Nonmolar Specimens Research Articles

Loss of p57 Expression in Conceptions Other Than Complete Hydatidiform Mole: A Case Series With Emphasis on the Etiology, Genetics, and Clinical Significance.

Combined p57 immunohistochemistry and DNA genotyping refines classification of products of conception specimens into specific types of hydatidiform moles and various nonmolar entities that can simulate them. p57 expression is highly correlated with genotyping and in practice can reliably be used to identify virtually all complete hydatidiform moles (CHM), but aberrant retained or lost p57 expression in rare CHMs and partial hydatidiform moles (PHM), as well as loss in some nonmolar abortuses, has been reported. Among a series of 2329 products of conceptions, we identified 10 cases for which loss of p57 expression was inconsistent with genotyping results (none purely androgenetic). They displayed a spectrum of generally mild abnormal villous morphology but lacked better developed features of CHMs/early CHMs, although some did suggest subtle forms of the latter. For 5 cases, genotyping (4 cases) and/or ancillary testing (1 case) determined a mechanism for the aberrant p57 results. These included 3 PHMs-2 diandric triploid and 1 triandric tetraploid-and 1 nonmolar specimen with loss of p57 expression attributable to partial or complete loss of the maternal copy of chromosome 11 and 1 nonmolar specimen with Beckwith-Wiedemann syndrome. For 5 cases, including 2 diandric triploid PHMs and 3 biparental nonmolar specimens, genotyping did not identify a mechanism, likely due to other genetic alterations which are below the resolution of or not targeted by genotyping. While overdiagnosis of a PHM as a CHM may cause less harm since appropriate follow-up with serum β-human chorionic gonadotropin levels would take place for both diagnoses, this could cause longer than necessary follow-up due to the expectation of a much greater risk of persistent gestational trophoblastic disease for CHM compared with PHM, which would be unfounded for the correct diagnosis of PHM. Overdiagnosis of a nonmolar abortus with loss of p57 expression as a CHM would lead to unnecessary follow-up and restriction on pregnancy attempts for patients with infertility. Genotyping is valuable for addressing discordance between p57 expression and morphology but cannot elucidate certain mechanisms of lost p57 expression. Future studies are warranted to determine whether chromosomal losses or gains, particularly involving imprinted genes such as p57, might play a role in modifying the risk of persistent gestational trophoblastic disease for PHMs and nonmolar conceptions that are not purely androgenetic but have some abnormal paternal imprinting of the type seen in CHMs.

"While there is p57, there is hope." The past and the present of diagnosis in first trimester abortions: Diagnostic dilemmas and algorithmic approaches. A review.

Distinction of hydatidiform moles (HM) from non-molar (NM) specimens and subclassification of HM as complete hydatidiform mole (CHM) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies. The issue of diagnostic reproducibility is still unsolved, the lack of diagnostic accuracy based on morphology is substantial with an important interobserver variability, even between experienced gynecologic pathologists. Many ancillary techniques have been investigated in the last years to refine HM diagnosis. p57 (a paternally imprinted, maternally expressed gene) immunohistochemistry, based on the unique genetics of CHM (purely androgenetic), PHM (diandric triploid), and NM specimens (biparental, with allelic balance) can identify CHMs, which lack p57 expression because of a lack of maternal DNA. However, although its role in HM diagnosis is pivotal, it does not allow the distinction of PHM from NM specimens, both of which express p57 due to the presence of maternal DNA. Molecular genotyping, which compares villous and decidual DNA patterns to determine the parental source and ratios of polymorphic alleles, distinguishes purely androgenetic CHM from diandric triploid PHM, and both of these from NM specimens. Beyond the claim of establishing a "diagnostic truth", exceptions and peculiar genetic scenarios in the origin of rare CHM and PHM should be kept in mind when approaching any ancillary technique. An algorithmic approach, even in settings with limited resources, can help the pathologists in the diagnostic dilemma of diagnosis of first trimester abortions.

Refined diagnosis of hydatidiform moles with p57 immunohistochemistry and molecular genotyping: updated analysis of a prospective series of 2217 cases.

