Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses.

The receptor for the luteinizing hormone (LH-R) is aberrantly over expressed in cancers of the reproductive system. To uncover whether LH-R over expression has a causative role in cancer, we generated a transgenic (TG) mouse which overexpresses the human LH-R (hLH-R) in the female reproductive tract, under the control of the oviduct-specific glycoprotein (OGP) mouse promoter (mogp-1). The transgene was highly expressed in the uterus, ovary and liver, but only in the uterus morphological and molecular alterations (increased proliferation and trans-differentiation in the endometrial layer) were detected. A transcriptomic analysis on the uteri of young TG mice showed an up regulation of genes involved in cell cycle control and a down regulation of genes related to the immune system and the metabolism of xenobiotics. Aged TG females developed tumor masses in the uteri, which resembled an Endometrial Cancer (EC). Microarray and immunohistochemistry data indicated the deregulation of signaling pathways which are known to be altered in human ECs. The analysis of a cohort of 126 human ECs showed that LH-R overexpression is associated with early-stage tumors. Overall, our data led support to conclude that LH-R overexpression may directly contribute to trigger the neoplastic transformation of the endometrium.

Detection of PIK3CA E545A mutation in circulating tumor DNA of a patient affected by uterine carcinosarcoma.

Uterine carcinosarcomas are biphasic neoplasms consisting of mixed epithelial and mesenchymal elements, representing less than 5% of all uterine malignancies. Carcinosarcomas are rare, although the most common cause of uterine cancer-specific death. Few information is available on the pathogenesis, and molecular characterization is poorly investigated. Consequently, the treatment has not changed over the last years and is far too being tailored, consisting of surgery and traditional chemotherapy and radiotherapy. Molecular characterization of liquid biopsy by circulating tumor DNA (ctDNA)/circulating cell-free DNA (ccfDNA) evaluation in a patient with uterine carcinosarcoma. Here, we describe a case report of an 83-year-old woman with carcinosarcomas, stage T3aN0M0. Cancer cells did not express estrogen nor progesterone receptors, while p53 and p16 were positive. Molecular characterization of ccfDNA and of ctDNA was performed by quantitative PCR, amplification-refractory mutation system technology. The presence of phosphatidylInositol-4,5-bisphosphate 3-Kinase catalytic subunit alpha p.E545A mutation was detected in plasma. This approach may suggest the use of liquid biopsy and the development of specific targeted therapy for precision personalized medicine even in rare carcinosarcomas.

Mutational profile in circulating tumor DNA in a patient affected by low-risk endometrial cancer: predictable tool of relapse?

Endometrial cancer is the commonest gynecological cancer, the majority is endometrioid type, diagnosed at an early stage with 69-88% 5-year survival. Low-grade endometrial cancers have low recurrence rates and often do not receive adjuvant therapy; however, a subset of these patients will have poor outcomes and would benefit from adjuvant treatment has been challenging. We evaluate the circulating cell-free DNA (ccfDNA) in a patient with low-risk endometrial cancer in order to identify the presence of molecular markers associated with risk of recurrence. The evaluation of mutation profile was performed by next-generation sequencing (NGS) in primary tumor formalin-fixed paraffin-embedded (FFPE) tissue and in circulating tumor DNA (ctDNA). We identified a specific mutational profile in ctDNA, different from primary tumor tissue suggesting that the clone involved in the relapse may be different in comparison to the most represented in the primary tumor. These findings open new prospective and new wonderings. The molecular characterization of tissue may be useful for setting new target personalized therapy even in the treatment of endometrial cancer, moreover, endometrial cancer at low risk should be not underestimated for the incidence of relapse, and for this evaluation the molecular characterization may be useful. Moreover, these results suggest that the single analysis of primary tumors may be not sufficient for setting a specific personalized therapy targeted to avoid the relapse but may be necessary to join the molecular characterization of liquid biopsy to primary tissue.

Identification of a Gene Panel for Endometrioid Endometrial Cancer: a Possible Prognostic Value?

The incidence of endometrial cancer (EC) is increasing in developed countries. The most frequent is the endometrioid subtype with usually good prognosis; nevertheless, some cases escape this paradigm and may have recurrence. A recent study from The Cancer Genome Atlas suggested to implement the EC analysis by molecular profile for improving diagnosis, prognosis, and therapeutic treatment. The present preliminary study was performed on 15 G3 endometrioid endometrial cancers (G3 EEC) for the identification of somatic mutations in a panel of specific exons in selected genes as ARID1A, CTNNB1, KRAS, PIK3CA, POLE, PTEN, and TP53. The combined procedure, based on the Sanger sequencing and PCR-high-resolution melting analysis, allowed the identification of variations of the selected gene panel in most of patients (93%) of our cohort. The overall evaluation of mutational load exhibited that the most frequent mutated genes were PTEN (93%), followed by PIK3CA (47%) suggesting a deep involvement of PI3K pathway alteration in G3 EEC. Mutations in TP53 (27%), ARID1A (27%), POLE (13%), and at the lower level in KRAS and CTNNB1 (7%) were also observed (exclusively in FIGO III stage patients). The evaluation of the mutations of our proposed panel (ARID1A, CTNNB1, KRAS, PIK3CA, POLE, PTEN, TP53) is suitable to improve the characterization of G3 EEC and could suggest targetable pathways for development of personalized treatments.

