutic approaches for HCC. These steps are crucial for confirming ZNF71’s potential as both a biomarker and a therapeutic target.