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Molecular Genotyping of Hydatidiform Moles

2009; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês

10.2353/jmoldx.2009.090039

1943-7811

Kathleen M. Murphy, Thomas G. McConnell, Michael J. Hafez, Russell Vang, Brigitte M. Ronnett,

Prenatal Screening and Diagnostics

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. 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. Distinction of hydatidiform moles (HM) from non-molar (NM) specimens and the subclassification of HMs 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 of patients, differs for CHMs, PHMs, and NMs.1Berkowitz RS Goldstein DP Diagnosis and management of the primary hydatidiform mole.Obstet Gynecol Clin North Am. 1988; 15: 491-503PubMed Google Scholar2Berkowitz RS Tuncer ZS Bernstein MR Goldstein DP Management of gestational trophoblastic diseases: subsequent pregnancy experience.Semin Oncol. 2000; 27: 678-685PubMed Google Scholar3Sebire NJ Fisher RA Foskett M Rees H Seckl MJ Newlands ES Risk of recurrent hydatidiform mole and subsequent pregnancy outcome following complete or partial hydatidiform molar pregnancy.Bjog. 2003; 110: 22-26Crossref PubMed Scopus (163) Google Scholar4Sebire NJ Foskett M Fisher RA Lindsay I Seckl MJ Persistent gestational trophoblastic disease is rarely, if ever, derived from non-molar first-trimester miscarriage.Med Hypotheses. 2005; 64: 689-693Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar5Garner EI Goldstein DP Feltmate CM Berkowitz RS Gestational trophoblastic disease.Clin Obstet Gynecol. 2007; 50: 112-122Crossref PubMed Scopus (70) Google Scholar However, diagnosis of HMs based solely on morphology suffers from poor interobserver reproducibility.6Javey H Borazjani G Behmard S Langley FA Discrepancies in the histological diagnosis of hydatidiform mole.Br J Obstet Gynaecol. 1979; 86: 480-483Crossref PubMed Scopus (45) Google Scholar,7Howat AJ Beck S Fox H Harris SC Hill AS Nicholson CM Williams RA Can histopathologists reliably diagnose molar pregnancy?.J Clin Pathol. 1993; 46: 599-602Crossref PubMed Scopus (103) Google Scholar Even among experienced pathologists, high interobserver and intraobserver variability exist.8Fukunaga M Katabuchi H Nagasaka T Mikami Y Minamiguchi S Lage JM Interobserver and intraobserver variability in the diagnosis of hydatidiform mole.Am J Surg Pathol. 2005; 29: 942-947Crossref PubMed Scopus (112) Google Scholar The unique genetic features of CHMs, PHMs, and NMs can be exploited to improve diagnosis of HMs. CHMs are most often diploid with both chromosomal complements being paternal in origin (androgenetic diploidy), whereas PHMs are triploid with one maternal chromosome complement and two paternal chromosome complements (diandric triploidy). NMs are typically diploid with one maternal and one paternal chromosome complement (biparental diploidy); some NMs can be triploid due to two maternal and one paternal chromosome complement (digynic triploidy) but these specimens do not have the morphological features of PHMs.9Redline RW Hassold T Zaragoza MV Prevalence of the partial molar phenotype in triploidy of maternal and paternal origin.Hum Pathol. 1998; 29: 505-511Abstract Full Text PDF PubMed Scopus (67) Google Scholar,10Zaragoza MV Surti U Redline RW Millie E Chakravarti A Hassold TJ Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole.Am J Hum Genet. 2000; 66: 1807-1820Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar A variety of ancillary techniques targeting these genetic differences have been used to improve diagnosis of HMs. These include formal cytogenetic analysis (karyotyping), determination of ploidy by flow cytometry,11Lage JM Mark SD Roberts DJ Goldstein DP Bernstein MR Berkowitz RS A flow cytometric study of 137 fresh hydropic placentas: correlation between types of hydatidiform moles and nuclear DNA ploidy.Obstet Gynecol. 1992; 79: 403-410Crossref PubMed Scopus (123) Google Scholar12Lage JM Popek EJ The role of DNA flow cytometry in evaluation of partial and complete hydatidiform moles and hydropic abortions.Semin Diagn Pathol. 1993; 10: 267-274PubMed Google Scholar13Lage JM Bagg A Berchem GJ Gestational trophoblastic diseases.Curr Opin Obstet Gynecol. 1996; 8: 79-82Crossref PubMed Scopus (2) Google Scholar fluorescent in situ hybridization,14Yver M Carles D Bloch B Bioulac-Sage P Martin Negrier ML Determination of DNA ploidy by fluorescence in situ hybridization (FISH) in hydatidiform moles: evaluation of FISH on isolated nuclei.Hum Pathol. 