The importance of demonstrating the reproducibility of biomarker studies to validate their utility for the clinic is increasingly well recognized . Our systematic review and meta-analysis is the first investigation of the overall worldwide evidence on the association of the actin-bundling protein fascin-1 with mortality, time-to-disease progression, lymph node metastasis and distant metastasis, in some of the most prevalent forms of carcinoma.
Our meta-analyses demonstrate that there is strong evidence that fascin-1 protein is associated with an up to two and a half-fold increased risk of mortality in breast, colorectal and esophageal carcinomas. At present, there is little evidence that fascin-1 is associated with mortality for gastric and lung carcinomas. Fascin-1 is correlated with increased risk of disease progression in breast and colorectal carcinomas, but not in lung carcinoma. Strong evidence for association of fascin-1 with increased risk of lymph node metastasis was found for colorectal and gastric carcinomas, but not for lung and esophageal carcinomas. Fascin-1 protein was also associated with a greater than 70% increased risk of distant metastasis in colorectal, gastric and esophageal carcinomas, although the statistical evidence for association with esophageal carcinoma metastasis was weak.
The potential for search and inclusion bias in our meta-analysis is considerably reduced as we conducted a comprehensive search of four databases for papers published in all languages. Publication bias could result from selective reporting of results by individual studies, because statistically insignificant results are often not published [68, 69]. Many authors did not respond to requests for information or clarification and we also identified several studies which did not report all their data. However, funnel plot analyses were not generally indicative of any strong publication bias because visual inspection of funnel plots did not show asymmetry (Figure 6). There was potential for misclassification of outcome, because some studies did not provide a clear definition of the cause of death. However, sensitivity analysis of studies that had provided a clear definition of cancer-specific mortality demonstrated that fascin-1 expression was associated with a 49% increased risk of cancer-specific death. This effect-estimate was similar to the pooled results from studies that did not have a clear definition of the cause of death (Table 2A).
All esophageal carcinoma studies included in our meta-analysis were carried out in Asia. Esophageal carcinoma shows marked geographic variation with exceptionally high rates in Asia . However, rates of esophageal adenocarcinomas are increasing rapidly in several Western countries . To exclude ethnicity and locality as modifying factors, more studies should be carried out to assess the association between fascin-1 and risk of esophageal cancer-specific mortality in Western countries.
All the included studies used immunohistochemical analysis of archived tumor specimens to quantify fascin-1. Immunohistochemistry is a pathologist-based scoring system that is the most commonly used and reliable technique in diagnostic histopathology . In addition, nearly all included studies used the same two antibodies to fascin-1, enabling comparability of prognostic effects in our meta-analysis. Only one cohort  was prospectively assembled. The remaining studies were retrospective in design using samples based on the availability of specimens with interpretable cores (which may have been non-random) and clinical histories, rather than specifying and recruiting a truly representative sample from a clearly defined target population. No studies provided an appropriate justification of the sample size or a power calculation. In addition, there was limited control for confounding in the analysis of most datasets and incomplete ascertainment of outcome data, due to loss to follow-up, which can lead to biased effect-estimates. Therefore, most of the studies included in our meta-analysis were assessed as having a risk of bias. For the 18 studies examining associations of fascin-1 with mortality, only 8 studies, [6, 7, 9, 24, 52, 55, 56, 58], were found to be of high methodological quality. We were somewhat reassured, however, that the high quality studies showed a positive association between fascin-1 expression and mortality (pooled fixed effects HR: 1.43 (1.26 to 1.63; P <0.001), that was similar in magnitude to the association observed when all 18 studies were pooled (pooled random effects HR: 1.44 (1.24 to 1.68; P <0.001).
The scoring of fascin-1 by immunohistochemistry is a continuous measurement and in most publications researchers categorized tumor specimens into high/positive fascin-1 or low/negative fascin-1 based on different semi-quantitatively assessed cut-off points. For example, two studies [58, 66] categorized 0 to 8 as low expression and 9 to 12 as high expression.
On the other hand, another study  dichotomized low expression as a score of <75% immunoreactive tumor cells and high expression as >75% immunoreactive tumor cells. These differences could possibly discard potentially important quantitative information and reduce statistical power to detect real associations . There was also qualitative evidence of heterogeneity between the individual studies, but based on the number of available studies for each carcinoma type, it was not possible to investigate this statistically using meta-regression (apart from stratifying by scoring method). However, a recent meta-analysis of other immunohistochemical prognostic markers demonstrated that the cut-off value of the percentage of positively-stained cells used in the scoring criteria appears unlikely to bias the underlying relationship between a prognostic biomarker and mortality . With regard to future studies of fascin-1, our analyses indicate that scoring of tumors as fascin-1 negative (taken either as completely absent [52, 61], below 5% fascin-1 positive cells within a tumor [21, 50, 53, 55–57, 60, 64], or below 10% fascin-1 positive cells within a tumor [19, 20, 51, 62]) versus fascin-1 positive (that is, either more than 5% or 10% fascin-1 positive cells within a tumor) is adequate to detect an association of fascin-1 with risk of mortality (Figure 2). The validity of this simple scoring metric should facilitate clinical application of fascin-1 as a biomarker.
