PD-L1 in gestational trophoblastic disease: an antibody evaluation.

INTRODUCTION: Treatment with antibodies directed against programed-cell death ligand 1 (PD-L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD-L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti-PD-L1 treatment. Multiple antibodies are available to detect PD-L1-expressing cells, and percentages of PD-L1-expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD-L1 antibody best reflects PD-L1 expression to select patients for treatment. MATERIAL AND METHODS: Seven commercially available antibodies for PD-L1 staining (E1L3N, 73-10, 22C3, CAL10, SP142, 28-8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD-L1, PD-L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists. RESULTS: All seven antibodies showed membranous staining in the PD-L1-transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD-L1-positive cells (70%-90% and 60%-70%, respectively). E1L3N stained the cytoplasm of non-transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD-L1-stained trophoblast cells and staining intensity varied substantially per used PD-L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94-0.96). CONCLUSIONS: Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD-L1-expressing trophoblast cells. All antibodies detected PD-L1-expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD-L1-expressing cells.

MicroRNA Expression Profiling in Hydatidiform Mole for the Prediction of Postmolar GTN : MicroRNA Profile in Postmolar GTN.

Objectives: The primary aim of the study was to identify miRNAs that were differentially expressed between complete hydatidiform moles (CHMs) that turned out to be gestational trophoblastic neoplasia (GTN) [GTN moles] and CHMs that regressed spontaneously after evacuation [remission moles]. The secondary aim was to study the profiles of miRNA expressions in CHMs. Methods: A case-control study was conducted on GTN moles and remission moles. We quantitatively assessed the expression of 800 human miRNAs from molar tissues using Nanostring nCounter. Results: From a pilot study, 21 miRNAs were significantly downregulated in GTN moles compared to the remission moles. Five of them (miR-566, miR-608, miR-1226-3p, miR-548ar-3p and miR-514a-3p) were downregulated for >4 folds. MiR-608 was selected as a candidate for further analysis on 18 CHMs (9 remission moles and 9 GTN moles) due to its striking association with malignant formation. MiR-608 expression was slightly lower in GTN moles compared to the remission moles, that is, 2.22 folds change [p = 0.063]. Conclusion: We identified 21 miRNAs that were differentially expressed between GTN moles and remission moles suggesting that miRNA profiles can distinguish between the two groups. Although not reaching statistically significant, miR-608 expression was slightly lower in GTN moles compared to remission moles.

Gestational trophoblastic neoplasia: Novelties and challenges.

Gestational trophoblastic diseases are a group of pregnancy-related disorders, originated from trophoblast cells. They include benign and aggressive tumors, such as the invasive mole, the choriocarcinoma, the placental site trophoblastic tumor (PSTT), and the epithelioid trophoblastic tumor (ETT). These malignancies are characterized as gestational trophoblastic neoplasm (GTN), rarer, although more dangerous. The diagnosis of GTN is made in most cases by monitoring serum chorionic gonadotropin (hCG) with histological confirmation. The use of specific tissue biomarkers has been increasingly employed as a differential diagnosis, leading to more accurate results and different therapy protocols and prognosis for each GTN. The treatment is based on the International Federation of Gynecology and Obstetrics anatomical staging system and the World Health Organization prognostic score system. If an accurate diagnosis is made and the guidelines followed, the cure for choriocarcinoma and invasive mole cases can reach 98%, whereas PSTT and ETT still present mild success rates. The improved knowledge about GTN and its peculiarities allows physicians to efficiently achieve the differential diagnosis and choose the best available therapy protocol, thus increasing the overall survival of affected women. Nevertheless, obtaining epidemiological data and improving knowledge through basic and translational research are essential to answer open questions on GTN physiopathology, their causes, and cellular behavior.

Expression pattern Nanog is associated with the development of gestational trophoblastic neoplasia in Chinese women from Fujian province: a case-control study.