Immunohistochemical analysis of p57 expression and molecular genotyping accurately subclassify molar specimens into complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM) and distinguish these from nonmolar specimens. Characteristics of a prospective series of potentially molar specimens analyzed in a large gynecologic pathology practice are summarized. Of 2217 cases (2160 uterine, 57 ectopic), 2080 (94%) were successfully classified: 571 CHMs (570 uterine, 1 ectopic), 498 PHMs (497 uterine, 1 ectopic), 900 nonmolar (including 147 trisomies, 19 digynic triploids, and 4 donor egg conceptions), and 56 androgenetic/biparental mosaics; 137 were complex or unsatisfactory and not definitively classified. CHMs dominated in patients aged < 21 and >45 years and were the only kind of molar conception found in the latter group. Of 564 successfully immunostained CHMs, 563 (99.8%) were p57-negative (1 p57-positive [retained maternal chromosome 11] androgenetic by genotyping). Of 153 genotyped CHMs, 148 (96.7%) were androgenetic (85% monospermic) and 5 were biparental, the latter likely familial biparental hydatidiform moles. Of 486 successfully immunostained PHMs, 481 (99%) were p57-positive (3 p57-negative [loss of maternal chromosome 11], 2 unknown mechanism). Of 497 genotyped PHMs, 484 (97%) were diandric triploid (99% dispermic) and 13 were triandric tetraploid (all at least dispermic). Of 56 androgenetic/biparental mosaics, 37 had a p57-negative complete molar component (16 confirmed as androgenetic by genotyping). p57 expression is highly correlated with genotyping, serving as a reliable marker for CHMs, and identifies molar components and androgenetic cell lines in mosaic conceptions. Correlation of morphology, p57 expression, genotyping data, and history are required to recognize familial biparental hydatidiform moles and donor egg conceptions, as the former can be misclassified as nonmolar and the latter can be misclassified as dispermic CHM on the basis of isolated genotyping results.

Hydatidiform Moles: Ancillary Techniques to Refine Diagnosis.

Distinction of hydatidiform moles from nonmolar specimens and subclassification of hydatidiform moles as complete hydatidiform mole versus partial hydatidiform mole are important for clinical practice and investigational studies. Risk of persistent gestational trophoblastic disease and clinical management differ for these entities. Diagnosis based on morphology is subject to interobserver variability and remains problematic, even for experienced gynecologic pathologists. To explain how ancillary techniques target the unique genetic features of hydatidiform moles to establish diagnostic truth, highlight the issue of diagnostic reproducibility and importance of diagnostic accuracy, and illustrate use of p57 immunohistochemistry and polymerase chain reaction-based DNA genotyping for diagnosis. Sources are the author's 10-year experience using ancillary techniques for the evaluation of potentially molar specimens in a large gynecologic pathology practice and the literature. The unique genetics of complete hydatidiform moles (purely androgenetic), partial hydatidiform moles (diandric triploid), and nonmolar specimens (biparental, with allelic balance) allow for certain techniques, including immunohistochemical analysis of p57 expression (a paternally imprinted, maternally expressed gene) and genotyping, to refine diagnoses of hydatidiform moles. Although p57 immunostaining alone can identify complete hydatidiform moles, which lack p57 expression because of a lack of maternal DNA, this analysis does not distinguish partial hydatidiform moles from nonmolar specimens because both express p57 because of the presence of maternal DNA. Genotyping, which compares villous and decidual DNA patterns to determine the parental source and ratios of polymorphic alleles, distinguishes purely androgenetic complete hydatidiform moles from diandric triploid partial hydatidiform moles, and both of these from biparental nonmolar specimens. An algorithmic approach to diagnosis using these techniques is advocated.

Accuracy of p57KIP2 compared with genotyping to diagnose complete hydatidiform mole: a systematic review and meta-analysis.

BackgroundDistinguishing hydatidiform moles (HMs) from nonmolar specimens and the subclassification of HM are important because complete hydatidiform mole (CHM) is associated with an increased risk of development of gestational trophoblastic neoplasia. However, diagnosis based solely on morphology has poor inter‐observer reproducibility. Recent studies have demonstrated that the use of p57KIP 2 immunostaining improves diagnostic accuracy for CHM.ObjectivesTo evaluate the accuracy of p57KIP 2 immunostaining compared with molecular genotyping for the diagnosis of CHM.Search strategyMajor databases were searched from inception to March 2017 using the terms ‘hydatidiform mole’, ‘p57’, and ‘genotyping’, with their variations, and the search limit for the relevant study design.Selection criteriaAny cross‐sectional study, case series, case–control study, cohort study, or clinical trial that evaluated the accuracy of p57KIP 2 immunostaining for the diagnosis of CHM compared with genotyping was included. Case reports, narrative reviews, expert opinions, and animal testing were excluded.Data collection and analysisExtracted accuracy data were tabulated and pooled using a hierarchical bivariate random effects model.Main resultsBivariate meta‐analysis produced a summary sensitivity of 0.984 (95% CI: 0.916–1.000) and specificity of 0.625 (95% CI: 0.503–0.736) with significant heterogeneity for specificity (I 2 = 71.8, chi‐square P = 0.029). The pooled summary diagnostic odds ratio was 56.54 (95% CI: 11.03–289.74) with no heterogeneity (I 2 = 0.00%, chi‐square P = 0.67). The diagnostic performance of the test was high with an area under the curve of (AUC) 0.980.Conclusionsp57KIP 2 immunostaining is accurate when diagnosing CHM. It can be used as an adjunct test in a combination algorithmic approach.Tweetable abstractA meta‐analysis to evaluate the accuracy of p57KIP 2 compared with genotyping to diagnose CHM.

