Distinct microRNA profiles for complete hydatidiform moles at risk of malignant progression.

BACKGROUND: MicroRNAs are small noncoding RNAs with important regulatory functions. Although well-studied in cancer, little is known about the role of microRNAs in premalignant disease. Complete hydatidiform moles are benign forms of gestational trophoblastic disease that progress to gestational trophoblastic neoplasia in up to 20% of cases; however, there is no well-established biomarker that can predict the development of gestational trophoblastic neoplasia. OBJECTIVE: This study aimed to investigate possible differences in microRNA expression between complete moles progressing to gestational trophoblastic neoplasia and those regressing after surgical evacuation. STUDY DESIGN: Total RNA was extracted from fresh frozen tissues from 39 complete moles collected at the time of uterine evacuation in Brazil. In the study, 39 cases achieved human chorionic gonadotropin normalization without further therapy, and 9 cases developed gestational trophoblastic neoplasia requiring chemotherapy. Total RNA was also extracted from 2 choriocarcinoma cell lines, JEG-3 and JAR, and an immortalized normal placenta cell line, 3A-subE. MicroRNA expression in all samples was quantified using microRNA sequencing. Hits from the sequencing data were validated using a quantitative probe-based assay. Significantly altered microRNAs were then subjected to target prediction and gene ontology analyses to search for alterations in key signaling pathways. Expression of potential microRNA targets was assessed by quantitative real-time polymerase chain reaction and western blot. Finally, potential prognostic protein biomarkers were validated in an independent set of formalin-fixed paraffin-embedded patient samples from the United States (15 complete moles progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed) using quantitative immunohistochemistry. RESULTS: In total, 462 microRNAs were identified in all samples at a threshold of <1 tag per million. MicroRNA sequencing revealed a distinct set of microRNAs associated with gestational trophoblastic neoplasia. Gene ontology analysis of the most altered transcripts showed that the leading pathway was related to response to ischemia (P<.001). Here, 2 of the top 3 most significantly altered microRNAs were mir-181b-5p (1.65-fold; adjusted P=.014) and mir-181d-5p (1.85-fold; adjusted P=.014), both of which have been shown to regulate expression of BCL2. By quantitative real-time polymerase chain reaction, BCL2 messenger RNA expression was significantly lower in the complete moles progressing to gestational trophoblastic neoplasia than the regressing complete moles (-4.69-fold; P=.018). Reduced expression of BCL2 was confirmed in tissue samples by western blot. Immunohistochemistry in the independent patient samples revealed significantly lower cytoplasmic expression of BCL2 in the villous trophoblasts from cases destined for progression to gestational trophoblastic neoplasia compared with those that regressed, both with respect to staining intensity (optic density 0.110±0.102 vs 0.212±0.036; P<.001) and to the percentage of positive cells (16%±28% vs 49.4%±28.05%; P=.003). CONCLUSION: Complete moles progressing to gestational trophoblastic neoplasia are associated with a distinct microRNA profile. miR-181 family members and BCL2 may be prognostic biomarkers for predicting gestational trophoblastic neoplasia risk.

Intensified therapies improve survival and identification of novel prognostic factors for placental-site and epithelioid trophoblastic tumours.