BACKGROUND: Gestational trophoblastic disease (GTD) is a group of interrelated but distinct diseases and has a serious impact on the reproductive health of women. To analyze the expression of Nanog in GTD and to evaluate its potential to predict the development of gestational trophoblastic neoplasia (GTN). METHODS: The study included 41 normal first-trimester placentas matched by gestational age to 53 regressed-hydatidiform-moles (rHMs), 56 malignant-HMs (mHMs) and 17 choriocarcinomas (CCAs) and evaluated the Nanog expression by immunohistochemistry. The chi-square test, ANOVA, Fisher's exact test and logistic regression were performed to assess the Nanog expression and clinical prognostic factors in GTD. RESULTS: Compared to normal placenta levels, the Nanog expression was increased in GTD samples (p < .05). In HMs, Nanog expression was positively correlated with serum ß-hCG levels,uterine size and theca-lutein cysts (p < .05). Compared with the low-risk metastatic group (Federation of Gynecology and Obstetrics (FIGO) score ≤ 6), the high-risk metastatic group (FIGO score >7) had higher Nanog expression (p = .030). Moreover, logistic regression analysis showed that the positive expression of Nanog had the highest risk of developing into GTN (OR = 4.764, p < .001). CONCLUSIONS: Nanog is an independent predictor of clinical outcomes. It can also be a reliable predictor for GTN development from GTD.

Efficacy of Oral Vitamin A in Reducing ß-hCG Levels in Low-Risk Gestational Trophoblastic Neoplasia Patients.

OBJECTIVE: Low-risk gestational trophoblastic neoplasia (GTN) is generally treated with single agent chemotherapy and methotrexate (MTX) as a first-line therapy. Vitamin A helps to increase trophoblast cell regression, as well as to decrease ß-hCG levels. Vitamin A also increases the effectiveness of MTX by inducing more malignant cell death than MTX alone. Therefore, the aim of the current study was to analyze the changes in ß-hCG levels in low-risk GTN patients following vitamin A administration. METHODS: This study was a randomized clinical trial, which examined initial serum vitamin A and ß-hCG levels in GTN patients before and after three cycles of MTX therapy. Patients were given vitamin A supplementation of 6,000 IU (1.8 mg RAEs) per day, and the changes in serum ß-hCG were observed after three cycles. Patients were grouped by ß-hCG levels (decreased or stagnant). RESULTS: A total of 32 low-risks GTN patients were divided into the intervention group (16 patients who received vitamin A supplementation) and the control group (16 patients who did not receive vitamin A supplementation). In the intervention group, the average initial ß-hCG level was 170,949.3 ± 354,452.1 mIU/mL, and the average ß-hCG post-cycle level was 1,611.9 ± 3,652.5 mIU/mL. In the control group, the average initial ß-hCG level was 178,834.1 ± 2913844.6 mIU/mL, and the average ß-hCG post-cycle level was 25,388.5 ± 58,437.7 mIU/mL. CONCLUSION: In patients with low-risk GTN who underwent MTX chemotherapy, the levels of ß-hCG and the incidence of chemo resistance in the intervention group were lower than those in the control group. Older age may also influence the incidence of chemo resistance in GTN patients. Oral administration of 6,000 IU vitamin A could help to reduce ß-hCG levels in low-risk GTN patients who receive MTX chemotherapy.
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Immune cells in normal pregnancy and gestational trophoblastic diseases.

A healthy pregnancy requires the development of maternal-fetal immune tolerance against the semi-allogeneic fetus. The interactions between the trophoblastic cells and the maternal immune cells (p.e., natural killer cells, T cells, macrophages, dendritic cells and B-cells) are important for the development of the maternal-fetal immune tolerance and the placental growth and function. These interactions are mediated by cell to cell contact and secreted molecules such as cytokines, chemokines, angiogenic factors and growth factors. The maternal immune cells are present in normal non-pregnant and pregnant endometrium and there are several lines of evidence based on immunohistochemical and RNA sequencing data that the decidual immune cells and immune-related pathways display alterations in GTD, which may have pathogenetic and clinical significance. The present review focuses on the usefulness of the immunohistochemical analysis which provides multiparametric in situ information regarding the numbers, the immunophenotypes and the immunotopographical distributions of the decidual immune cells in tissue sections from normal pregnancy and GTD. We also discuss the significance of the immunohistochemical information in order to gain insight in the putative mechanisms explaining the alterations of the decidual immune cells in GTD and the potential implications of these alterations in the pathogenesis and the clinical behavior of GTD.