Accuracy of p57KIP2 compared with genotyping for the diagnosis of complete hydatidiform mole: protocol for a systematic review and meta-analysis.

BackgroundDistinguishing hydatidiform moles (HMs) from non-molar specimens and the subclassification of HM are important because complete hydatidiform mole (CHM) is associated with an increased risk of gestational trophoblastic neoplasia. However, diagnosis based solely on morphology has poor interobserver reproducibility. Recent studies have demonstrated that the use of p57KIP2 immunostaining improves diagnostic accuracy for CHM.MethodsWe will conduct a systematic review of prospective and retrospective studies to evaluate the accuracy of p57KIP2 immunostaining compared with molecular genotyping for the diagnosis of CHM. A high-sensitivity search strategy will be employed in MEDLINE, EMBASE, LILACS, The Grey Literature Report, OpenGrey, OAIster, and Cochrane CENTRAL. Two reviewers will independently screen all identified references for eligibility and extract data. The methodological quality and bias of the included studies will be assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool, and the overall quality of evidence will be assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. If a meta-analysis is possible, pooled estimates of sensitivity, specificity, and positive and negative likelihood ratios will be calculated using bivariate random-effects models. Statistical heterogeneity will be evaluated with I 2 statistics and explored through sensitivity analysis.DiscussionThere is considerable overlap between the histological features of molar and non-molar pregnancies and between complete and partial HMs, which results in significant interobserver variability in the diagnosis of CHM and its mimics. Therefore, molecular techniques are used to correctly diagnosis and treat CHM. However, these molecular diagnostic methods are technically difficult to perform, relatively costly, and unavailable in most pathology laboratories. According to our results, p57KIP2 immunostaining appears to be a practical and accurate adjunct for the diagnosis of CHM and its mimics because this technique is relatively simple, reliable, cost-efficient, and rapid. This systematic review will help to determine whether p57KIP2 immunostaining is an adequate alternative diagnostic test for CHM.Systematic review registrationPROSPERO CRD42015024181 Electronic supplementary materialThe online version of this article (doi:10.1186/s13643-016-0349-7) contains supplementary material, which is available to authorized users.

Characteristics of hydatidiform moles: analysis of a prospective series with p57 immunohistochemistry and molecular genotyping

Immunohistochemical analysis of cyclin-dependent kinase inhibitor 1C (CDKN1C, p57, Kip2) expression and molecular genotyping accurately classify hydatidiform moles into complete and partial types and distinguish these from non-molar specimens. Characteristics of a prospective series of all potentially molar specimens encountered in a large gynecologic pathology practice are summarized. Initially, all specimens were subjected to both analyses; this was later modified to triage cases for genotyping based on p57 results: p57-negative cases diagnosed as complete hydatidiform moles without genotyping; all p57-positive cases genotyped. Of the 678 cases, 645 were definitively classified as complete hydatidiform mole (201), partial hydatidiform mole (158), non-molar (272), and androgenetic/biparental mosaic (14); 33 were unsatisfactory, complex, or problematic. Of the 201 complete hydatidiform moles, 104 were p57-negative androgenetic and an additional 95 were p57-negative (no genotyping), 1 was p57-positive (retained maternal chromosome 11) androgenetic, and 1 was p57-non-reactive androgenetic; 90 (85%) of the 106 genotyped complete hydatidiform moles were monospermic and 16 were dispermic. Of the 158 partial hydatidiform moles, 155 were diandric triploid, with 154 p57-positive, 1 p57-negative (loss of maternal chromosome 11), and 1 p57-non-reactive; 3 were triandric tetraploid, with 2 p57-positive and 1 p57-negative (loss of maternal chromosome 11). Of 155 diandric triploid partial hydatidiform moles, 153 (99%) were dispermic and 2 were monospermic. Of the 272 non-molar specimens, 259 were p57-positive biparental diploid, 5 were p57-positive digynic triploid, 2 were p57-negative biparental diploid (no morphological features of biparental hydatidiform mole), and 6 were p57-non-reactive biparental diploid. Of the 14 androgenetic/biparental mosaics with discordant p57 expression, 6 were uniformly mosaic and 8 had a p57-negative androgenetic molar component. p57 expression is highly correlated with genotyping, serves as a reliable marker for diagnosis of complete hydatidiform moles, and identifies androgenetic cell lines in mosaic conceptions. Cases with aberrant and discordant p57 expression can be correctly classified by genotyping.

