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Review of Immunohistochemistry Biomarkers in Pancreatic Cancer Diagnosis

Pancreatic cancer is one of the cancer types with poor prognosis and high rate of mortality. Diagnostic modalities for early detection of pancreatic cancer have been among the academic concerns. On account of the potential role of immunohistochemistry (IHC) biomarkers in overcoming certain limitations of imaging diagnostic tools in discriminating pancreatic cancer tissues from benign ones, a growing scholarly attention has been given to the diagnostic efficacy of IHC biomarkers for pancreatic cancer. This review will analyze and synthesize published articles to provide an insight into potential IHC biomarkers for pancreatic cancer diagnosis.


INTRODUCTION
In the developed countries, pancreatic cancer is presently the fourth leading cancer cause of mortality (1). Age-standardized incident rate of pancreatic cancer is 7.2 per 100,000 in developed countries versus 2.8 in less developed regions (2). Pancreatic cancer incident rate has gradually accelerated and it is anticipated, within a decade, to increase to the second, behind lung cancer, among the leading cancer-related causes of mortality (3). Over half of the pancreatic cancer cases have been diagnosed at an advanced stage, which can partially explain its five-year survival rate being under 9% (4).
The most prevalent (around 85%) among all pancreatic cancer types is pancreatic ductal adenocarcinoma (PDAC) (5). Precise diagnosis of PDAC is required for optimal patient management. Nonetheless, current PDAC diagnosis modalities, such as endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), demonstrate clinical limitations (6)(7)(8). Due to the potential resemblance of PDAC and benign diseases of the pancreas through imaging (9), certain challenges are encountered in differentiating them (6). Additionally, difficulties in gathering sufficient diagnostic samples as well as false positive and false negative diagnoses have limited the clinical utility of the EUS-FNA technique (6).
Endeavors to surmount such limitations in screening tests for early diagnosis of PDAC have concentrated on immunohistochemistry (IHC) biomarkers (3). Regardless of the proved efficacy of many IHC biomarkers for PDAC diagnosis (6) in terms of IHC images (see a representative IHC image in Figure 1), sensitivity, and specificity, as well as their ease of use, accessibility, and low costs (10), IHC biomarkers have demonstrated lack of consistency in diagnostic value. For instance, used as the standard pancreatic cancer biomarker, CA19-9 level elevation can be found not merely in PDAC but likewise in pancreatitis (11).
Our study hence aims to analyze and synthesize the findings from published articles in terms of the diagnostic role of IHC biomarkers for pancreatic cancer. Our review is crucial since it can suggest IHC biomarkers that are useful and efficacious for the early detection of pancreatic cancer in the clinical practices.

Search Strategy
A search was performed through the PubMed database to find fulltext English language journal articles relevant to our research question published from 2013 up to August 2021. We chose 2013 as the lower time limit since the most comprehensive systematic review of IHC biomarkers as a diagnostic tool for pancreatic cancer was conducted by Liu and colleagues (12) in 2012. The terms used for this search strategy comprised: (cancer.mp., or neoplasms/) AND (pancreatic or pancreas) AND (biomarkers.mp., or biomarkers/, or biomarkers, tumor/) AND (immunohistochemistry/, or immunohistochemical.mp.).

Exclusion and Inclusion Criteria
Articles that were excluded from the study consist of ones that: (1) are letters, case reports, theses, or conference proceedings; (2) merely examined IHC biomarkers in animal samples; (3) did not utilize apposite control groups; (4) demonstrated unclear clinical (diagnostic) implications; and (5) contained duplicate data or no reliable data (13,14). No limit applied to study design, geographical area, population, or race. An article was included into the study if it contained: (1) pancreatic cancer biospecimen; (2) IHC analytical implementation; (3) clinical significance in biomarker expression; (4) apposite biomarker metrics (specificity, sensitivity); (5) univariate or multivariate analysis as statistical estimation of biomarkers for differentiation of pancreatic cancer from benign conditions of the pancreas (13).
The quality of the included studies regarding IHC biomarkers was assessed by the principal researcher and an oncologist, in light of STARD (Standards for Reporting of Diagnostic Accuracy) guideline comprising 30 items (15). An article obtained 1 point for an item if we found all its facets, 0.5 points if we found some facets, and 0 point if we did not find it. The total score of all items determines the article quality in terms of three levels: high quality: 20-30; average quality: > 10, < 20; and low quality: ≤ 10 points (14).
Journal articles included in the review met inclusion and exclusion criteria as well as the quality of average rating or above. Analyzing each included article entailed deriving the data, for each type of IHC biomarker, on the sample size, pathologically positive results, test accuracy parameters, and the link of each IHC biomarker's expression to level of clinical evidence.