BACKGROUND: Placental-site trophoblastic (PSTT) and epithelioid trophoblastic tumours (ETT) are the rarest malignant forms of gestational trophoblastic disease (GTD). Our prior work demonstrated that an interval of ≥48 months from the antecedent pregnancy was associated with 100% death rate, independent of the stage. Here, we assess whether modified treatments for these patients have increased survival and identify new prognostic factors. METHODS: The United Kingdom GTD database was screened to identify all PSTT/ETT cases diagnosed between 1973 and 2014. Data and survival outcomes from our prior patient cohort (1976-2006) were compared to our new modern cohort (2007-2014), when intensified treatments were introduced. RESULTS: Of 54,743 GTD patients, 125 (0.23%) were diagnosed with PSTT and/or ETT. Probability of survival at 5 and 10 years following treatment was 80% (95% CI 72.8-87.6%) and 75% (95% CI 66.3-84.3%), respectively. Univariate analysis identified five prognostic factors for reduced overall survival (age, FIGO stage, time since antecedent pregnancy, hCG level, mitotic index) of which stage IV disease (HR 6.18, 95% CI 1.61-23.81, p = 0.008) and interval ≥48 months since antecedent pregnancy (HR 14.57, 95% CI 4.17-50.96, p < 0.001) were most significant on multivariable analysis. No significant differences in prognostic factors were seen between the old and new patient cohort. However, the new cohort received significantly more cisplatin-based and high-dose chemotherapy, and patients with an interval ≥48 months demonstrated an improved median overall survival (8.3 years, 95% CI 1.53-15.1, versus 2.6 years, 95% CI 0.73-4.44, p = 0.·005). CONCLUSION: PSTT/ETT with advanced FIGO stage or an interval ≥48 months from their last known pregnancy have poorer outcomes. Platinum-based and high-dose chemotherapy may help to improve survival in poor-prognosis patients.

A case of intraplacental gestational choriocarcinoma; characterised by the methylation pattern of the early placenta and an absence of driver mutations.

BACKGROUND: Gestational choriocarcinoma is a rare malignancy believed to arise from the trophoblast cells of the placenta. Despite the frequently aggressive clinical nature, choriocarcinoma has been routinely curable with cytotoxic chemotherapy for over 50 years. To date little is known regarding the route to oncogenesis in this malignancy. METHODS: In a case of intraplacental choriocarcinoma, we have performed detailed genetic studies including microsatellite analysis, whole genome sequencing (WGS) and methylation analysis of the tumour and surrounding mature placenta. RESULTS: The results of the WGS sequencing indicated a very low level of mutation and the absence of any driver mutations or oncogene activity in the tumour. The methylation analysis identified a distinctly different profile in the tumour from that of the mature placenta. Comparison with a panel of reference methylation profiles from different stages of placental development indicated that the tumour segregated with the first trimester samples. CONCLUSIONS: These findings suggest that gestational choriocarcinoma is likely to arise as a result of aberrations of methylation during development, rather than from DNA mutations. The results support the hypothesis that gestational choriocarcinoma arises from a normally transient early trophoblast cell. At this point in development this cell naturally has a phenotype of rapid division, tissue invasion and sensitivity to DNA damaging chemotherapy that is very similar to that of the mature choriocarcinoma cell.

Diploid/triploid mixoploidy: A consequence of asymmetric zygotic segregation of parental genomes.

Triploidy is the presence of an extra haploid set of chromosomes and can exist in complete or mosaic form. The extra haploid set of chromosomes in triploid cells can be of maternal or paternal origin. Diploid/triploid mixoploidy is a unique form of triploid mosaicism that requires the aberrant segregation of entire parental genomes into distinct blastomere lineages (heterogoneic cell division) at the earliest zygotic divisions. Here we report on eight cases of diploid/triploid mixoploidy from our institution and conduct a comprehensive review of the literature. The parental origin of the extra set of chromosomes was determined in two cases; and, based on phenotypic evidence we propose the parental origin in the other cases. One case with complex mixoploidy appears to have a digynic origin in addition to the involvement of two different sperm. Of our eight cases, only one resulted in the birth of a live healthy child. The other pregnancies ended in miscarriage, elective termination of pregnancy, intrauterine fetal demise or neonatal death. A review of the literature and the results of our cases show that a preponderance of recognized cases of diploid/triploid mixoploidy has a digynic origin.

Molecular genotyping of placental site and epithelioid trophoblastic tumours; female predominance.