Ki-67 Expression in Hydatidiform Moles and Hydropic Abortions

BackgroundDifferential diagnosis of hydatidiform moles from non-molar specimens as well as their sub-classification such as complete and partial hydatidiform moles are important for clinical management and accurate risk assessment for persistent gestational trophoblastic disease, but diagnosis based solely on histomorphology suffers from poor interobserver and intraobserver reproducibility.ObjectivesThis study was undertaken to determine whether the expression of Ki-67 protein could differentiate these entities.Materials and MethodsWe performed Ki-67 immunohistochemical staining in 19 molar (8 partial and 11 complete moles) and 10 non-molar (hydropic abortions) formalin-fixed, paraffin-embedded tissue samples. Ploidy analysis using flow cytometry had confirmed diploidy in hydropic abortions and complete moles and triploidy in partial moles.ResultsKi-67 immunoreactivity was assessed in villous cytotrophoblasts, syncytiotrophoblasts and stromal cells. Positive cells were found to be restricted mostly to the villous cytotrophoblasts, while syncytiotrophoblasts showed an absence of immunostaining for Ki-67, and occasional weak nuclear staining was seen in the stromal cells. There was a significant difference in Ki-67 immunoreactivity of cytotrophoblastic cells between hydropic abortions and complete moles (P < 0.001), hydropic abortions and partial moles (P = 0.002) and also between complete and partial moles (P < 0.001). On the other hand, there is significant overlap in the Ki-67 immunoreactivity between complete and partial moles (++ staining category) and between partial moles and hydropic abortions (+ staining category).ConclusionsDespite the significant differences , Ki-67 immunostaining could not be helpful in distinguishing molar placentas from hydropic abortions as well as partial from complete hydatidiform moles, because there are considerable overlaps between results in different categories.

Characterization of Androgenetic/Biparental Mosaic/Chimeric Conceptions, Including Those With a Molar Component

Recent studies have demonstrated the value of ancillary techniques, including p57 immunohistochemistry and short tandem repeat genotyping, for distinguishing hydatidiform moles (HM) from nonmolar specimens and for subtyping HMs as complete hydatidiform moles (CHM) and partial hydatidiform moles (PHM). With rare exceptions, CHMs are p57-negative and androgenetic diploid; partial hydatidiform moles are p57-positive and diandric triploid; and nonmolar specimens are p57-positive and biparental diploid. Androgenetic/biparental mosaic/chimeric conceptions can have morphologic features that overlap with HMs but are genetically distinct. This study characterizes 11 androgenetic/biparental mosaic/chimeric conceptions identified in a series of 473 products of conception specimens subjected to p57 immunohistochemistry and short tandem repeat genotyping. Fluorescence in situ hybridization was performed on 10 to assess ploidy. All cases were characterized by hydropically enlarged, variably sized and shaped villi. In 5 cases, the villi lacked trophoblastic hyperplasia, whereas in 6 there was a focal to extensive villous component with trophoblastic hyperplasia and features of CHM. The villi lacking trophoblastic hyperplasia were characterized by discordant p57 expression within individual villi (p57-positive cytotrophoblast and p57-negative stromal cells), whereas the villous components having trophoblastic hyperplasia were uniformly p57-negative in both cell types. Short tandem repeat genotyping of at least 2 villous areas in each case demonstrated an excess of paternal alleles in all regions, with variable paternal:maternal allele ratios (usually >2:1); pure androgenetic diploidy was identified in those cases with a sufficiently sized villous component having trophoblastic hyperplasia and features of CHM. Fluorescence in situ hybridization demonstrated uniform diploidy in 7 cases, including 4 of 5 tested cases with trophoblastic hyperplasia and 3 of 5 cases without trophoblastic hyperplasia. Two cases without trophoblastic hyperplasia had uniformly diploid villous stromal cells but 1 had triploid and 1 had tetraploid cytotrophoblast; 1 case with trophoblastic hyperplasia had uniformly diploid villous stromal cells but a mixture of diploid, triploid, and tetraploid cytotrophoblast. In 3 cases with a CHM component, persistent gestational trophoblastic disease developed. These results indicate that androgenetic/biparental mosaic/chimeric conceptions are most often an admixture of androgenetic diploid (p57-negative) and biparental diploid (p57-positive) cell lines but some have localized hyperdiploid components. Recognition of their distinctive p57 expression patterns and genotyping results can prevent misclassification as typical CHMs, PHMs, or nonmolar specimens. The presence of androgenetic cell lines, particularly in those with a purely androgenetic CHM component, warrants follow-up because of some risk of persistent gestational trophoblastic disease.