Knockdown of lncRNA OGFRP1 Inhibits Proliferation and Invasion of JEG-3 Cells Via AKT/mTOR Pathway.

Increasing evidence indicates the pivotal role of long noncoding RNAs in a variety of cancers, but there is limited focus on the link between long noncoding RNAs and gestational choriocarcinoma. This study aimed to examine the role of long noncoding RNA OGFRP1 in JEG-3 and JAR cells. Small interfering RNA was used to downregulate long noncoding RNA OGFRP1 level. Cell proliferation was measured by cell counting kit-8 and clone formation assays. Cell cycle and apoptosis were analyzed by flow cytometry. Cell invasion was examined by transwell assay. Protein expression was determined by Western blot. A double-effect inhibitor (BEZ235) that inhibits AKT and mTOR phosphorylation was used as a positive control. Knockdown of long noncoding RNA OGFRP1 significantly inhibited the proliferation of JEG-3 and JAR cells. Knockdown of long noncoding RNA OGFRP1 induced cell cycle arrest in G1 phase and apoptosis. On the other hand, knockdown of long noncoding RNA OGFRP1 inhibited the invasion of JEG-3 and JAR cells. Finally, knockdown of long noncoding RNA OGFRP1 resulted in the inactivation of AKT/mTOR signaling pathway. In addition, knockdown of long noncoding RNA OGFRP1 caused changes in the expression of intracellular cell cycle-related proteins and apoptosis-related proteins, including downregulation of CDK4, CDK6, Cyclin D1, Nusap1, and Bcl2 protein expression and upregulation of Bax protein expression. In conclusion, we found that downregulation of long noncoding RNA OGFRP1 inhibited cell proliferation, cell cycle progression, and invasion of JEG-3 and JAR cells and induced apoptosis through AKT/mTOR pathway. This study extends the understanding of the function of long noncoding RNA OGFRP1 in tumorigenesis, and these findings may be important for developing a potential therapeutic target for gestational choriocarcinoma therapy.

Differential expression of SALL4 in CTCs derived from hydatidiform moles and gestational trophoblastic neoplasms.

PROBLEM: To investigate EMT phenotype and SALL4 expression of circulating tumour cells (CTCs) in patients with gestational trophoblastic neoplasm (GTN). METHOD OF STUDY: CanPatrol CTC detection system in combination with SALL4 RNA in situ hybridization was used to investigate the profile of CTCs in different types of gestational trophoblastic disease (GTD). Circulating CTCs were phenotyped and annotated with SALL4 expression in 41 GTD patients, including 12 HM and 29 GTN, as well as 22 pregnant volunteers. RESULTS: A positive correlation between the number of CTC and serum ß-hCG concentration was found among the GTN patients. The number of E/M-CTC was positively correlated with serum ß-hCG, while M-CTC was positively correlated with prognostic score. Comparison among malignant GTD, benign GTD and healthy pregnant women revealed a significant difference in the number of total CTC, E/M-CTC, and M-CTC but not in E-CTC. ROC analysis was conducted to evaluate the performance of CTC phenotypes in distinguishing GTD patients from healthy pregnant women yielding an AUC as 0.826. Youden's index was maximal at the cutoff value of 8.5/4 mL with sensitivity and specificity at 53.66% and 100%, respectively. SALL4 expression was evaluated in GTD patients with CTC count greater than cutoff value. SALL4 high expressing CTCs (>2 signal dots) were detected in 66.67% (10/15) of malignant GTD patients but not in benign patients (0/5). CONCLUSION: Differential expression of SALL4 was seen in CTCs derived from hydatidiform moles and GTN. CTC profiling may be developed as an adjunct marker to diagnose GTN.

Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.

BRCA1 promoter hypermethylation in human placenta: a hidden link with ß-hCG expression.

Gestational trophoblastic diseases (GTD) are group of pregnancy-related tumors characterized by abnormal levels of 'ß-hCG' with higher incidence in South-East Asia, especially India. Our laboratory has reported that wild-type BRCA1 transcriptionally regulates ß-hCG in triple negative breast cancers (TNBCs). These factors culminated into analysis of BRCA1 status in GTD, which would emanate into elucidation of BRCA1- ß-hCG relationship and unraveling etio-pathology of GTD. BRCA1 level in GTD is down-regulated due to the over-expression of DNMT3b and subsequent promoter hypermethylation, when compared to the normal placentae accompanied with its shift in localization. There is an inverse correlation of serum ß-hCG levels with BRCA1 mRNA expression. The effects of methotrexate (MTX), which is the first-line chemotherapeutic used for GTD treatment, when analyzed in comparison with plumbagin (PB) revealed that PB alone is efficient than MTX alone or MTX-PB in combination, in showing selective cytotoxicity against GTD. Interestingly, PB increases BRCA1 levels post-treatment, altering DNMT3b levels and resultant BRCA1 promoter methylation. Also, cohort study analyzed the incidence of GTD at Sree Avittom Thirunal (SAT) Hospital, Thiruvananthapuram, which points out that 11.5% of gestational trophoblastic neoplasia (GTN) cases were referred to Regional Cancer Centre, Thiruvananthapuram, for examination of breast lumps. This has lend clues to supervene the risk of GTD patients towards BRCA1-associated diseases and unveil novel therapeutic for GTD, a plant-derived naphthoquinone, PB, already reported as selectively cytotoxic against BRCA1 defective tumors.

When to stop human chorionic gonadotrophin (hCG) surveillance after treatment with chemotherapy for gestational trophoblastic neoplasia (GTN): A national analysis on over 4,000 patients.

OBJECTIVE: To determine the optimal duration of human chorionic gonadotrophin (hCG) surveillance following treatment for low and high risk gestational trophoblastic neoplasia (GTN) and establish whether the current surveillance protocol that recommends life-long hCG monitoring requires revision. METHODS: A population-based cohort study was undertaken using a national registry, comprising patients from both tertiary trophoblastic disease treatment units in the UK (London and Sheffield). All patients who received chemotherapy for low or high risk GTN in the UK between 1958 and 2014 in London and 1973 and 2015 in Sheffield (n = 4201) were included in the study. Patients with placental site trophoblastic tumours and epithelioid trophoblastic tumours were excluded due to their distinct clinical behavior, treatment and follow-up requirements. The risk of recurrence with time following completion of chemotherapy for low or high risk GTN was measured. RESULTS: The overall risk of relapse in this national cohort of 4201 patients was 4.7% (198/4201) with a median time to recurrence of 117.5 days (range 9 days to 6.54 years). The greatest risk of recurrence occurred in the first year after completing treatment for either low or high risk GTN measuring 72.7% (n = 112) or 86.4% (n = 38), respectively. The subsequent recurrence risk reduced over time with none observed beyond 7 years. CONCLUSIONS: The absence of any recurrences beyond seven years following completion of chemotherapy for GTN indicates that the UK policy of life-long hCG surveillance is unnecessary. Our revised conservative protocol recommends stopping after 10 years.

Can Ki67 and Caspase Predict Molar Progression?

