Fine needle aspiration cytology of metastatic choriocarcinoma presenting as a breast lump. A case report

BACKGROUND: While choriocarcinoma is a rapidly invasive, widely metastasizing malignancy, it responds well to chemotherapy, so it is important to obtain an early diagnosis. We report the fine needle aspiration cytology (FNAC) of a case of choriocarcinoma metastatic to the breast. CASE: A 48-year-old female presented with a cough, hemoptysis and epistaxis. Chest computed tomography revealed multiple nodules in both lung fields. Also, a firm, slightly tender mass in the lower outer quadrant of the left breast was palpated. The breast mass was clinically suspected to be a metastatic lung cancer. FNAC of the breast showed a malignant tumor that had been misdiagnosed as a metastatic non-small cell carcinoma of the lung. Histologic examination of a nasal biopsy revealed metastatic choriocarcinoma. CONCLUSION: The cytologic features of choriocarcinoma are quite characteristic, with side-by-side, malignant, mononucleated cells and multinucleated giant cells corresponding to cytotrophoblasts and syncytiotrophoblasts, respectively. The disease is possible to diagnose by a careful examination of FNAC samples.     

Choriocarcinoma metastatic to the breast diagnosed by fine needle aspiration.

Fine needle aspiration (FNA) was used to study nodules in the left breast of a patient with a previous history of uterine choriocarcinoma. The FNA smears contained numerous malignant mononucleated cells and multinucleated giant cells. The cytologic diagnosis was metastatic choriocarcinoma, which was confirmed by histologic study of excised tissue. That diagnosis would have been difficult to make cytologically if the previous history had not been known; the differential diagnosis of multinucleated giant cells in an aspirate from a breast mass is discussed.     

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.     

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.     

Fine needle aspiration of pleomorphic giant-cell carcinoma of the pancreas. Case report with ultrastructural observations

The fine needle aspirate in a case of pleomorphic giant-cell carcinoma of the pancreas, an unusual but highly malignant variant of ductal carcinoma of the pancreas, was characterized by bizarre tumor giant cells, "osteoclastlike" giant cells and abundant mitoses. The differential diagnostic possibilities include sarcoma (rhabdomyosarcoma, malignant fibrous histiocytoma and liposarcoma), melanoma, choriocarcinoma, metastatic giant-cell carcinoma of the lung and giant-cell tumor of the pancreas. A combination of clinical history, imaging findings and fine needle aspiration biopsy with transmission electron microscopy could lead to the appropriate diagnosis and help differentiate this entity from the other possible considerations.     

Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 β1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.     

NECC1, a candidate choriocarcinoma suppressor gene that encodes a homeodomain consensus motif

We isolated a candidate choriocarcinoma suppressor gene from a PCR-based subtracted fragmentary cDNA library between normal placental villi and the choriocarcinoma cell line CC1. This gene comprises an open reading frame of 219 nt encoding 73 amino acids and contains a homeodomain as a consensus motif. This gene, designated NECC1 (not expressed in choriocarcinoma clone 1), is located on human chromosome 4q11-q12. NECC1 expression is ubiquitous in the brain, placenta, lung, smooth muscle, uterus, bladder, kidney, and spleen. Normal placental villi expressed NECC1, but all choriocarcinoma cell lines examined and most of the surgically removed choriocarcinoma tissue samples failed to express it. We transfected this gene into choriocarcinoma cell lines and observed remarkable alterations in cell morphology and suppression of in vivo tumorigenesis. Induction of CSH1 (chorionic somatomammotropin hormone 1) by NECC1 expression suggested differentiation of choriocarcinoma cells to syncytiotrophoblasts. Our results suggest that loss of NECC1 expression is involved in malignant conversion of placental trophoblasts.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.     