Diagnostic Utility of Microsatellite Genotyping for Molar Pregnancy Testing

Molecular genotyping by analysis of DNA microsatellites, also known as short tandem repeats (STRs), is an established method for diagnosing and classifying hydatidiform mole. Distinction of both complete hydatidiform mole and partial hydatidiform mole from nonmolar specimens is relevant for clinical management owing to differences in risk for persistent gestational trophoblastic disease. To determine the technical performance of microsatellite genotyping by using a commercially available multiplex assay, and to describe the application of additional methods to confirm other genetic abnormalities detected by the genotyping assay. Microsatellite genotyping data on 102 cases referred for molar pregnancy testing are presented. A separate panel of mini STR markers, flow cytometry, fluorescence in situ hybridization, and p57 immunohistochemistry were used to characterize cases with other incidental genetic abnormalities. Forty-eight cases were classified as hydatidiform mole (31, complete hydatidiform mole; 17, partial hydatidiform mole). Genotyping also revealed 11 cases of suspected trisomy and 1 case of androgenetic/biparental mosaicism. Trisomy for selected chromosomes (13, 16, 18, and 21) was confirmed in all cases by using a panel of mini STR markers. This series illustrates the utility of microsatellite genotyping as a stand-alone method for accurate classification of hydatidiform mole. Other genetic abnormalities may be detected by genotyping; confirmation of the suspected abnormality requires additional testing.

Diagnostic Reproducibility of Hydatidiform Moles

Distinction of hydatidiform moles from nonmolar specimens (NMs) and subclassification of hydatidiform moles as complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM) are important for clinical practice and investigational studies; however, diagnosis based solely on morphology is affected by interobserver variability. Molecular genotyping can distinguish these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to diagnose CHMs, PHMs, and NMs, respectively. Eighty genotyped cases (27 CHMs, 27 PHMs, 26 NMs) were selected from a series of 200 potentially molar specimens previously diagnosed using p57 immunohistochemistry and genotyping. Cases were classified by 6 pathologists (3 faculty level gynecologic pathologists and 3 fellows) on the basis of morphology, masked to p57 immunostaining and genotyping results, into 1 of 3 categories (CHM, PHM, or NM) during 2 diagnostic rounds; a third round incorporating p57 immunostaining results was also conducted. Consensus diagnoses (those rendered by 2 of 3 pathologists in each group) were also determined. Performance of experienced gynecologic pathologists versus fellow pathologists was compared, using genotyping results as the gold standard. Correct classification of CHMs ranged from 59% to 100%; there were no statistically significant differences in performance of faculty versus fellows in any round (P-values of 0.13, 0.67, and 0.54 for rounds 1 to 3, respectively). Correct classification of PHMs ranged from 26% to 93%, with statistically significantly better performance of faculty versus fellows in each round (P-values of 0.04, <0.01, and <0.01 for rounds 1 to 3, respectively). Correct classification of NMs ranged from 31% to 92%, with statistically significantly better performance of faculty only in round 2 (P-values of 1.0, <0.01, and 0.61 for rounds 1 to 3, respectively). Correct classification of all cases combined ranged from 51% to 75% by morphology and 70% to 80% with p57, with statistically significantly better performance of faculty only in round 2 (P-values of 0.69, <0.01, and 0.15 for rounds 1 to 3, respectively). p57 immunostaining significantly improved recognition of CHMs (P<0.01) and had high reproducibility (κ=0.93 to 0.96) but had no impact on distinction of PHMs and NMs. Genotyping provides a definitive diagnosis for the ∼25% to 50% of cases that are misclassified by morphology, especially those that are also unresolved by p57 immunostaining.

Diagnostic value of lectins in differentiation of molar placentas.

Distinction of hydatidiform moles from non-molar specimens and subclassification of hydatidiform moles as complete and partial are important for clinical practice, but diagnosis based solely on histomorphology suffers from poor interobserver reproducibility. Nowadays, pathologists rely on molecular techniques, however these methods are technically difficult, relatively expensive, and time consuming, and cannot be applied in all laboratories. Therefore, a relatively easy, time- and cost-effective ancillary tool, would be helpful. This study aimed to assess the role of lectins in differential diagnosis of molar placentas. Lectin histochemistry with a panel of HRP-conjugated lectins comprising SBA, DBA, MPA, PNA, VVA, UEA-1, LTA, GS-І (B4), and WGA were performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions) and 20 molar (partial and complete moles), formalin-fixed paraffin-embedded tissue samples. On the basis of staining intensity, sections were graded and Kruskal-Wallis non-parametric statistical test was used to compare differences between samples. There was a significant difference between the reactivities of LTA and UEA-І with syncytiotrophoblasts of molar and non-molar specimens (P<0.001). These lectins generally showed a moderate reactivity with syncytiotrophoblasts of molar group but did not react with this cell population in non-molar group. Furthermore, WGA showed relatively increased reaction with syncytiotrophoblasts of molar tissues compared with abortions, however, this did not reach to statistical significance (P=0.07). No major differences were seen in other lectins reactivities between the studied groups. The present study showed that UEA-1 and LTA lectins may be used as cytochemical probes in differentiating molar from non-molar placentas, but did not differentiate partial moles from complete moles.