Background: A clinical risk score has been introduced into the management of persistent trophoblastic disease to allow individualized therapy. However, this risk scoring system lacks histopathologic predictors. The hypothesis is that there are prognostic histological markers that might contribute to the detection of those cases that will have persistent trophoblastic disease. Methods: Trophoblastic proliferation and apoptosis were investigated via immunohistochemical expression of Ki67 and caspase in 24 complete moles. These were divided into two groups; group A represented cases with persistent trophoblastic disease and group B represented cases with no persistent trophoblastic disease. Sections were immunostained with a monoclonal antibody for both caspase and Ki67. Results: No statistically significant difference was found between either group regarding the expression of Ki67 or caspase. Conclusion: Neither proliferation or apoptosis are reliable markers for progression of molar pregnancy.

PD-L1, B7-H3 and VISTA are highly expressed in gestational trophoblastic neoplasia.

AIMS: The B7 family check-point molecules are potential therapeutic targets in cancer immunotherapy. However, their expression status in human gestational trophoblastic neoplasia (GTN) remains unknown. We investigated the expression profiles of the B7 family check-point proteins PD-L1, PD-L2, B7-H3, B7-H4, VISTA and B7-H6 in GTN and their clinical significance. METHODS AND RESULTS: We identified 112 patients with GTN, including 68 with choriocarcinoma, 33 with placental-site trophoblastic tumour (PSTT) and 11 with epithelioid trophoblastic tumour (ETT). Immunohistochemical staining was performed on whole-tissue GTN sections using anti-B7 family antibodies. VISTA expression was immunohistochemically analysed using microarrays of normal human tissues and of 20 common cancers. PD-L1 and B7-H3 were highly expressed in all GTN tumours, while PD-L2 was expressed in 87.5% of the samples. B7-H4 and B7-H6 were negative in 100% and 98.2% of the samples, respectively. PD-L1, B7-H3 and VISTA levels were significantly higher in choriocarcinomas and PSTTs than in ETTs. There was no association between B7 family check-point expression in tumour cells and disease stage, prognostic score or patient outcomes (complete remission versus death). VISTA protein was widely overexpressed in 98.2% of all the GTN, but its expression varied in other cancer types and was negative in normal adult and fetal tissues except placental trophoblasts and splenic lymphocytes. CONCLUSIONS: The GTN trophoblast cells show high expression of PD-L1, B7-H3 and VISTA in a manner that is independent of clinical outcomes. These proteins may be potential immunotherapeutic targets when treating GTN.

Proteomic identification of predictive biomarkers for malignant transformation in complete hydatidiform moles.

INTRODUCTION: Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole. METHODS: Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from -70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (ß-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l-lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group. DISCUSSION: This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD_DB/Supplementary_Data.xlsx.

Versican silencing in BeWo cells and its implication in gestational trophoblastic diseases.

Versican is a proteoglycan known to interact with cells to influence their ability to proliferate, differentiate, migrate, invade and assemble extracellular matrix, with all of these cell functions present during placentation. In the placenta, cytotrophoblast cells have the ability to differentiate into the syncytiotrophoblast, a mechanism that is greatly increased in gestational trophoblastic diseases (GTD). Nevertheless, the molecular signaling underlying the increased syncytiotrophoblast differentiation are still being unveiled and may result in novel therapeutic targets for GTD. Versican expression was investigated to establish its differential expression among GTD (partial moles, complete moles, invasive moles and choriocarcinoma) and the possible functional outcomes from versican gene silencing. Tissue samples had their versican expression evaluated using immunohistochemistry and RT-PCR. BeWo cells were employed for versican silencing with siRNA and the efficiency was confirmed by RT-PCR, immunofluorescence and flow cytometry. Cell death and forskolin-induced syncytialization were analyzed by a morphological analysis and human chorionic gonadotropin (hCG) production using immunofluorescence. Versican V0 and V1 isoforms were mainly expressed in the syncytiotrophoblast and they were the most expressed in benign rather than in malignant tumors. BeWo cells also expressed V0 and V1 isoforms, but only in cells undergoing syncytial fusion. After versican silencing, cell death was greatly increased, whereas spontaneous and forskolin-induced syncytialization decreased as well as hCG production. Versican is differentially expressed in GTD and is important for hydatidiform moles pathophysiology, protecting trophoblast cells from death and playing a role in their differentiation and functionality.