Choriocarcinoma cells increase the number of differentiating human cytotrophoblasts through an in vitro interaction

The human placenta arises from the zygote through single cell intermediates called cytotrophoblasts that in turn give rise to a syncytium. In culture, mononucleated cytotrophoblasts exhibit little, if any, cell division but are converted to multinucleated cells. Choriocarcinoma, the malignant tumor of placenta trophoblast, comprises a mixed population of dividing cellular intermediates that resemble cytotrophoblasts but are less differentiated. Because the choriocarcinoma intermediates arise from dividing cells, the tumor may contain one or more cell types in abundance not present in the population of isolated placental cells. To study placental differentiation through cell-cell interaction, choriocarcinoma cell lines were co-cultured with placenta-derived cytotrophoblasts, and placental hormone biosynthesis, as a marker of differentiation was examined. We reasoned that intermediates formed by the tumor might interact with and complement those intermediates in the placenta-derived cytotrophoblast population. Co-culturing either the JAr or JEG choriocarcinoma cell lines with cytotrophoblasts elevated the synthesis of the chorionic gonadotropin alpha and beta subunits 10-20 fold, and human placental lactogen 5-fold. The effect was specific for these trophoblast-derived cells, since comparable quantities of Chinese hamster ovary or HeLa cells did not affect the placental cytotrophoblast culture. Further experiments suggested that the source of enhanced synthesis was the cytotrophoblasts. We propose that an interaction between cytotrophoblasts and choriocarcinoma cells occurs, which results in an increased number of differentiating cytotrophoblasts. Such co-cultures may represent a model system for examining choriocarcinoma cell interaction with normal cells, a process known to occur in vivo. The data are also consistent with the hypothesis that the regulated chorionic gonadotropin production in the placenta is determined by interaction among trophoblast cells at different stages of differentiation.     

Downregulation of the Gli Transcription Factors Regulator Kif7 Facilitates Cell Survival and Migration of Choriocarcinoma Cells

The kinesin protein Kif7 has been recognized as an integral component of hedgehog signalling. Aberrant activation of hedgehog signalling has been implicated in many human solid tumours. Gestational trophoblastic disease includes frankly malignant choriocarcinoma and potentially malignant hydatidiform mole. Here we investigated the hedgehog signalling components expression profiles in gestational trophoblastic disease. Downregulation of Gli1, Gli2, Gli3 and Kif7 was demonstrated in clinical samples of choriocarcinoma and hydatidiform moles as well as choriocarcinoma cell lines when compared with normal placentas. Ectopic expression of Kif7 in two choriocarcinoma cell lines JAR and JEG-3 led to a decrease in cell growth and increase in apoptosis demonstrated by MTT and TUNEL assays, respectively. Overexpression of Kif7 also led to suppressed cell migration through transwell assay. In contrast, knocking down Kif7 in HTR-8/SVneo, an immortalized trophoblast cell line, increased cell number over time and increased the migratory ability of the cells. Taken together, Kif7 may contribute to pathogenesis of gestational trophoblastic disease through enhancing survival and promoting dissemination of trophoblasts.     

Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers

Summary Adhesive interactions of trophoblast cells with the endometrium are essential for embryo implantation in the uterus. Choriocarcinoma cells, the malignant counterpart of trophoblast, show pronounced invasiveness and are of interest for model studies. We describe here an in vitro model system for the study of adhesion of human JAR choriocarcinoma multicellular spheroids to different human endometrial epithelial cell lines (RL95-2, HEC-1A, KLE, AN3-CA) grown as monolayers. Cell characterization showed JAR spheroids to secrete the placental hormones human chorionic gonadotropin and progesterone into the culture medium; distinct patterns of keratin, vimentin, and uvomorulin expression were seen in the endometrial cell lines. Spheroid attachment to endometrial monolayers was quantified using a centrifugal force-based adhesion assay, and morphology was examined by light and electron microscopy. Results showed the JAR spheroids to attach to three of the endometrial monolayers (RL95-2, HEC-1A, KLE) progressively over a 24-h period (by which time ≥80% of the spheroids attached). Significant differences in spheroid attachment were most pronounced at 5 h (RL95-2 > HEC-1A > KLE and poly-d-lysine control, i.e. 90:45:17:17% attached). JAR spheroids did not attach to the endometrial cell line AN3-CA. Morphology revealed choriocarcinoma cells to begin to intrude between the uterine RL95-2 epithelial cells at 5 h. At 24 h, this intrusive type of penetration continued to be seen only with the RL95-2 monolayer. The assay system thus identifies differences in attachment properties between choriocarcinoma cells and various endometrial cell lines and forms the basis for further studies on the molecular interactions involved.     