Diagnostic Reproducibility of Hydatidiform Moles

Distinction of hydatidiform moles (HMs) from nonmolar specimens (NMs) and subclassification of HMs as complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs) are important for clinical practice and investigational studies; yet, diagnosis based solely on morphology is affected by interobserver variability. Molecular genotyping can distinguish these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to diagnose CHMs, PHMs, and NMs, respectively. Eighty genotyped cases (27 CHMs, 27 PHMs, and 26 NMs) were selected from a series of 200 potentially molar specimens previously diagnosed using p57 immunostaining and genotyping. Cases were classified by 3 gynecologic pathologists on the basis of H&E slides (masked to p57 immunostaining and genotyping results) into 1 of 3 categories (CHM, PHM, or NM) during 2 diagnostic rounds; a third round incorporating p57 immunostaining results was also conducted. Consensus diagnoses (those rendered by 2 of 3 pathologists) were determined. Genotyping results were used as the gold standard for assessing diagnostic performance. Sensitivity of a diagnosis of CHM ranged from 59% to 100% for individual pathologists and from 70% to 81% by consensus; specificity ranged from 91% to 96% for individuals and from 94% to 98% by consensus. Sensitivity of a diagnosis of PHM ranged from 56% to 93% for individual pathologists and from 70% to 78% by consensus; specificity ranged from 58% to 92% for individuals and from 74% to 85% by consensus. The percentage of correct classification of all cases by morphology ranged from 55% to 75% for individual pathologists and from 70% to 75% by consensus. The κ values for interobserver agreement ranged from 0.59 to 0.73 (moderate to good) for a diagnosis of CHM, from 0.15 to 0.43 (poor to moderate) for PHM, and from 0.13 to 0.42 (poor to moderate) for NM. The κ values for intraobserver agreement ranged from 0.44 to 0.67 (moderate to good). Addition of the p57 immunostain improved sensitivity of a diagnosis of CHM to a range of 93% to 96% for individual pathologists and 96% by consensus; specificity was improved from a range of 96% to 98% for individual pathologists and 96% by consensus; there was no substantial impact on diagnosis of PHMs and NMs. Interobserver agreement for interpretation of the p57 immunostain was 0.96 (almost perfect). Even with morphologic assessment by gynecologic pathologists and p57 immunohistochemistry, 20% to 30% of cases will be misclassified, and, in particular, distinction of PHMs and NMs will remain problematic.

Tetraploid Partial Hydatidiform Mole

Distinction of hydatidiform moles from nonmolar specimens and their subclassification as complete (complete hydatidiform mole) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies to refine ascertainment of risk of persistent gestational trophoblastic disease, which differs among these entities. Immunohistochemical analysis of p57 expression, a paternally imprinted maternally expressed gene on 11p15.5, and molecular genotyping are useful for improving diagnosis. Here, we describe a first trimester abortus with morphologic features consistent with a hydatidiform mole and p57 expression pattern supporting a diagnosis of PHM. Short tandem repeat (STR) genotyping and fluorescent in-situ hybridization analysis showed tetraploidy with 3 paternal and 1 maternal chromosome complements. To our knowledge, this is the first description of a tetraploid PHM confirmed to be triandric by STR analysis, and the first description of p57 immunostaining in a confirmed triandric tetraploid PHM. This case highlights the complex nature of the genetics that can be encountered in molar specimens and illustrates that STR genotyping, in contrast to fluorescent in-situ hybridization or ploidy analysis, offers the advantage of determining the parental origin of chromosome complements for refined diagnosis of hydatidiform moles.

Diandric Triploid Hydatidiform Mole With Loss of Maternal Chromosome 11

Distinction of hydatidiform moles (HM) from nonmolar specimens and their subclassification as complete (CHM) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies to refine ascertainment of risk of persistent gestational trophoblastic disease (GTD), which differs among these entities. Immunohistochemical analysis of p57 expression, a paternally imprinted maternally expressed gene on 11p15.5, and molecular genotyping are useful for improving diagnosis. CHMs are characterized by androgenetic diploidy, with loss of p57 expression due to lack of maternal DNA. Loss of p57 expression distinguishes CHMs from both PHMs (diandric triploidy) and nonmolar specimens (biparental diploidy), which retain expression. We report a unique HM characterized by morphologic features suggesting an early CHM, including lack of p57 expression by immunohistochemistry, but with genetic features more in keeping with a PHM. Specifically, molecular genotyping by short tandem repeat markers provided evidence to support interpretation as a PHM by demonstrating allele patterns and ratios most consistent with diandric triploidy, with evidence of loss of the maternal copy of chromosome 11 to explain the lack of p57 expression. This case illustrates the value of combined traditional pathologic and ancillary molecular techniques for refined diagnosis of molar specimens. It also raises questions regarding which modalities should be used to ultimately define the subtypes of HMs and whether chromosomal losses or gains, particularly involving imprinted genes such as p57, might play a role in modifying risk of persistent GTD.