Analytical Procedure
As aforementioned, the articles were assessed based the inclusion and exclusion criteria and the rating scores. The studies were incorporated into the review if they investigated IHC biomarkers for pancreatic cancer diagnosis and met the selection criteria as well as whether their results were inclusive or negative. The data from the articles selected were transferred into Excel spreadsheet. The characteristics of the studies embraced: (1) research title, (2) the name of the first author, (3) publication year, (4) studied IHC biomarker(s), (5) sample size (total sample size and individual sample size for pancreatic cancer, benign pancreatic diseases, and healthy/normal cases), (6) analytic techniques (IHC staining, ELISA, and/or Western blotting), and (7) statistical findings related to IHC biomarker expression (sensitivity/ specificity). The articles were further analyzed in light of the findings regarding the efficacy of IHC biomarkers in discriminating pancreatic cancer from benign pancreatic diseases and normal/ healthy cases.

Included Studies
Systematic Reviews and Meta-analyses (PRISMA) flow chart was utilized for identifying, screening, and assessing articles in light of the inclusion and exclusion criteria. PRISMA flow chart is portrayed in Figure 2. 2896 studies were identified at initial literature search for the period of 2013-present. Eliminating duplicates led to 2174 studies. Screening titles and abstracts yielded 483 studies that underwent the assessment of full texts for eligibility. After further excluding studies that reflected low quality based on the criteria from STARD, as well as    Table 1, the number of the publications that have investigated IHC biomarkers as a diagnostic tool for pancreatic cancer has increased in the past five years. 11 out of the 17 articles (64.7%) have been published in the last five years. Across the 17 publications analyzed, there are 14 articles (82.4%) studying one IHC biomarker, two studies (11.8%) examining two IHC biomarkers, and one study (5.9%) examining four IHC biomarkers. While most articles have examined IHC biomarkers in PDAC, benign pancreatic diseases (especially chronic pancreatitis), and healthy/normal cases (70.6%), three (17.6%) out of 17 studies have assessed PDAC and healthy cases only.

Summary of the Key Characteristics of the Included Studies
In the 17 articles analyzed, total sample size that consisted of both pancreatic cancer and controls ranged from 67 to 497 with 88.2% of the studies whose sample size surpassed 100 cases. When it comes to sample size for pancreatic cancer cases only, the sample size varied between 33 and 298 with 76.5% of the studies whose sample size exceeded 90 cases of pancreatic cancer. Additionally, in terms of the efficacy to detect pancreatic cancer, 15 (88.2%) out of the 17 selected studies estimated sensitivity and specificity, whereas one study employed c-statistic (17) and the other study merely assessed the elevation of IHC expression (26). Furthermore, amongst the 17 studies analyzed, all studies utilized immunohistochemistry, 10 studies (58.8%) utilized ELISA, two studies (11.8%) utilized Western blot analysis, two studies (11.8%) used these three techniques, 10 studies (58.8%) used both immunohistochemistry and ELISA, and two studies (11.8%) used both immunohistochemistry and Western blot analysis. In terms of IHC staining, two studies (11.8%) reported cytoplasmic staining only, four studies (23.5%) demonstrated cytoplasmic and membranous staining, two studies (11.8%) reported cytoplasmic and nuclear staining, and one study (5.9%) demonstrated membranous and nuclear staining.
As for IHC biomarkers that the current review shares with Liu et al.'s (12) review, some consistencies and disparities are identified. Consistent with Liu et al.'s (12) review, the present review analysis unveils high discriminatory value for maspin (sensitivity/specificity: 87.5/100), S100P (sensitivity/specificity: 83/100), and IMP3 (sensitivity/specificity: 85/90) in diagnosis of pancreatic cancer. While Liu et al.'s (12) review indicated that MUC5AC was expressed in 67% of the PDAC cases, the current review mirrored a rather high sensitivity and specificity (83/80) for this IHC biomarker in pancreatic cancer diagnosis. Moreover, in Liu et al.'s (12) review, MUC1 expression was positive in 95% of the PDAC cases but in 50% of the normal pancreatic ducts cases, whereas our analysis indicates a high sensitivity (89) and an acceptable specificity (63). Furthermore, our review analysis identifies pVHL as a highly sensitive and specific diagnostic IHC biomarker for pancreatic cancer (sensitivity/specificity: 100/81.8), the review by Liu et al. (12) reported negative expression of pVHL in almost all PDAC cases and its positive expression in all cases of nonneoplastic acini and ducts.

