Methods
Design
The methodological approach for evidence searching and synthesis described in this protocol will conform to the Cochrane Collaboration’s methods for assessing diagnostic test accuracy [
5]. We will also follow the recommendations of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) included as an additional file (Additional file
1) [
6].
The study is registered at PROSPERO, the International Prospective Register of Systematic Reviews, at the University of York (CRD42015024181).
PIRO question
For analyses of the diagnostic accuracy of research questions, the acronym PIRO is used, corresponding to P (population), I (index test), R (reference standard), and O (outcome).
The definitions of the components of the PIRO acronym for this systematic review are as follows:
-
Population: women of reproductive age (14–50 years)
-
Index test: p57KIP2 immunohistochemistry
-
Reference standard: genotyping
-
Outcome: MHC idenification.
Search methods for identifying studies
Keywords and Medical Subject Headings related to HM, p57KIP2 and molecular genotyping will be used alone or in combination (together with synonyms and closely related words) to retrieve the relevant articles.
We will conduct searches in the Excerpta Medica Database (EMBASE), Centro Latinoamericano y del Caribe de Información en Ciencias de la Salud (LILACS), Medical Literature Analysis and Retrieval System Online (MEDLINE), Cochrane Central Register of Controlled Trials (CENTRAL), and Web of Science. We will also screen the reference lists of relevant studies and reviews for additional articles and will search the grey literature websites The Grey Literature Report, OpenGrey, and the Open Archives Initiative (OAIster). If necessary (in the case of unclear data, missing data or extractable data), we will attempt to contact the corresponding authors of the included studies for missing data and for clarification. The search strategy developed for MEDLINE (see Additional file
2) will be adapted for the other databases. There will be no language or publication year restriction.
Eligibility criteria for the included studies
Any cross-sectional study, case series, case-control study, cohort study, or clinical trial that evaluates the accuracy of p57KIP2 immunostaining for the diagnosis of CHM compared with genotyping will be included. Case reports, narrative reviews, and expert opinions will be excluded. Animal testing will be excluded.
Data collection
Two independent researchers will evaluate the titles and abstracts arising from the combined search and will independently extract all data from the retrieved articles using a predefined data extraction sheet. A third author will adjudicate any discrepancies.
In the case of duplicate publications or more than one publication from a preliminary study, we will attempt to maximize the use of the information by simultaneously evaluating all of the available data, but we will not include the same group of patients in the analysis more than once.
The data will be extracted in the form of a data sheet specifically developed for this analysis (Tables
1 and
2). The following information will be extracted from each study, with the possibility of adding further information during the extraction process when appropriate:
-
Study characteristics: title, author, country, design, language of publication, year of publication, sample size, and number of centres
-
Population characteristics: total number of patients, number of patients in groups for comparison, and age of the patients
-
Index test: type of test and diagnostic criteria
-
Standard test: type of test and diagnostic criteria
-
Outcomes: number of true positives, false positives, true negatives, and false negatives, sensitivity and specificity, negative predictive value (NPV) and positive predictive value (PPV), and the positive likelihood ratio (LR+) and negative likelihood ratio (LR−)
Table 1
Characteristics of the studies included
If there are any missing or insufficient data in the included studies, we will contact the corresponding authors of the studies via email to obtain additional information. When more than one threshold is available, all data will be recorded. A sensitivity analysis will be conducted to assess the impact of including studies with 20 % or more missing data.
Risk of bias assessment
We will assess the quality of the studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool [
7]. If necessary, QUADAS-2 will be adapted to fit different study designs included in accordance with our research question. Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will be used to rate the quality of the body of evidence retrieved in the search [
8].
Outcomes
The primary outcome will be the diagnostic accuracy of p57KIP2 immunostaining for the diagnosis of CHM, which will be described based on sensitivity and specificity, negative and positive predictive values, and positive and negative likelihood ratios wherever possible.
Statistical analysis
Where the data permit, we will compare the index test against the reference test. For each study, we will extract the number of true positives, true negatives, false positives, and false negatives. When the raw data are provided, contingency tables will be built to display the results of the tests. The test results will be treated as positive or negative for the cut-off values of the index test. The sensitivity, specificity, positive predictive value, false-positive rate, and positive likelihood ratio will be calculated from the cut-off values of the index test. Forest plots will be generated to illustrate sensitivity and specificity and the 95 % confidence intervals (CI). The diagnostic odds ratio (DOR) and the area under the curve (AUC) of the summary ROC (summary receiver operating characteristic (SROC)) will be calculated because the summary statistics indicate the power of the overall assessment for each of the two tests. A hierarchical model, using the correlation between sensitivity and specificity across the studies to provide a summary of the model, will be employed to calculate the pooled estimation using the R software 3.3.1 packages metafor [
9], mada [
10], and HSROC [
11]. The choice between the bivariate random-effects model [
12] and the HSROC model from Rutter e Gatsonis [
13] will be based on the presence of different thresholds. Both approaches can be used to compute estimates of the summary ROC curve and the average operating point and allow us to determine the extent of heterogeneity in the estimated pooled measure. If heterogeneity is detected, we will conduct a subgroup analysis and meta-regression to evaluate the impact of the covariates in the pooled estimation. The two models are mathematically equivalent when no covariates are included in the model.