Hydatidiform Moles

Distinction of hydatidiform moles from nonmolar specimens and subclassification of hydatidiform moles as complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), or early CHM are important for both clinical practice and investigational studies. The risk of persistent gestational trophoblastic disease and hence, clinical management, differs for CHMs, PHMs, and nonmolar specimens. However, diagnosis based solely on morphology suffers from poor interobserver reproducibility and remains problematic even for experienced gynecologic pathologists. The unique genetic features of CHMs (androgenetic diploidy), PHMs (diandric triploidy), and nonmolar specimens (biparental diploidy) allow for certain molecular techniques, including immunohistochemical analysis of p57 expression (a paternally imprinted maternally expressed gene) and molecular genotyping, to refine the diagnosis of hydatidiform moles. Although p57 immunostaining alone can identify CHMs, which lack p57 expression because of the lack of maternal DNA, this analysis cannot distinguish PHMs from nonmolar specimens as both express p57 because of the presence of maternal DNA. Short tandem repeat genotyping, which can determine the parental source of polymorphic alleles, can distinguish among all of these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to rigorously diagnose CHMs, PHMs, and nonmolar specimens, respectively. An algorithmic approach using these techniques to refine morphologic diagnosis has been developed for routine practice. This review discusses current issues in the diagnosis of hydatidiform moles, including the limitations of morphologic diagnosis, the need for refined diagnosis to assure accurate ascertainment of risk of persistent gestational trophoblastic disease associated with the different subtypes of hydatidiform moles, the use of ancillary immunohistochemical and molecular techniques for providing such refined diagnosis, and problems that can be encountered with these techniques.

Molecular Analysis of Hydatidiform Moles

Distinction of hydatidiform moles from nonmolar specimens and subclassification of hydatidiform moles as complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), or early CHM are important for both clinical practice and investigational studies. The risk of persistent gestational trophoblastic disease and hence, clinical management differs for CHMs, PHMs, and nonmolar specimens. However, diagnosis based solely on morphology suffers from poor interobserver reproducibility and remains problematic even for experienced gynecologic pathologists. The unique genetic features of CHMs (androgenetic diploidy), PHMs (diandric triploidy), and nonmolar specimens (biparental diploidy) allow for certain molecular techniques, including immunohistochemical analysis of p57 expression (a paternally imprinted maternally expressed gene) and molecular genotyping, to refine the diagnosis of hydatidiform moles. While p57 immunostaining alone can identify CHMs, which lack p57 expression due to the lack of maternal DNA, this analysis cannot distinguish PHMs from nonmolar specimens since both express p57 due to the presence of maternal DNA. Short tandem repeat genotyping, which can determine the parental source of polymorphic alleles, can distinguish among all of these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to rigorously diagnose CHMs, PHMs, and nonmolar specimens, respectively. An algorithmic approach using these techniques to refine morphologic diagnosis has been developed for routine practice. This review discusses current issues in the diagnosis of hydatidiform moles, including the limitations of morphologic diagnosis, the need for refined diagnosis to assure accurate ascertainment of risk of persistent gestational trophoblastic disease associated with the different subtypes of hydatidiform moles, and the utility of ancillary immunohistochemical and molecular techniques for providing such refined diagnosis.

Molecular Genotyping of Hydatidiform Moles: Analytic Validation of a Multiplex Short Tandem Repeat Assay

Distinction of hydatidiform moles from non-molar (NM) specimens, as well as their subclassification as complete (CHM) versus partial hydatidiform moles (PHM), is important for clinical management and accurate risk assessment for persistent gestational trophoblastic disease. Because diagnosis of hydatidiform moles based solely on morphology suffers from poor interobserver reproducibility, a variety of ancillary techniques have been developed to improve diagnosis. Immunohistochemical assessment of the paternally imprinted, maternally expressed p57 gene can identify CHMs (androgenetic diploidy) by their lack of p57 expression, but cannot distinguish PHMs (diandric monogynic triploidy) from NMs (biparental diploidy). Short tandem repeat genotyping can identify the parental source of polymorphic alleles and thus discern androgenetic diploidy, diandric triploidy, and biparental diploidy, which allows for specific diagnosis of CHMs, PHMs, and NMs, respectively. In this study, a retrospectively collected set of morphologically typical CHMs (n = 8), PHMs (n = 10), and NMs (n = 12) was subjected to an analytic validation study of both short tandem repeat genotyping and p57 immunohistochemistry. Several technical and biological problems resulted in data that were difficult to interpret. To avoid these pitfalls, we have developed an algorithm with quantitative guidelines for the interpretation of short tandem repeat genotyping data.