The Validity and Credibility of Our Findings
The validity and credibility of the findings in our review might have been influenced by some factors especially from primary studies. First, patient populations demonstrated high heterogeneity in terms of ethnicity, age, and disease stage. Second, lack of uniformity in measuring the expression of IHC biomarkers in the specimens might function as a potential bias. Heterogeneity in immunostaining scoring is found among the articles incorporated into this review. The review reflects a lack of a consensus-based standard in reporting cut-offs for positivity. Staining intensity (weak, moderate, and strong) is the scoring system most commonly employed in the primary studies (18,20,21,23,(26)(27)(28)(29)(30). IHC biomarker expression levels in pancreatic cancer have also been estimated through gauging ratios of positive cells in some primary studies (18,20,25,26,28). Third, inappropriateness in matching between cases and controls as well as small sample sizes might affect sensitivity and specificity of IHC biomarkers in some selected articles (22,28), which might affect the quality of our review analysis.

Strengths and Limitations
This review analysis contains some strengths and limitations. This review is amongst the few in the area of IHC biomarkers that detect early pancreatic cancer through their differentiation of pancreatic cancer from benign pancreatic diseases. By identifying and comparing 22 IHC biomarkers in terms of their sensitivity and specificity, this review can indicate to clinicians sensitive and specific IHC biomarkers for pancreatic cancer diagnosis. Our analytic review can be generalized due to the clarity and rigor in its inclusion and exclusion criteria and article quality scoring, as well as no limits applied geographical area or race. Numerous limitations can be found in this study. First, by virtue of our search strategy that involves including merely published articles and excluding abstracts or unpublished studies, publication bias in this analysis is inevitable. Second, some IHC biomarkers in some confirmatory investigations might be missed due to our incorporation of IHC biomarker studies merely from 2013 up to present. Third, this analysis does not have strong available data on account of a small number of articles that examined specific IHC biomarkers. Fourth, results from the studies that combined IHC staining technique with other techniques might not have undergone appropriate analyses due to our focus on IHC approach to biomarkers for pancreatic cancer. Fifth, the generalizability of our analysis can be enhanced if similar findings in IHC differentiation of pancreatic cancer from benign pancreatic diseases can be reproduced from largerscale investigations into these 22 IHC biomarkers.

CONCLUSION
Several attempts to improve the current diagnostic techniques for pancreatic cancer and in turn its prognosis. Our systematic review of articles in relation to pancreatic cancer identifies 22 IHC biomarkers reported to increase in plasma of the tissues of pancreatic cancer. The studies in our review indicate a practical certainty of the precise diagnosis of pancreatic cancer by utilizing these IHC biomarkers by virtue of their sensitivity and specificity that are comparable to or higher than those of CA19-9 as a current biomarker standard. Our review may hence advance the strand of research on pancreatic cancer IHC biomarkers regardless of a need for further standardizations as well as validation for the use of IHC biomarkers alone or their panels for early diagnosis of pancreatic cancer. Further investigations should be conducted in more rigorous designs with multi-center scale, large sample sizes, appropriate control groups, annotated specimens, and standardization of immunostaining scoring and IHC threshold for positivity in pathological specimens.