Analysis of PD-L1 expression in trophoblastic tissues and tumors.

The immune checkpoint proteins, programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1), are crucial for maintaining fetomaternal immune tolerance and immune escape in cancers. In this study, we performed a comprehensive immunohistochemical study of PD-L1 expression in a large cohort of trophoblastic tissues and tumors. We found that normal villi and hydatidiform moles showed a heterogeneous PD-L1 staining among trophoblast (strong in syncytiotrophoblast, moderate in intermediate trophoblast, and weak/negative in cytotrophoblast). Eleven exaggerated placental sites (100%) showed variable PD-L1 staining, whereas 7 (36.8%) of 19 placental site nodules/plaques were weakly positive for PD-L1 (P < .001). All gestational choriocarcinomas (CCs; n = 63), epithelioid trophoblastic tumors (n = 12), and placental site trophoblastic tumors (n = 41) were PD-L1 positive, with most showing strong staining. However, PD-L1 expression was lower in epithelioid trophoblastic tumors compared with placental site trophoblastic tumors and CCs (P = .004). Three presumably germ cell-derived pure CCs, the CC elements in 13 mixed germ cell tumors, and 4 gastric/rectal CCs were also positive for PD-L1, with widespread staining. The background nontrophoblastic tissues, such as endometrial glands, squamous cells, and adenocarcinomas, were PD-L1 negative. Western blot analysis showed that PD-L1 was expressed in all 3 trophoblastic cell lines. We conclude that PD-L1 is a sensitive but nonspecific marker for trophoblast and related tumors. The frequent strong PD-L1 expression suggests that immune checkpoint blockade could be a promising approach in treating trophoblastic tumors that merits further investigation.

Human Placental Growth Hormone Variant in Pathological Pregnancies.

Growth hormone (GH), an endocrine hormone, primarily secreted from the anterior pituitary, stimulates growth, cell reproduction, and regeneration and is a major regulator of postnatal growth. Humans have two GH genes that encode two versions of GH proteins: a pituitary version (GH-N/GH1) and a placental GH-variant (GH-V/GH2), which are expressed in the syncytiotrophoblast and extravillous trophoblast cells of the placenta. During pregnancy, GH-V replaces GH-N in the maternal circulation at mid-late gestation as the major circulating form of GH. This remarkable change in spatial and temporal GH secretion patterns is proposed to play a role in mediating maternal adaptations to pregnancy. GH-V is associated with fetal growth, and its circulating concentrations have been investigated across a range of pregnancy complications. However, progress in this area has been hindered by a lack of readily accessible and reliable assays for measurement of GH-V. This review will discuss the potential roles of GH-V in normal and pathological pregnancies and will touch on the assays used to quantify this hormone.

PD-L1 Expression in Human Placentas and Gestational Trophoblastic Diseases.

One of the major immune checkpoints responsible for immune evasion in cancer cells is the interaction between programmed cell death-1 (PD-1) and its ligand (PD-L1). As human trophoblastic cells display many of the features of malignant cells such as the ability to invade normal tissue including blood vessels and are apparently not eradicated by the host immune system, we undertook the present study to determine whether PD-L1 was upregulated in different types of trophoblastic cells during normal pregnancy and in gestational trophoblastic diseases. Immunohistochemistry using an anti-PD-L1-specific antibody demonstrated that in early and term normal placentas, PD-L1 was highly expressed in syncytiotrophoblast and to a much lower extent in intermediate trophoblastic cells located in the chorion laeve and implantation site. PD-L1 immunoreactivity was undetectable in cytotrophoblastic cells. This staining pattern in normal placenta was recapitulated in various types of gestational trophoblastic disease. PD-L1 was highly expressed by syncytiotrophoblast in complete moles and choriocarcinomas. The intermediate trophoblastic neoplasms, placental site trophoblastic tumors, and epithelioid trophoblastic tumors showed variable PD-L1 immunoreactivity but at a lower intensity than in the neoplastic syncytiotrophoblast in choriocarcinoma. In addition, we observed PD-1-positive lymphocytes located within the implantation site and in trophoblastic tumors. In summary, this study describes a novel mechanism for trophoblastic cells to create a tolerogenic feto-maternal interface by upregulating PD-L1 in syncytiotrophoblast and in intermediate trophoblast. Trophoblastic tumors may also use PD-L1 expression to evade the host immune response thereby promoting their survival.

