Prognostic value of LAT-1 status in solid cancer: A systematic review and meta-analysis

Background The expression of the L-type amino acid transporter 1 (LAT1) plays a significant role in tumor progression. However, it remains unclear whether high LAT1 expression correlates with poor prognosis of solid tumor patients. Here, we conducted a meta-analysis to assess the potential of LAT1 in predicting the prognosis of tumor patients. Methods and findings A total of 4,579 cases were analyzed from 35 qualified studies. In patients with solid tumors, elevated expression of LAT1 is associated with poor prognosis (overall survival [OS]: pooled hazard ratio (HR) = 1.848, 95% confidence interval (CI) = 1.620–2.108, P < 0.001; disease free survival [DFS]: pooled HR = 1.923, 95% CI = 1.585–2.333, P < 0.001; progression free survival [PFS]: pooled HR = 1.345, 95% CI = 1.133–1.597, P = 0.001). Furthermore, in subgroup analysis, we found an association between high LAT1 expression and poor OS in non-small cell lung cancer (HR = 1.554, 95% CI = 1.345–1.794, P < 0.001), pancreatic cancer (HR = 2.052, 95% CI = 1.613–2.724, P < 0.001) and biliary tract cancer (HR = 2.253, 95% CI = 1.562–3.227, P < 0.001). Conclusion The results of this meta-analysis indicate the reliability and potential of using LAT1 expression as a predictive biomarker in solid cancers prior to treatment. However, further studies with larger sample sizes would be beneficial for fully evaluating the predictive value of LAT1 expression for clinical applications.


Introduction
The L-type amino acid transporter 1 (LAT1) is a membrane protein responsible for transporting neutral amino acids, including phenylalanine leucine, valine, etc., as a part of system L for cellular intake of nutrients [1]. LAT1, also known as 4F2 light chain, is connected through a disulfide bond to the membrane-spanning 4F2 heavy chain (CD98) for its functional expression as part of a heterodimeric complex in the plasma membrane [2,3]. It has been previously reported that LAT1 is associated with the prognosis of tumors.
However, it remains unclear whether LAT1 expression is associated with a worse outcome across solid cancer patients. These conflicting results may be due to the small sample size among individual studies and limitations of current technology. Therefore, we conducted this meta-analysis to evaluate the prognostic value of LAT1 expression as a predictive biomarker with the goal of guiding clinicians and patients in determining the best course of treatment. third author (Chen) independently assessed the data from the initial article to review the discrepancy and agreement was achieved through discussion. The study was based on three outcome endpoints: OS, DFS and PFS. We extracted the following information from the selected papers according to the criteria above, including the name of the first author, country of origin, year of publication, number of patients analyzed, antibody, cancer types, LAT1 detection method, cut-off value, endpoints and Newcastle-Ottawa Scale (NOS). The summary of the included studies can be found in Table 1. For articles plotting prognosis only with Kaplan-Meier curve, survival data was extracted with Engauge Digitiser V4.1, and then HRs and 95% CIs were estimated by Tierney's method [23]. NOS was used to assess the quality of all studies [24]. Studies with NOS scores ranging from 6 to 9 were used for quality control of methodology.

Statistical analysis
The correlation between LAT1 expression and prognosis was assessed by meta-analysis using studies determined eligible by the inclusion criteria. The outcome endpoints (OS, DFS and PFS) were calculated for pooled HRs and 95% CIs. Subgroup analysis was conducted when each subgroup has or exceeds three studies. Higgins's I 2 statistic and Cochran's Q test were used to evaluate statistical heterogeneity [25], and lack of heterogeneity among studies was indicated when P value >0.10 and I 2 <40%. In accordance with heterogeneity status, we used the random effects model or fixed effects model to merge HR.
This analysis uses Funnel plots and the Egger's test to assess any potential publication bias [26]. In the event of publication bias, the Duval and Tweedie's trim and fill method was used to evaluate their impact on the overall effect [27]. Sensitivity analysis also identified potential outliers by omitting each study or specific studies. Stata 14.0 (StataCorp, College Station, TX) was used to conduct statistical analyses. The P values were two-tailed and statistical significant when p<0.05, except for those with statistical heterogeneity.