Metastasis is the major source of cancer-related mortality and, at the cellular level, a major effect of fascin-1 is in promoting carcinoma cell migration and invasion. Promotion of metastasis by fascin-1 has been reported in several mouse tumor xenograft models [10–13]. Pooling across all the included carcinomas in our dataset, we obtained strong evidence that fascin-1 is associated with increased risk of mortality, lymph node metastasis and distant metastasis. However, there was evidence of heterogeneity between different carcinomas. This could be expected due to the biological differences in mechanisms of carcinoma progression for different organs. For example, the weak association between fascin-1 and metastasis in esophageal carcinomas could be due to the rapid progression to metastatic disease that occurs in most patients with esophageal carcinoma. This is due to the unique anatomy of the esophagus which lacks serosa, thus making it easier for early rapid tumor cell spread through the extensive network of lymph nodes [75, 76]. This disease course is very different from that of breast or colon carcinomas that develop through distinct stages to an aggressive phenotype over many years.
Heterogeneity was also apparent within carcinoma types. The three breast carcinoma studies showed evidence of heterogeneity for mortality (I2 = 31.4%). However, it should be noted that each study included different histological types of breast carcinomas. Yoder et al.  studied primary node-positive and node-negative invasive breast carcinomas, which included infiltrating ductal carcinomas, infiltrating lobular carcinomas, mucinous carcinomas and medullary carcinomas. Al-Alwan et al.  studied invasive ductal carcinoma only, and Rodriguez-Pinilla et al.  investigated node-negative sporadic and hereditary invasive breast carcinomas. As breast cancer is a clinically heterogeneous disease , the heterogeneity identified in the meta-analysis could be due to the pooling of results across different subtypes of breast carcinomas. Further analyses of the association of fascin-1 with breast cancer mortality or metastasis will need to specifically evaluate different sub-types of breast carcinomas. In contrast, all the esophageal carcinoma studies had analyzed squamous cell carcinomas (Additional file 2); thus the between study heterogeneity is not due to pooling of results across biologically different tumors within the same organ.
Heterogeneity was also evident between the lung carcinoma studies (I2 = 41.9%). All the studies in our dataset had analyzed only non-small cell lung carcinomas, which include both squamous cell carcinomas and adenocarcinomas. Patients with pulmonary squamous cell carcinoma have a higher mortality rate than those with adenocarcinomas, which might be attributable to confounding factors, such as smoking status and age-related co-morbidities [78, 79]. Adenocarcinomas include different subtypes such as acinar, papillary, bronchioalveolar carcinoma and adenocarcinomas with mixed subtypes, which could account for the histological heterogeneity. One study reported that fascin-1 immuno-staining was more common in adenocarcinomas with prevalent invasive components of the acinar, papillary and solid types compared to adenocarcinomas with a prevalent bronchiolo-alveolar component . Thus, the observed heterogeneity in these studies could be due to differences in fascin-1 immunostaining among different subtypes of adenocarcinomas.
Differences in patient selection and disease etiology could also explain the heterogeneity between studies of colorectal carcinomas. Most analyses included all stages of colorectal carcinomas in their specimen sets [6, 11, 48, 54, 61–63], nevertheless, multiple studies reported important correlations of fascin-1 expression with increased mortality for stage III and IV tumors [6, 24, 51, 52], as confirmed by our sensitivity analysis results (Table 2B). Historically, the majority of colon adenocarcinomas originate in the left (distal) colon, yet the prevalence of right-sided (proximal) tumors is increasing  and multiple studies correlated high fascin-1 expression with right-sided (proximal) colonic tumors [6, 24, 61, 62]. In addition, rectal carcinomas have different biological, clinicopathologic and therapeutic implications from colon carcinomas [52, 81]. It would be preferable for future studies to analyze the association of fascin-1 with colorectal carcinomas specifically within stages and according to tumor location [24, 52].
For our meta-analysis of the association of fascin-1 with mortality, we extracted multivariable analysis results from most studies, except one, , which presented univariable analysis results and seven studies, [19–21, 50, 53, 57, 60], which presented results as univariable Kaplan-Meier curves. Subgroup analysis of studies that presented multivariable analysis results showed that fascin-1 protein was associated with a 44% increased risk of mortality. Ideally, meta-analysis would be based on the data of individual patients to allow for standardized control for confounding across all studies . As the hazard ratios from different studies have been adjusted for different confounders, we were unable to assess the independent role of fascin-1 as a new marker over existing markers .