Complete Hydatidiform Mole With Retained Maternal Chromosomes 6 and 11

Distinction of hydatidiform moles from nonmolar specimens and their subclassification as complete hydatidiform mole (CHM) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies to refine ascertainment of risk of persistent gestational trophoblastic disease which differs among these entities. Immunohistochemical analysis of p57 expression, a paternally imprinted maternally expressed gene on 11p15.5, and molecular genotyping are useful for improving diagnosis. CHMs are characterized by androgenetic diploidy, with the loss of p57 expression owing to lack of maternal DNA. Loss of p57 expression distinguishes CHMs from both PHMs (diandric triploidy) and nonmolar specimens (biparental diploidy) which retain expression. In the process of evaluating molar specimens in our laboratory with p57 immunohistochemistry and molecular genotyping, we identified a morphologically typical androgenetic diploid CHM with aberrant diffuse p57 expression. Molecular genotyping by short tandem repeat markers and genome-wide copy number analysis by single nucleotide polymorphism array established androgenetic diploidy with retained maternal copies of chromosomes 6 and 11, with aberrant p57 expression attributable to the latter. This case, only the second reported to date, illustrates the value of combined traditional pathologic and ancillary molecular techniques for refined diagnosis of molar specimens. Specimens with morphologic features suggestive of CHM yet retaining p57 expression should be subjected to molecular genotyping to establish a definitive diagnosis because misclassification as PHM underestimates the risk of persistent gestational trophoblastic disease. We recommend use of p57 immunohistochemistry and molecular genotyping to evaluate all products of conception specimens for which there is any consideration of a diagnosis of hydatidiform mole. Genome-wide analysis has the potential to assist in localizing imprinted genes critical for determining the morphologic and behavioral phenotypes of hydatidiform moles.

Diagnosis and Subclassification of Hydatidiform Moles Using p57 Immunohistochemistry and Molecular Genotyping: Validation and Prospective Analysis in Routine and Consultation Practice Settings With Development of an Algorithmic Approach

Distinction of hydatidiform moles (HM) from nonmolar specimens and subclassification of HMs as complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), or early CHM (eCHM) are important for clinical practice and investigational studies but diagnosis based solely on morphology suffers from poor interobserver reproducibility. Recent studies have demonstrated the use of p57 immunostaining and molecular genotyping for improving diagnosis of HMs. After performing a validation study of both techniques on 24 archival products of conception specimens (7 CHMs, 8 PHMs, 9 nonmolar), we prospectively analyzed 42 cases, largely obtained from a gynecologic pathology consultation practice, for which there was any consideration of a diagnosis of HM. After satisfactory experience with prospective cases, a modified approach was adopted, with p57 immunostaining used in conjunction with morphology to triage cases for molecular genotyping. Final diagnoses for the prospective cases based on combined morphology and ancillary testing were 24 CHMs (including 7 eCHMs), 7 PHMs, and 11 nonmolar specimens. P57 immunostaining, performed on all 66 cases, was negative in all CHMs, with the exception of 1 case of molecularly confirmed CHM with diffuse p57 expression, and positive in all PHMs and nonmolar specimens, with the exception of 3 cases of molecularly confirmed PHMs with an equivocal extent of p57 expression. Molecular genotyping of 51 cases (24 validation, 27 prospective) yielded data consistent with p57 results in the 47 cases with unequivocal p57 expression patterns and was used to establish the diagnoses for the 4 cases with aberrant or equivocal p57 results. All 17 genotyped CHMs demonstrated androgenetic diploidy, including the CHM with retained p57 expression; this case also demonstrated trisomy of chromosome 11 (retained maternal allele), accounting for the aberrant p57 expression. The remaining 14 CHMs were diagnosed by morphology and negative p57 results alone. All 15 PHMs demonstrated diandric triploidy. All genotyped nonmolar specimens demonstrated biparental diploidy. This study validates p57 immunostaining as a prospectively applicable triage assay for the diagnosis of CHMs based on morphology and a negative p57 result. Molecular genotyping is validated as a method to confirm a diagnosis of CHM by demonstrating androgenetic diploidy and to resolve p57-positive cases into diandric triploid PHMs, biparental diploid nonmolar specimens, and the rare CHM with aberrant p57 expression.