Pilot investigation of the mutation profile of PIK3CA/PTEN genes (PI3K pathway) in grade 3 endometrial cancer.

Endometrial cancer (EC) comprises a biological and clinical heterogeneous group of tumors. Several genetic alterations are involved in the development and progression of EC, and may be used for targeted therapy, particularly in patients with advanced­stage EC. In the present study, a combined procedure was developed based on polymerase chain reaction (PCR)­high resolution melting analysis (HRMA) and Sanger sequencing for the evaluation of somatic mutations in selected phosphoinositide 3­kinase (PI3K) catalytic subunit α (PIK3CA; exons 1, 9 and 21) and phosphatase and tensin homolog (PTEN; exons 5, 6, 7 and 8) exons. This combined procedure has the specificity and sensitivity of the two techniques, and overcomes their limitations. A pilot study was performed on 18 selected homogenous EC samples, of grade 3 endometrioid subtype (G3 EEC). First, the feasibility of the combined procedure was investigated to properly identify the presence of somatic mutations on PIK3CA and PTEN, the variations identified were analyzed using Catalogue of Somatic Mutations in Cancer, PolyPhen­2 and Mutation Taster software, and the frequency of mutations/variations was determined in the selected samples. The evaluation of mutational load revealed that the majority of the G3 EEC samples exhibited PIK3CA mutations (39%) and PTEN mutations (67%), and the majority of the samples (83%) had mutations in at least one of the two genes, and 33% had mutations in the two genes. The results of the present pilot study suggested that the cost­effective combined PCR­HRMA and Sanger sequencing procedure may be suitable for identification of PTEN and PIK3CA mutations in G3 EEC and that their frequency was consistent in G3 EEC, indicating that the PI3K pathway serves a pivotal function that may have potential for defining targeted therapy for the treatment of G3 EEC.

Spontaneous Resolution of an Acquired Uterine Arteriovenous Malformation in an Elderly Primigravida.

BACKGROUND Uterine arteriovenous malformation (AVM) is an uncommon lesion characterized by an abnormal connection between arterial and venous circulation that can be congenital or acquired. Acquired uterine AVMs are generally traumatic and follow delivery, abortion, curettage, or uterine surgery. CASE REPORT A 45-year-old female who was gravida 1 para 0 presented to our hospital with severe vaginal bleeding. Two weeks before, the patient underwent therapeutic abortion. At admission, a transvaginal ultrasound showed an unclear intrauterine lesion that spread out to the myometrium. Color Doppler evaluation demonstrated an elevated color score. Beta human chorionic gonadotropin (beta-hCG) levels were measured at admission and daily repeated, with a progressive decrease of values up to a negative level. A pelvic magnetic resonance imaging described an area of tubular and tortuous structures involving the myometrium. A computed tomography angiography confirmed the presence of a lesion infiltrating the endometrium and myometrium containing arteriovenous structures with a highly enhanced effect. Despite these findings, the patient was clinically stable. A diagnosis of uterine AVM was made and, after accurate counselling with the patient, she was discharged and underwent "watch and wait" management. After 35 days, the patient had a follow-up ultrasound that showed a complete resolution of the uterine lesion. CONCLUSIONS AVM should be considered in the presence of heavy and sudden vaginal bleeding in a patient with risk factors for acquired AVM. A color Doppler ultrasound scan should be performed as the first approach and an expectant management should be taken into account especially with a patient of childbearing age and hemodynamic instability.

Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Immunohistochemical Score Associated with Poor Prognosis in Endometrial Cancer Patients.

The aim of this study was to develop a scoring system of the immunohistochemical (IHC) expression of luteinizing hormone/human chorionic gonadotropin receptor (LHCG-R) in endometrial cancer (EC) patients. Nonconsecutive hysterectomy specimens containing EC collected from April 2013 to October 2015 were selected. Hematoxylin-eosin stained sections from each case were reviewed and representative sections from each tumor were selected. IHC staining was performed for the detection of LHCG-R. The percentage of stained cells and the staining intensity were assessed in order to develop an immunohistochemical score. Moreover, we examined the correlation of the score with grading and lymphovascular space invasion (LVSI). There was a statistically significant positive correlation between grading and IHC scoring (p = 0.01) and a statistically significant positive correlation between LVSI and IHC score (p < 0.01). In conclusion, we suggest that the immunohistochemical score presented here could be used as a marker of bad prognosis of EC patients. Nevertheless, further studies are needed in order to validate it. The study was registered in the Careggi Hospital public trials registry with the following number: 2013/0011391.