2004; 35: 752-758Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,15Lescoat D Jouan H Loeuillet-Olivo L Le Calve M Fluorescent in situ hybridization (FISH) on paraffin-embedded placental tissues as an adjunct for understanding the etiology of early spontaneous abortion.Prenat Diagn. 2005; 25: 314-317Crossref PubMed Scopus (14) Google Scholar PCR amplification of short tandem repeat (STR) loci,16Bell KA Van Deerlin V Addya K Clevenger CV Van Deerlin PG Leonard DG Molecular genetic testing from paraffin-embedded tissue distinguishes nonmolar hydropic abortion from hydatidiform mole.Mol Diagn. 1999; 4: 11-19Crossref PubMed Scopus (50) Google Scholar17Lai CY Chan KY Khoo US Ngan HY Xue WC Chiu PM Tsao SW Cheung AN Analysis of gestational trophoblastic disease by genotyping and chromosome in situ hybridization.Mod Pathol. 2004; 17: 40-48Crossref PubMed Scopus (53) Google Scholar18Popiolek DA Yee H Mittal K Chiriboga L Prinz MK Caragine TA Budimlija ZM Multiplex short tandem repeat DNA analysis confirms the accuracy of p57(KIP2) immunostaining in the diagnosis of complete hydatidiform mole.Hum Pathol. 2006; 37: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar19Bifulco C Johnson C Hao L Kermalli H Bell S Hui P Genotypic analysis of hydatidiform mole: an accurate and practical method of diagnosis.Am J Surg Pathol. 2008; 32: 445-451Crossref PubMed Scopus (69) Google Scholar and immunohistochemistry for the paternally imprinted gene p57.18Popiolek DA Yee H Mittal K Chiriboga L Prinz MK Caragine TA Budimlija ZM Multiplex short tandem repeat DNA analysis confirms the accuracy of p57(KIP2) immunostaining in the diagnosis of complete hydatidiform mole.Hum Pathol. 2006; 37: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,20Hoffner L Dunn J Esposito N Macpherson T Surti U P57KIP2 immunostaining and molecular cytogenetics: combined approach aids in diagnosis of morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions.Hum Pathol. 2008; 39: 63-72Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Most of these techniques, including karyotyping and ploidy analyses, have limitations beyond the known technical and interpretive difficulties in that they cannot specifically discern the maternal and paternal chromosomal contributions in a specimen. Thus, while diploid and triploid specimens can be identified to improve both recognition of PHMs and distinction of PHMs from CHMs, CHMs (particularly the morphologically subtle early forms) cannot be distinguished from NMs (both yield nonspecific diploid results), and PHMs cannot be distinguished from digynic triploid NMs (both yield nonspecific triploid results). CHMs (including early forms) can be distinguished from PHMs and NMs by immunohistochemical assessment of expression of the paternally imprinted p57 gene. CHMs are characterized by lack of p57 expression in villous stromal cells and cytotrophoblast due to the lack of maternal DNA. However, p57 immunohistochemistry cannot distinguish PHMs from NMs since both retain expression of p57 due to the presence of maternal DNA. Molecular genetic analysis of the type provided by STR genotyping offers greater diagnostic discriminatory capability than these other techniques in that CHMs, PHMs, and NMs can be specifically distinguished from one another based on identification of the parental source of polymorphic alleles and their ratios. In particular, this analysis can discern androgenetic diploidy, diandric triploidy, and biparental diploidy to rigorously diagnose and distinguish CHMs, PHMs, and NMs, respectively. Such analysis is particularly important for the diagnosis of PHMs, which continue to pose diagnostic difficulty and cannot be distinguished from NMs, especially those exhibiting abnormal villous morphology of the type associated with other (non-molar type) genetic abnormalities, due to shared p57 expression patterns.21Chew SH Perlman EJ Williams R Kurman RJ Ronnett BM Morphology and DNA content analysis in the evaluation of first trimester placentas for partial hydatidiform mole (PHM).Hum Pathol. 2000; 31: 914-924Abstract Full Text PDF PubMed Scopus (52) Google Scholar Several studies have demonstrated the proof of concept and utility of STR genotyping for distinction of CHMs, PHMs, and NMs.16Bell KA Van Deerlin V Addya K Clevenger CV Van Deerlin PG Leonard DG Molecular genetic testing from paraffin-embedded tissue distinguishes nonmolar hydropic abortion from hydatidiform mole.Mol Diagn. 