Demographic characteristics
As shown in the search flowchart (Fig 1), we initially retrieved 107 records in document retrieval databases using a predefined search approach. After reviewing the retrieved titles and abstracts, we excluded 32 articles that described irrelevant endpoints of the 75 articles downloaded as full-text, we excluded 40 studies that were unrelated to prognosis (15 articles), had no prognosis data (11 articles), where the prognosis was related to CD98 (3 articles), LAT2 (1 article), LAT1 and ASCT2 co-expression (3 articles), status of LAT1 RNA in cancers(2 articles), or were a description about LAT1 detection technology(5 articles). The remaining 35 articles that reached our meta-analysis criteria were selected to evaluate LAT1 expression and solid cancer prognosis of patients. To assess the relevance between LAT1 and the prognosis of solid cancer, 4,579 eligible patients were selected for this analysis. Sample cases ranged from 21 to 321. Of the 35 prognostic studies, 35 were on OS, 5 on DFS, and 7 on PFS. Most studies were performed in Japan (n = 33), followed by China (n = 1) and Thailand (n = 1), respectively. As for the cancer type, 11 studies evaluated lung cancer, of which 10 were non-small cell lung cancer, pancreatic cancer and biliary tract cancer each have 3 studies, hepatocellular carcinoma, ovarian tumor, gastric cancer, prostatic cancer and colorectal cancer each have 2 studies, breast cancer, multiple myeloma, tongue cancer, laryngeal squamous cell carcinoma, cutaneous melanoma, cutaneous angiosarcoma, malignant pleural mesothelioma and adenoid cystic carcinoma each have 1 study. Records idenƟfied through databases search (n=107)7) Full text arƟcles for further assessment (n=75) Studies included in this meta-analysis (n=35) Records excluded because Ɵtle and/or abstract were not relevant to the endpoint of the study (n=32) Full-text arƟcles excluded: Have no relaƟonship with the prognosis (n=15) Without prognosis data (n=11) CD98 as predicƟve factor (n=3) LAT2 as predicƟve factor (n=1) LAT1 and ASCT2 co-expression as predicƟve factor (n=3) LAT1 RNA as predicƟve factor (n=2) LAT1 detecƟon technology (n=5)

Evidence synthesis
This meta-analysis focused on the relationship between LAT1 status and the prognosis of cancer patients. In this study, the p value of heterogeneity test for OS was 0.004, and the I 2 value was 43.0%. The random effect model was used. The results showed that there was a significant

PLOS ONE
Prognostic value of LAT-1 status in solid cancer correlation between LAT1 high expression solid cancers and poor OS (pooled HR = 1.848, 95% CI = 1.620-2.108, P < 0.001) (Fig 2). Examining five DFS, the p value and I 2 value of heterogeneity test report were 0.052 and 57.4%, respectively. The pooled HR and 95% confidence interval (CI) were calculated using the random effect model. The results suggested that LAT1 high expression was associated with poor DFS (pooled HR = 1.923, 95% CI = 1.585-2.333, P < 0.001) (Fig 3). Seven PFS databases were included in the meta-analysis, and the p value and I 2 value of heterogeneity test report were 0.182 and 32.2%, respectively. The pooled HR and 95% confidence interval (CI) were calculated by using the fixed effect model. The results suggested that high LAT1 expression was associated with poor PFS (pooled HR = 1.345, 95% CI = 1.133-1.597, P = 0.001) (Fig 4).
To explore the source of heterogeneity, subgroup analysis was performed when more than 3 articles participated in each type. The results of subgroup analysis are shown in Table 2. In the cancer subgroups, there was a significant relationship between high LAT1 expression and poor OS in solid tumors observed in patients with lung cancer (HR = 1.573, 95% CI = 1.309-1.890, P < 0.001), non-small cell lung cancer (HR = 1.554, 95% CI = 1.345-1.794, P < 0.001), pancreatic cancer (HR = 2.052, 95% CI = 1.613-2.724, P < 0.001) and biliary tract cancer (HR = 2.253, 95% CI = 1.562-3.227, P < 0.001). When analyzed by the statistical method of

Publication bias and sensitivity analysis
Begg's funnel plot and Egger's test were used to detect publication bias of the above literature. There was no evidence of significant asymmetry on the funnel plot, and Egger's test found no publication bias (p<0.05) (Fig 5). Our sensitivity analysis confirmed the impact of these results. None of the individual studies had guided this meta-analysis and eliminating any single study had no significant effect on the final results (Fig 6).

Discussion
There is increasing evidence showing that LAT1 plays a significant role in promoting tumor cell growth and proliferation [2,3,28,29]  In our paper, we also used subgroup analysis to illustrate the relationship between high expression of LAT1 and prognosis. The subgroup analysis was conducted by at least 3 articles participated in each type including cancer type, HR estimation, cut off value and ethnicity. The significant relationship was still found in these subgroup.
The results of our current analysis may be affected by a number of limitations. First, the meta-analysis may be influenced by the exclusion of articles that are not published in English as well as articles that are proceedings or books. Despite our rigorous search and attempts to obtain unpublished information, some data loss was inevitable. Second, most of the studies involved were retrospective, with positive results more likely to be published than negative. Third, although the expression of LAT1 was detected by immunohistochemistry in all studies, the antibody concentration and cut-off value were different for each study, which could lead to some deviation in the combined analysis. Lastly, LAT1 is typically co-expressed with CD98, which may play a synergistic role in the development and progression of tumors.
Despite these limitations, this meta-analysis has several advantages. This is the first comprehensive statistical study of LAT1 that evaluates the prognostic significance of LAT1 status in solid cancers before treatment. We collected 4,579 patients from the limited subsets, which represented a large number of cases and significantly improved the statistical value in this analysis. Additionally, no publication biases were detected, suggesting that the results of the analysis are impartial.
The results suggest that before tumor treatment, the expression of LAT1 can be used to predict tumor patient outcome. Nevertheless, to verify our finding more accurately, more comprehensive studies will be required to evaluate the predictive value of high LAT1 expression for future clinical practice.    (6)