OBJECTIVE: To investigate a large series of placental site trophoblastic tumours (PSTT) and epithelioid trophoblastic tumours (ETT) and determine the relationship between their development and the type and sex of both the immediately antecedent and causative pregnancies. METHODS: The antecedent pregnancy was determined from patient records in 92 cases with a confirmed diagnosis of PSTT, ETT or mixed PSTT/ETT. In a subset of 57 cases, type and sex of the causative pregnancy was established by molecular genotyping of tumour tissue microdissected from formalin-fixed, paraffin-embedded blocks. RESULTS: The antecedent pregnancy was a normal live birth in 59 (64%) cases, a hydatidiform mole in 19 (21%) and other pregnancy loss in 14 (15%). Where the sex was recorded, 36 (78%) of 46 antecedent normal pregnancies were female, a significantly greater proportion than expected (p<0.0001). Genotyping of 57 cases found 15 (26%) to derive from hydatidiform moles while 42 (74%) arose in non-molar pregnancies. Where the causative pregnancy was non-molar, 38 (91%) tumours arose in female conceptions, significantly greater than expected (p<0.0001). Analysis of short tandem repeats on the X chromosome in three tumours with an XY chromosomal constitution confirmed that the X chromosome was maternal in origin. CONCLUSIONS: PSTT and ETT predominantly arise in female pregnancies but can develop in male pregnancies. A male derived X chromosome is not required for the development of these tumours. While these tumours are predominantly female it is not because most originate in complete hydatidiform moles.

Atypical placental site nodule (APSN) and association with malignant gestational trophoblastic disease; a clinicopathologic study of 21 cases.

The WHO Classification of Gestational Trophoblastic Tumors classifies placental site nodule (PSN) as a benign tumor-like trophoblastic neoplasm. Cases of PSN with atypical features were described [atypical placental site nodule (APSN)] and we started registering APSN in our unit in 2005. The aim of this study is to present our initial experience with these lesions. The Trophoblastic Disease Unit database was searched to identify all patients who were either referred with, or on review were diagnosed with, APSN from September 2005 to May 2013. Case notes and the pathology findings for these patients were retrieved and reviewed. A total of 21 cases of APSN were included, 3 of which were associated with gestational trophoblastic neoplasm on follow-up or review. Malignant gestational trophoblastic disease was associated with 3/21 (14%) cases of APSN, either concurrently or developing/manifesting within 16 mo of APSN diagnosis. None of these patients had raised serum hCG levels either at presentation or follow-up. Presence of APSN should indicate a thorough clinical and radiologic investigation and follow-up if diagnosed on curettage specimens. With increased recognition of this entity and corresponding larger series with longer follow-up, more accurate patient counseling will be possible.

Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Histopathological and immunohistochemical features of early hydatidiform mole in relation to subsequent development of persistent gestational trophoblastic disease.

OBJECTIVE: To examine histomorphological and immunohistochemical findings in hydatidiform moles to determine whether any features can reliably predict clinical behavior. STUDY DESIGN: Blinded semiquantitative review of histological and immunohistochemical findings in cases of partial hydatidiform mole (PHM) (N = 50) and complete hydatidiform mole (CHM) which either spontaneously resolved (N = 50) or required chemotherapy (N = 50). Immunostains assessed included MLH1, MSH2, nm23, TERT, p53, EGFR, and CerbB2 based on previous data. RESULTS: There were marked morphological differences in various criteria between CHMs and PHMs, including the proportion of villi with abnormal trophoblast hyperplasia (29% vs. 6%, respectively). However, there were no significant differences in any morphological parameters between CHMs that spontaneously resolved and those that subsequently required chemotherapy. Similarly, there were no clinically useful differences regarding any immunostaining scores between CHM groups. CONCLUSION: Neither morphological nor immunohistochemical features can reliably predict subsequent requirement of chemotherapy in CHMs.

Management and survival of patients with FIGO high-risk gestational trophoblastic neoplasia: the U.K. experience, 1995-2010.