From pathogenesis to clinical practice: Emerging medical treatments for endometriosis.

Endometriosis is a chronic disease, and a lifelong management plan should be developed by using pharmacological treatment and surgical procedures. The pathogenesis of endometriosis is complicated and has not been definitively established. The mechanisms involved are numerous, and their understanding is constantly evolving. Currently, the first-line drugs act by blocking ovarian function, creating an hypoestrogenic environment. The blockade of estrogen secretion and receptor activity and the activation of progesteron receptors are the main target of several current drugs, as well as those under development. The oral GnRH antogonists, the aromatase inhibitors, SERMs, and SPRMs are the hormonal drugs currently studied for treating endometriosis. The increasing knowledge of the pathogenesis has allowed the development of new treatments. The most studied are the anti-inflammatory drugs, starting from the new NSAIDs to the monoclonal antibodies and the statins. Among the antiangiogenic compounds, a role is suggested for Icon, PPARs, and HDACIs. A new class of drugs is the cannabinoids. The aim of this review was to investigate the new therapeutic hormonal and non-hormonal alternatives to standard treatments.

Severe Fetal Distress and Placental Damage might be Associated with High Troponin I (cTnI) Levels in Mothers.

BACKGROUND Troponin I is the gold standard for the diagnosis of adult acute coronary syndrome. Although it is known that a hypoxic fetus may produce cTnI, fetal cTnI passage in maternal blood has never been documented. CASE REPORT We report a case where the rise of cTnI in the blood of a pregnant woman was not related to maternal heart disease. Instead, it might be suggestive of a fetal cardiac origin, as there was a severe placental insufficiency with a fetal intrauterine growth restriction. CONCLUSIONS This study suggests that the rise of cTnI in maternal blood in a cardiovascular healthy pregnant woman might have a fetal origin. After having excluded any maternal causes, cTnI elevation could be explained with the transfer of fetal cTnI through an injured placenta.

LH/hCG-Receptor Expression May Have a Negative Prognostic Value in Low-Risk Endometrial Cancer.

INTRODUCTION: A 51 year-old woman was diagnosed with endometrial cancer (EC) and underwent surgical staging. Pathological evaluation showed a 2 cm × 1 cm G2 endometrioid EC with a 30% myometrial deep invasion (FIGO Stage 1A). The patient was classified as low risk of recurrence, and no adjuvant treatment was offered. Six months after surgery, the patient developed an early vescico-vaginal recurrence, and chemotherapy treatment was started. Few months later, a subsequent involvement of vaginal wall, ileum, and omentum was detected, and the patient underwent second surgery. BACKGROUND: LH/hCG-receptor (LH/hCG-R) expression has been previously reported to be associated with an invasive phenotype in EC cells. Moreover, in a preclinical mouse model of EC behaves as a prometastatic molecular device. DISCUSSION: We analyzed the expression level of LH/hCG-R in cancer specimens collected during surgeries. Molecular and immunohistochemical analyses showed a strong expression of both mRNA and protein for LH/hCG-R in all specimens. CONCLUSION: LH/hCG-R expression may be assessed together with other clinicopathological parameters in order to better predict the risk of recurrence in low-risk EC patients. Further clinical trials are warranted in order to validate LH/hCG-R as biomarker in EC.

Over-Expression of the LH Receptor Increases Distant Metastases in an Endometrial Cancer Mouse Model.

OBJECTIVE: The aim of the present study was to define the role of luteinizing hormone receptor (LH-R) expression in endometrial cancer (EC), using preclinical mouse models, to further transfer these data to the clinical setting. MATERIALS AND METHODS: The role of LH-R over-expression was studied using EC cells (Hec1A, e.g., cells with low endogenous LH-R expression) transfected with the LH-R (Hec1A-LH-R). In vitro cell proliferation was measured through the WST-1 assay, whereas cell invasion was measured trough the matrigel assay. The effects of LH-R over-expression in vivo were analyzed in an appropriately developed preclinical mouse model of EC, which mimicked postmenopausal conditions. The model consisted in an orthotopic xenograft of Hec1A cells into immunodeficient mice treated daily with recombinant LH, to assure high levels of LH. RESULTS: In vitro data indicated that LH-R over-expression increased Hec1A invasiveness. In vivo results showed that tumors arising from Hec1A-LH-R cells injection displayed a higher local invasion and a higher number of distant metastases, mainly in the lung, compared to tumors obtained from the injection of Hec1A cells. LH withdrawal strongly inhibited local and distant metastatic spread of tumors, especially those arising from Hec1A-LH-R cells. CONCLUSION: The over-expression of the LH-R increases the ability of EC cells to undergo local invasion and metastatic spread. This occurs in the presence of high LH serum concentrations.

hLH/hCG-receptor expression correlates with in vitro invasiveness in human primary endometrial cancer.