ERV3 human endogenous provirus mRNAs are expressed in normal and malignant tissues and cells, but not in choriocarcinoma tumor cells

Messenger RNA expression of a human endogenous provirus, ERV3, has been characterized in 170 specimens of normal and malignant human tissues and cells. In contrast to the high expression in first-trimester and full-term placental chorionic villi, most other human tissues expressed ERV3 mRNAs at a level of 2-30% of placenta. However, ERV3 mRNAs were not detected in choriocarcinoma tumor cell lines. These studies suggest that the ERV3 provirus may have been preempted for a biological function and disruption of its mRNA expression results in choriocarcinoma.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation.     

Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.     

Nodal signals via β-arrestins and RalGTPases to regulate trophoblast invasion

Placentation is critical for establishing a healthy pregnancy. Trophoblasts mediate implantation and placentation and certain subtypes, most notably extravillous cytotrophoblast, are highly invasive. Trophoblast invasion is tightly regulated by microenvironmental cues that dictate placental morphology and depth. In choriocarcinomas, malignant trophoblast cells become hyperinvasive, breaching the myometrium and leading to major complications. Nodal, a member of the TGF-β superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. Nodal promotes the invasion of numerous types of cancer cells. However, Nodal's role in trophoblast and choriocarcinoma cell invasion is unclear. Here we show that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells in a dose-dependent manner. We found that endogenous β-arrestins and Ral GTPases, key regulators of the cell cytoskeleton, are constitutively associated with Nodal receptors (ALK4 and ALK7) in trophoblast cells and that RalA is colocalized with ALK4 in endocytic vesicles. Nodal stimulates endogenous β-arrestin2 to associate with phospho-ERK1/2, and knockdown of β-arrestin or Ral proteins impairs Nodal-induced trophoblast and choriocarcinoma cell invasion. These results demonstrate, for the first time, that β-arrestins and RalGTPases are important regulators of Nodal-induced invasion.     

Restoration of TGF-beta regulation of plasminogen activator inhibitor-1 in Smad3-restituted human choriocarcinoma cells

Proliferation, migration, and invasiveness of the normal placental extravillous trophoblast (EVT) cells are negatively regulated by transforming growth factor-beta (TGF-beta), whereas malignant EVT (JAR and JEG-3 choriocarcinoma) cells are resistant to TGF-beta. These malignant cells were found to have lost the expression of Smad3. Present study examined whether Smad3 restitution in JAR cells could restore TGF-beta response. We produced a stable Smad3 cDNA-transfected clone (JAR-smad3/c) which exhibited further upregulation of Smad3 in the presence of TGF-beta1. Since anti-invasive effects of TGF-beta in the normal EVT cells were shown to be mediated in part by plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA), we compared the expression of PAI-1 and uPA in the normal EVT, JAR, and JAR-smad3/c cells in the presence or absence of TGF-beta1. The basal levels of PAI-1 mRNA and secreted PAI-1 and uPA proteins were found to be very low in JAR and JAR-smad3/c cells, as compared to the normal EVT cells. However, TGF-beta1 upregulated PAI-1 and downregulated uPA in JAR-smad3/c cells, but not in JAR cells. Thus, resistance of choriocarcinoma cells to anti-invasive effects of TGF-beta may, at least in part, be due to loss of Smad3 expression.