1999; 4: 11-19Crossref PubMed Scopus (50) Google Scholar,17Lai CY Chan KY Khoo US Ngan HY Xue WC Chiu PM Tsao SW Cheung AN Analysis of gestational trophoblastic disease by genotyping and chromosome in situ hybridization.Mod Pathol. 2004; 17: 40-48Crossref PubMed Scopus (53) Google Scholar,19Bifulco C Johnson C Hao L Kermalli H Bell S Hui P Genotypic analysis of hydatidiform mole: an accurate and practical method of diagnosis.Am J Surg Pathol. 2008; 32: 445-451Crossref PubMed Scopus (69) Google Scholar However, none of these reports provides a technical validation that includes specific details regarding how STR data were interpreted, criteria for accepting or rejecting data, and sources of technical and interpretive problems. In the current report we have used a set of retrospective, morphologically typical cases of HMs and NMs to develop an algorithm for the interpretation of STR genotyping data, including specific quantitative criteria for interpretation of results. To fully validate the utility of both STR genotyping and p57 immunohistochemistry and to implement these ancillary tests into routine clinical practice, this algorithm was then applied prospectively to all cases encountered on the Johns Hopkins Gynecologic Pathology Service for which a diagnosis of hydatidiform mole was considered (overwhelmingly morphologically challenging consultation cases); this prospective analysis is described in a separate report.22McConnell TG Murphy KM Hafez M Vang R Ronnett BM 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.Am J Surg Pathol. 2009; 33: 805-817Crossref PubMed Scopus (97) Google Scholar Thirty cases were selected from the routine Gynecologic Pathology Service files of The Johns Hopkins Hospital, Baltimore, MD. These included 8 CHMs, 10 PHMs, and 12 NMs. All cases were reviewed by two pathologists (B.M.R., R.V.) at a multiheaded microscope to select only those cases for which there was complete agreement that the morphological features were fully diagnostic of the assigned diagnostic category (CHM, PHM, NM). The material from these cases was collected between the years 1995 and 2006. This study is covered under Institutional Review Board approval NA 00002948 for the use of de-identified clinical samples to evaluate new methods/technologies. Formalin-fixed, paraffin-embedded tissue sections (5-micron) were stained using a Ventana BenchMark XT automated immunostaining system (Tucson, AZ) with mouse monoclonal antibodies against p57 protein (Neomarkers, Fremont, CA) from a ready to use preparation without dilution. The presence or absence of nuclear positivity was assessed in villous stromal cells, cytotrophoblast, intermediate trophoblast, and maternal decidua. Based on the staining patterns reported in the literature indicating that virtually all CHMs entirely lack p57 expression in villous stromal cells and cytotrophoblast whereas PHMs and NMs diffusely express p57 in these cell types, the quantity of these cells expressing p57 was estimated by routine light microscopic assessment of all of the villi present in the stained sections, without resorting to actual cell counting. Staining was semiquantitatively assessed as follows: negative (no staining in these cell types), limited (staining in 10% but 50% of these cell types). The p57 immunostain result was then interpreted as "positive" when the extent of staining was diffusely positive in these cell types. This pattern of expression is consistent with all forms of NMs as well as PHMs and cannot distinguish among these entities. The p57 immunostain was interpreted as "negative" and satisfactory when maternal decidua and/or intermediate trophoblastic cells exhibited nuclear expression of p57 (serving as internal positive control) but villous stromal cells and cytotrophoblast were either entirely negative or demonstrated only limited expression ( 10% but 300 bases) are produced for some loci, which may not yield sufficient amplification for interpretation. Regardless of which STR loci are used, rigorous validation of the assay is required if the results are to be reported for clinical use. Communication between the pathologist choosing tissue for analysis and the laboratory performing the assay is also essential for quality results.