Factors related to treatment outcomes in low-risk gestational neoplasia.

OBJECTIVE: To define the factors associated with methotrexate (MTX) resistance in patients with low-risk gestational trophoblastic neoplasia (GTN). METHODS: A total of 63 patients with low-risk GTN according to International Federation of Gynecology and Obstetrics (FIGO) criteria were included. A total of 37 (58.7%) patients were treated with successive doses of 1 mg/kg intramuscular (IM) MTX on days 1, 3, 5, and 7, and 0.1 mg/kg IM folinic acid (FA) on days 2, 4, 6, and 8, until ß-human chorionic gonadotropin (hCG) levels were normalized. After the ß-hCG value dropped to the normal level, an additional cycle of MTX/FA was administered. This protocol is defined as the standard protocol. In a watchful waiting protocol, the same 8-day IM MTX/FA regimen was given only once (n = 8) or twice (n = 18) to 26 (41.3%) patients and patients in whom ß-hCG values declined were subjected to follow-up and no additional cycles were administered as long as there was a decrease in ß-hCG value. Clinical response and factors affecting therapeutic outcomes were analyzed retrospectively. RESULTS: Of 63 patients, 47 (74.3%) were cured with primary MTX/FA treatment irrespective of any protocol. Of the 16 patients who were not able to be treated with primary MTX/FA, 3 were treated with single-agent actinomycin-D and 11 were treated with multi-agent chemotherapy. Univariate analysis showed that a pretreatment ß-hCG level of ≥5000 IU/L was related to reduced therapeutic response (p = 0.001). The FIGO score, antecedent gestational pathology, and treatment with standard or watchful waiting protocol were not related to treatment response. CONCLUSIONS: The level of ß-hCG prior to therapy is an important factor for predicting therapeutic outcomes. It should be noted that the success of the therapy decreases notably in case of high ß-hCG level.

Imbalance between matrix metalloproteinases and their tissue inhibitors in preeclampsia and gestational trophoblastic diseases.

The invasion cascade exhibited by placental trophoblasts and cancerous cells bears many similarities, and it is attributed to extracellular matrix degradation mediated by matrix metalloproteinases (MMPs). Although proper and controlled invasion by trophoblasts into the maternal uterus is an essential requirement for maintenance of normal pregnancy, any abnormality in this phenomenon results in the development of invasion-related disorders such as gestational trophoblastic diseases (GTDs) and preeclampsia. We studied the epigenetic basis of differential expression of two placental MMPs (MMP2 and MMP9) and tissue inhibitors of metalloproteinases (TIMP2 and TIMP1) during normal gestation and invasion-related disorders, i.e., preeclampsia and GTDs. Our study suggests the association of H3K9/27me3 with differential expression of these MMPs and their inhibitors, which regulate the placental invasion during normal pregnancy, whereas no role of CpG methylation was observed in the differential expression of MMPs/TIMPs. Further, development of GTDs was associated with abnormally higher expression of these MMPs and lower levels of their inhibitors, whereas the reverse trends were observed for MMPs and their TIMPs in case of preeclampsia, in association with abnormal changes in H3K9/27me3. These results suggest the involvement of higher levels of MMPs in an aggressive invasive behavior depicted by GTDs, whereas lower levels of these MMPs in shallow and poor invasive phenotype associated with preeclampsia. Thus, our study shows the significance of a proper balance regulated by histone trimethylation between differential expression of MMPs and their TIMPs for maintaining normal pregnancy and its deregulation as a contributing factor for pathogenesis of invasive disorders during pregnancy.