OBJECTIVE: To present survival rates of high-risk gestational trophoblastic neoplasia (GTN) (FIGO score > 7) patients treated between 1995 and 2010 in the U.K. Death due to GTN is largely confined to patients with high-risk disease. In the U.K. a national system ensures that all patients are treated at only 2 specialist centers: Charing Cross Hospital (CXH) in London and Weston Park Hospital (WPH) in Sheffield. STUDY DESIGN: A total of 196 high-risk patients were identified using the CXH and WPH GTN databases, based on the risk score at the time of presentation. RESULTS: In all, 140 CXH and 56 WPH high-risk patients were treated with EMA/CO (etoposide, methotrexate, actinomycin D alternating with cyclophosphamide and vincristine) and MEA (methotrexate, etoposide, actinomycin D), respectively. The FIGO score at presentation ranged from 6-23. Eight patients (7from WPH and 1 from CXH) who were treated prior to 2002 as high-risk based on their pre-2002 scoring scored a 6 using FIGO 2002. Two (1%) patients died within 4 weeks of starting treatment (early death), 12 (6%) relapsed, and 9 patients subsequently died due to drug resistance. The overall survival was 94%, with a median follow-up of 4.69 years. CONCLUSION: In the context of a national trophoblastic disease service, patients with high-risk GTN have an excellent prognosis with EMA/CO or MEA.

Mutations in NLRP7 and KHDC3L confer a complete hydatidiform mole phenotype on digynic triploid conceptions.

Digynic triploidy is classically associated with a severely growth restricted fetus and a small nonmolar placenta. However, in genotyping hydatidiform moles as part of clinical practice, we identified two digynic triploid conceptions presenting with histopathological features of classical complete hydatidiform mole (CHM). Both cases occurred in women with a history of previous molar pregnancies and no normal pregnancies. Pathological review and genotyping of other molar pregnancies in these cases showed them to be typical CHM with negative p57(KIP2) immunostaining of the cytotrophoblast cells and villous stroma and to be diploid but biparental, confirming a diagnosis of familial recurrent hydatidiform mole (FRHM). Mutation screening of NLRP7 had identified a homozygous duplication, leading to a truncated protein, in case 1 whereas mutation screening of KHDC3L (C6orf221) in case 2 showed both the proband and her sister to be compound heterozygotes for mutations in KHDC3L. The observation of a single digynic, triploid conception presenting as a CHM in women with FRHM, where other pregnancies are diploid and biparental, supports the hypothesis that the role of both NLRP7 and KHDC3L in pregnancy is in setting and/or maintaining the maternal imprint. Clinically, a diagnosis of FRHM should be considered in women with genetically unusual conceptions that are phenotypically CHM.

First confirmation by genotyping of transplacental choriocarcinoma transmission.

A mother developed multimetastatic gestational choriocarcinoma 13 months after delivery, and her infant died aged 11 months from the same tumor. The transplacental choriocarcinoma transmission was confirmed by genotyping. Henceforth, we recommend a 2-year maternal human chorionic gonadotropin follow-up after neonatal choriocarcinoma and extensive imaging if the human chorionic gonadotropin rises.

Mutations in NLRP7 are associated with diploid biparental hydatidiform moles, but not androgenetic complete moles.

BACKGROUND: NLRP7 (NALP7) has been identified as the major gene involved in the inherited predisposition to recurrent molar pregnancies, a rare recessive condition in which affected individuals have complete hydatidiform moles of diploid biparental origin (BiCHM). The role of NLRP7 in other types of molar pregnancy and reproductive wastage has not been conclusively demonstrated. The purpose of this study was to clarify this by identifying NLRP7 variation in two clinically well-defined groups of patients: women with recurrent BiCHM, and women with three or more recurrent complete hydatidiform moles of proven androgenetic origin (AnCHM). METHODS: Fluorescent microsatellite genotyping of molar tissue was used to establish a diagnosis of recurrent BiCHM (four novel cases) or recurrent AnCHM (nine women with multiple CHM). These two groups were subsequently screened for mutations in NLRP7 using DNA sequencing. Additional screening for non-pathological variants was performed in 21 previously published cases of recurrent BiCHM. Taqman genotyping was used to determine the frequency of novel NLRP7 variants in two control cohorts of Caucasian and Asian women with no adverse reproductive outcomes. RESULTS: Of the four novel cases with recurrent BiCHM, two were homozygous for mutations in NLRP7 while one was a compound heterozygote for a nonsense mutation and a pathological variant. No NLRP7 mutations or pathological variants were identified in the fourth case. None of the women with AnCHM carried any mutations or pathological variants of NLRP7. A single case of AnCHM was found to be heterozygous for a novel variant (R413Q). CONCLUSION: NLRP7 mutations do not represent a major cause of AnCHM.

