The Prognostic Significance of Hsp70 in Patients with Colorectal Cancer Patients: A PRISMA-Compliant Meta-Analysis

Background Hsp70 (heat shock protein 70) plays a key role in carcinogenesis and cancer progression. However, the relationship between the Hsp70 expression level and the colorectal cancer patient survival is unknown. This study is aimed at investigating the relationship between Hsp70 and the prognosis of colorectal carcinoma patients. Methods PubMed, Web of Science, and Embase were used for systematic computer literature retrieval. Stata SE14.0 software was used for quantitative meta-analysis. Besides, data was extracted from selected articles. Relationships between Hsp70 expression level and prognosis were further studied. The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were also computed. Results A total of 11 potentially eligible studies with 2269 patients were identified in 10 tumors from PubMed, Web of Science, and Embase. Hsp70 overexpression was associated with poor overall survival (OS) and disease-free survival (DFS) in colorectal carcinoma patients (HRs, 0.65 (95% CI: 0.52-0.78) and 0.77 (95% CI: 0.23-1.32), respectively). Conclusions Hsp70 overexpression can predict poor survival in colorectal cancer patients.


Introduction
Colorectal cancer (CRC) is a serious health problem worldwide. The response and overall survival of advanced colorectal cancer patients are very poor compared with the early stage. While colorectal cancer can be treated through surgery, tumor recurrence develops in about 25% to 40% of patients [1]. Surgical tumor resection is the major treatment method for local advanced colorectal cancer. However, there is no effective treatment for metastatic tumors, especially those that cannot be surgically removed and those with poor chemotherapy and radiotherapy effects [2].
Hsps are well-maintained molecules overexpressed in cells subjected to various stress stimuli (heat shock or disruption of homeostasis). Hsps act as molecular chaperones assisting protein folding in normal metabolic conditions and promoting protein repair and stabilization during molecular stress [3]. Hsps are essential in cellular defense against carcinogenesis [4]. Their tumorigenesis role includes oncogenic protein stabilization, programmed cell death, replicative senescence inhibition, tumor angiogenesis induction, invasion, and metastasis activation [5]. Interestingly, most Hsp70 expression studies are based on colorectal cancer cell lines and xenografts and not actual biopsy samples [6].

Methods
2.1. Literature Retrieval. Studies published between July 9, 1989 and July 9, 2020 were downloaded from PubMed, Web of Science, and Embase. Text word and MeSH strategy were used for the retrieval. The following terms were used: heat shock protein 70 OR Hsp70 AND colorectal AND cancer OR carcinoma OR malignant OR neoplasm OR tumor. Besides, the reference lists of the retrieved studies were manually screened for additional literature retrieval.

Selection
Criteria. Two researchers independently extracted the studies. The inclusion criteria were as follows: (a) studies showing the relationship between the Hsp70 expression level and OS or DFS in colorectal carcinoma, (b) studies describing the relationship between clinicopathological parameters and Hsp70 expression levels in human carcinoma tissues, (c) studies grouping patients (two groups) based on Hsp70 expression level, and (d) if hazard ratios and 95% confidence intervals could be calculated. The exclusion criteria were as follows: (a) studies with insufficient information to compute hazard ratios and 95% confidence intervals or (b) letters, case reports, expert opinions, and reviews or (c) in vitro or animal studies. Besides, only English researches were included.

Data Extraction.
Two authors (GY and ZY) extracted data from identified articles that complied with the standards, and a third researcher resolved different opinions. The first author, country, year of publication, tumor type, sample size, study design, cut-off value, follow-up time, methods used, and the outcome were recorded. Hazard ratios with 95% confidence intervals for overall survival or diseasefree survival were also determined. Engauge Digitizer version 4.1 was used to calculate prognosis after drawing the Kaplan-Meier curves [24].

Statistical Methods.
The prognostic value of the Hsp70 expression level in colorectal cancer patients was determined by estimating the hazard ratios between the upregulated and downregulated Hsp70 tissue groups for overall survival or disease-free survival. The 95% confidence intervals and the heterogeneity were also calculated and evaluated based on P value and I 2 . Furthermore, I 2 > 50% indicated significant heterogeneity. The random-effects model was used to determine the total hazard ratios. A fixed-effects model was used when I 2 ≤ 50% (moderate heterogeneity). The Stata 14.0 software was used for all data analyses.

Included Studies and Characteristics.
The research procedure is shown in Figure 1. A total of 1085 articles were selected after screening Web of Science, Embase, and PubMed. A total of 890 studies were excluded after the title and abstract search. Moreover, 142 articles were excluded since they did not meet the inclusion criteria. Another 42 records had insufficient data for HR calculation. Finally, 11 articles with 2269 patients were used [21][22][23][25][26][27][28][29][30][31][32]. The 11 articles had a sample size of 167 to 256 (average 206) ( Table 1). Besides, the 11 articles were published between 2009 and 2020 from various countries, including Hungary (4), Korea (2), and Britain, Greece, and Jordan (1 each). Most articles used immunohistochemistry (IHC) to determine the Hsp70 expression level.

High Hsp70
Expression Is Associated with Poor OS. Systematic meta-analysis was used to assess the relationship between the Hsp70 expression level and OS in colorectal cancer patients. Meta-analysis of nine articles with 1917 colorectal cancer patients showed that Hsp70 expression was significantly associated with OS (HR 0.65; 95% CI: 0.52-0.78; Figure 2), indicating that Hsp70 overexpression is associated with poor survival. A fixed-effects model was used since no significant heterogeneity was found (I 2 = 0%) in the identified studies.

High Hsp70
Expression Is Associated with Poor DFS. The relationship between Hsp70 expression and disease-free survival of 352 cancer patients was also investigated (Figure 3). A fixed-effects model was used since there was no significant heterogeneity (I 2 = 0). The upregulated Hsp70 was associated with poor disease-free survival (HR = 0:77, 95% CI: 0.23-1.32).

Publication Bias.
Begg's and Egger's tests were used to evaluate the publication bias via funnel plot symmetry estimation. There was no significant asymmetry of the funnel plots. Therefore, this study has no significant publication bias [33] (Figures 4 and 5). A sensitivity analysis was also conducted to verify the credibility of hazard ratios for overall survival. There were no significant impacts on HRs after excluding any one study, suggesting result reliability ( Figure 6).

Discussion
In recent years, potential prognostic biomarkers have been identified to improve the efficacy and survival rate. However, the prognosis of most cancers is relatively low. Therefore, high/positive tissue Hsp70 expression could be associated with the prognosis of colorectal carcinoma patients. Furthermore, previous studies have concluded that Hsp70 expression has biological and clinical significance. However, various cancers have different results. Hsp70 is significantly upregulated in gastric, breast, and prostate carcinoma compared with the normal tissues. In contrast, some articles have indicated that Hsp70 is significantly downregulated in cancer tissues compared with normal tissues in malignant     However, a large study should be conducted to verify these results.
Notably, this study has several limitations. First, some survival statistics from digital computer technology should be carefully considered. Besides, non-English articles were excluded. Second, only 11 articles with 2269 patients were included. There were no enough articles for subgroup analyses on disease-free survival. Third, different studies had different definitions of Hsp70 expression. Therefore, more researches are needed for more inclusive conclusions.

Conclusion
Hsp70 overexpression is significantly associated with poor overall survival and disease-free survival in colorectal carcinoma patients. Several such studies can provide mechanistic, pathophysiological, and epidemiological evidence complementing the findings of the current clinical trials on Hsp70 inhibitors.

Data Availability
All data included in this study are available from the corresponding author upon request.