The magnitude of heterogeneity will be assessed using Cochran’s Q statistic and Higgins
I2 statistic, where an
I2 greater than 50 % indicates the presence of significant heterogeneity. The
I2 statistic will be calculated according to the following equation:
I2 = 100 % × (
Q −
df)/Q, where
Q is the Cochran heterogeneity statistic [
14]. If quantitative synthesis is not appropriate, a descriptive analysis might be undertaken.
We will perform a sensitivity analysis to examine the effect of sample size and missing data on the results of the review. If there are adequate studies (no less than three studies), we will conduct a sensitivity analysis to check the robustness of the conclusions and assess the impact of the methodological quality.
The presence of publication bias will be assessed by performing a regression of lnDOR and the effective sample size (ESS) based on methods described by Deeks et al. [
15].
Discussion
There is considerable overlap in histological features between molar and non-molar pregnancies and between CHMs and PHMs, which results in significant interobserver variability in the diagnosis of HM and its mimics. Therefore, correct diagnosis of these difficult cases may require molecular techniques that examine the differences in DNA content between CHM and PHM, including flow or image cytometric DNA analysis, chromosome in situ hybridization, polymerase chain reaction-based genotyping, or HLA typing. However, these molecular diagnostic methods are technically difficult to perform, relatively costly, and unavailable in most pathology laboratories [
1].
Banet et al. established that immunohistochemical analysis of p57
KIP2 expression is highly correlated with genotyping results and demonstrated that CHM is almost always p57-negative, with only rare examples (0.5 %) displaying aberrant (positive) p57
KIP2 expression, which is attributable to retention of the maternal copy of chromosome 11. CHMs are androgenetic conceptions by definition, and the vast majority are monospermic (85 %) [
4].
The rare examples of aberrant p57
KIP2 expression in both CHM and PHM can be correctly classified using genotyping. The findings of Banet et al. demonstrated that p57
KIP2 IHC is extremely reliable for the diagnosis of CHM. Therefore, the algorithmic approach for the diagnosis of HM proposed in this study advocates that p57
KIP2 results be used to triage cases for genotyping because this technique provides a highly reliable method for accurately diagnosing CHM in routine practice using a single immunohistochemical stain, with very little risk of misclassification of CHM. Consequently, genotyping for CHM is not necessary in routine practice and can be reserved for problematic cases, such as when p57
KIP2 immunostaining is suboptimal or unsatisfactory or when there is a discrepancy between morphology and p57
KIP2 results. One exception would be the case of recurrent HM, which raises the possibility of familial biparental HM. Patients with this disorder can have multiple/recurrent CHMs that are morphologically, immunophenotypically, and clinically similar to conventional CHMs; specifically, they are p57-negative and appear to show a similar risk of persistent gestational trophoblastic disease but are characterized by biparental diploidy rather than androgenetic diploidy [
4].
Therefore, genotyping is useful in any patient with recurrent HMs to determine if they represent the familial form. It is important to recognize that the biparental form exists so that the genotyping results of biparental diploidy are not used to reject a diagnosis of CHM when the morphology and/or p57
KIP2 results support a diagnosis of CHM [
4].
Some studies [
1,
4] have confirmed that p57
KIP2 immunostaining is a practical and accurate adjunct for the diagnosis of CHM and its mimics because this technique is a relatively simple, reliable, cost-efficient, and rapid procedure. Therefore, the most ideal method for correctly classifying all HMs and non-molar specimens is a combined approach that includes the correlation of morphological features, p57
KIP2 IHC, and molecular genotyping [
4]. This combined approach is particularly important when evaluating difficult and challenging cases with discordant positive p57
KIP2 staining, when molecular techniques are still necessary [
1]. The findings of Banet et al. also confirmed that p57
KIP2 IHC analysis is useful for identifying androgenetic/biparental mosaic/chimeric conceptions, which include uniformly androgenetic/biparental mosaic specimens without molar features (probably early forms of placental mesenchymal dysplasia, which is characterized by androgenetic/biparental mosaicism and a lack of trophoblastic hyperplasia), androgenetic/biparental mosaic specimens with a molar component (typically CHMs), and twin gestations composed of CHM and non-molar specimen components. Recognition of the discordant and divergent staining patterns in these specimens is key to correctly interpreting these complex specimens and is necessary for specific microdissection of the different components to assure accurate molecular genotyping [
4].
Therefore, we decided to perform this systematic review and meta-analysis using the most definitive method to assess the accuracy of p57KIP2 IHC compared with molecular genotyping for the diagnosis of CHM.
Acknowledgements
Not applicable.