Partial molar pregnancy after intracytoplasmic sperm injection occurring as a result of diploid sperm usage.

PURPOSE: Partial molar pregnancies are rare conceptions characterized by having 69 rather than 46 chromosomes, the additional chromosome complement usually occurring as a result of fertilization of the ovum by two sperm. Although assisted conception with intracytoplasmic sperm injection (ICSI) should prevent the development of a partial molar pregnancy, occasional cases have been described after assisted conception using ICSI. The objective of this study was to investigate the cause of partial molar pregnancy in a couple who had undertaken assisted conception with ICSI. METHODS: Fluorescent microsatellite genotyping of DNA from the couple and tissue from their partial molar pregnancy was performed in order to confirm diagnosis and investigate the origin of the additional chromosome set. RESULTS: Genotyping confirmed that the partial molar tissue was triploid with an additional chromosome complement from the father. Genotyping of additional loci proximal to the centromere demonstrated that the two paternal sets of chromosomes originated in a single sperm with a double complement of paternal DNA resulting from non-reduction at the second meiotic division. CONCLUSIONS: This study confirms that partial molar pregnancy may occur after assisted conception with ICSI and that this occurs as a result of fertilization with a diploid sperm.

Sensitive screening of single nucleotide polymorphisms in cell free DNA for diagnosis of gestational tumours.

Tumours expressing human chorionic gonadotropin (hCG), the majority of which are difficult to biopsy due to their vascularity, have disparate prognoses depending on their origin. As optimal management relies on accurate diagnosis, we aimed to develop a sensitive cell free DNA (cfDNA) assay to non-invasively distinguish between cases of gestational and non-gestational origin. Deep error-corrected Illumina sequencing of 195 common single nucleotide polymorphisms (SNPs) in cfDNA and matched genomic DNA from 36 patients with hCG-secreting tumours (serum hCG 5 to 3,042,881 IU/L) and 7 controls with normal hCG levels (≤4 IU/L) was performed. cfDNA from confirmed gestational tumours with hCG levels ranging from 1497 to 700,855 IU/L had multiple (n ≥ 12) 'non-host' alleles (i.e. alleles of paternal origin). In such cases the non-host fraction of cfDNA ranged from 0.3 to 40.4% and correlated with serum hCG levels. At lower hCG levels the ability to detect non-host cfDNA was variable, with the detection limit dependent on the type of causative pregnancy. Patients with non-gestational tumours were identifiable by the absence of non-host cfDNA, with copy number alterations detectable in the majority of cases. Following validation in a larger cohort, our sensitive assay will enable clinicians to better inform patients, for whom biopsy is inappropriate, of their prognosis and provide optimum management.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

667C>T and 1298A>C polymorphisms of MTHFR do not predict response to methotrexate in patients with gestational trophoblastic neoplasia.

OBJECTIVE: Approximately one third of patients treated with methotrexate for gestational trophoblastic neoplasia (GTN) following a molar pregnancy are reported to develop resistance to methotrexate and need to change to different chemotherapeutic agents. Previous studies, in other clinical settings, have suggested that polymorphisms in key folate metabolising enzymes such as 5,10-methylenetetrahydrofolate reductase (MTHFR) influence both toxicity and efficacy of methotrexate. Our objective was to investigate the impact of two common functional MTHFR polymorphisms, 677C>T and 1298A>C, on the efficacy of methotrexate in women treated for GTN following a molar pregnancy. METHODS: DNA from 121 women treated with methotrexate for GTN was genotyped for the 677C>T and 1298A>C polymorphisms using TaqMan SNP Genotyping Assays. In 64 cases these polymorphisms were also genotyped in the antecedent molar pregnancy, using DNA extracted from archival blocks of tissue. Response to methotrexate was evaluated with reference to serial human chorionic gonadotrophin (hCG) levels in patient serum. RESULTS: No significant association was found between the genotype of the patient, or presence of the variant allele, and clinical response to methotrexate therapy for either the 677C>T or the 1298A>C SNP. No significant association was found between the genotypes of the molar tissue and response to methotrexate. In molar tissue there was a significant reduction in the expected number with the 677TT genotype suggesting the 677C>T SNP may identify a subgroup of molar pregnancies less likely to progress to GTN. CONCLUSION: Neither the genotype for the 677C>T SNP or the 1298A>C SNP in MTHFR predict the therapeutic outcomes of women treated with single agent methotrexate for GTN.

Genetics of gestational trophoblastic disease.

The abnormal pregnancies complete and partial hydatidiform mole are genetically unusual, being associated with two copies of the paternal genome. Typical complete hydatidiform moles (CHMs) are diploid and androgenetic, while partial hydatidiform moles (PHMs) are diandric triploids. While diagnosis can usually be made on the basis of morphology, ancillary techniques that exploit their unusual genetic origin can be used to facilitate diagnosis. Genotyping and p57 immunostaining are now routinely used in the differential diagnosis of complete and partial hydatidiform moles, for investigating unusual mosaic or chimeric products of conception with a molar component and identifying the rare diploid, biparental HMs associated with an inherited predisposition to molar pregnancies. Genotyping also plays an important role in the differential diagnosis of gestational and non-gestational trophoblastic tumours and identification of the causative pregnancy where tumours are gestational. Recent developments include the use of cell-free DNA for non-invasive diagnosis of these conditions.

Prognostic markers and long-term outcome of placental-site trophoblastic tumours: a retrospective observational study.

BACKGROUND: Placental-site trophoblastic tumours are a rare form of gestational trophoblastic disease and consequently information about optimum management or prognostic factors is restricted. We aimed to assess the long-term outcome of stage-adapted management by surgery, chemotherapy, or both for patients with the disorder. METHODS: 35 550 women were registered with gestational trophoblastic disease in the UK (1976-2006), of whom 62 were diagnosed with placental-site trophoblastic tumours and included, retrospectively, in the study. Patients were treated by surgery, chemotherapy, or both. We estimated the probabilities of overall survival and survival without recurrence of disease 5 and 10 years after the date of first treatment, and calculated the association of these endpoints with prognostic factors, including time since antecedent pregnancy, serum concentration of beta-human chorionic gonadotropin, and stage of disease, with both univariate and multivariate analyses. FINDINGS: Probabilities of overall and recurrence-free survival 10 years after first treatment were 70% (95% CI 54-82) and 73% (54-85), respectively. Patients with stage I disease had a 10-year probability of overall survival of 90% (77-100) and did not benefit from postoperative chemotherapy. By contrast, patients with stage II, III, and IV disease required combined treatment with surgery and chemotherapy; probability of overall survival at 10 years was 52% (3-100) for patients with stage II disease and 49% (26-72) for stage III or IV disease. Outcome for patients who had recurrent or refractory disease was poor: only four (22%) patients achieved long-term survival beyond 60 months. Multivariate analysis showed that the only significant independent predictor of overall and recurrence-free survival was time since antecedent pregnancy. A cutoff point of 48 months since antecedent pregnancy could differentiate between patients' probability of survival (<48 months) or death (>/=48 months) with 93% specificity and 100% sensitivity, and with a positive predictive value of 100% and a negative predictive value of 98%. INTERPRETATION: Stage-adapted management with surgery for stage I disease, and combined surgery and chemotherapy for stage II, III, and IV disease could improve the effectiveness of treatment for placental-site trophoblastic tumours. Use of 48 months since antecedent pregnancy as a prognostic indicator of survival could help select patients for risk-adapted treatment. FUNDING: National Commissioning Group.

ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942.

The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.