OBJECTIVE: Endometrial cancer (EC) is the most frequent cancer of the female genital tract. It has been hypothesized that those ECs that occur in the postmenopausal period, might be sensitive to elevated levels of luteinizing hormone/human chorionic gonadotropin (LH/hCG). Based on previous indications, we analyzed the functional expression of LH/hCG receptors (LH/hCG-R) in primary ECs. METHODS: We studied a cohort of primary ECs, in which both the LH/hCG-R mRNA and the LH/hCG-R protein were analyzed. Results were correlated with both clinical-pathological data and the effects of LH addition on cell invasion in vitro. RESULTS: The LH/hCG-R mRNA levels ranged from 4.67 e(-02) to 2.36 e(+03). The transcript was properly translated into a functional LH/hCG-R protein. The analysis of cell invasion in vitro in response to LH/hCG allowed us to divide the EC samples into two groups, one with a null or very low response (non-responders=NR) and the other with a significant response to LH (responders=R). The two groups had significantly different levels of LH/hCG-R mRNA expression: the NR group had a median value of 1.40 e(+)(00), while the R group of 7.42 e(+)(01) (p=0.043). CONCLUSION: In primary ECs a statistically significant correlation emerged between the levels of LH/hCG-R mRNA and the LH-induced cell invasion in vitro. These results suggest that therapies aimed at decreasing LH levels, through Gonadotropin Releasing Hormone (Gn-RH) analogues, could produce benefits in the treatment of recurrent or metastatic EC, especially in patients displaying high LH/hCG-R levels.

The sialoglycoconjugates in the oviducts of fertile and postmenopausal women.

OBJECTIVES: The aim of the study was to investigate the distribution of the sialoderivatives of the glycoconjugates in the oviduct of normally menstruating and postmenopausal women. METHODS: Biopsies of ampullary and isthmic portions of the oviduct were obtained from fertile women, in proliferative and secretive phases, and from postmenopausal subjects. The study was carried out using digoxigenin-labelled lectins (MAA, SNA and PNA) in addition to the use of enzymatic and chemical treatments to characterize the different glycosidic linkages of the sialoderivatives and to obtain information on their structure. RESULTS: No appreciable difference in sialoderivatives distribution was observed among the oviducts, particularly at the epithelium luminal surface, of the fertile women in the two menstrual cycle phases or among those of the fertile and some postmenopausal women, independently from age. Moreover, no appreciable difference of distribution was observed between the ampullary and the isthmic portions in both the study groups. CONCLUSIONS: In the fertile women sialoderivatives present at the luminal surface of the epithelial cells could play a role in sperm capacitation and mobility, and facilitate the transit of the egg and of the early embryo along the oviducts. The similar distribution of sialoderivatives in the oviduct epithelium of some postmenopausal women of different age, compared to the fertile ones, suggests a maintaining of some functions of the organ, independently from the age of the woman. This could be due, in some cases, to the influence of remaining still sufficient sex hormonal levels.

Luteinizing hormone increases human endometrial cancer cells invasiveness through activation of protein kinase A.

Endometrial cancer (EC) is a hormone-dependent cancer that currently represents the most frequent malignancy of the female reproductive tract. The involvement of steroid hormones in its etiology and progression has been reported. The possibility that even gonadotropins (GT) could play a role in the genesis and establishment of EC is supported by the fact that specific receptors for the GT luteinizing hormone/human chorionic GT (LH/hCG) have been detected in a high percentage of ECs, and their expression is apparently related to the cancer grading. However, the precise mechanisms by which GTs might exert their effect on EC is still obscure. The aim of this study was to determine the effects of LH/hCG on the invasion potential of EC cell lines and primary human EC cells. Human recombinant (hr) LH (and hCG) induced a significant increase in cell invasiveness through Matrigel-coated porous membranes in an EC human cell line Hec1A, which expresses the LH/hCG receptor. This effect turned out to depend on hrLH binding to its specific receptors and to the subsequent activation of protein kinase A (PKA). Moreover the hrLH-induced increase in Hec1A invasiveness relied upon a PKA-dependent functional activation of beta(1) integrin receptors, as well as the subsequent induction of matrix metalloproteinase-2 secretion in its active form. The same mechanisms were also found to be operative in primary EC cells. In fact, a significant percentage of primary ECs expressed the LH/hCG receptor, and hrLH addition to primary EC cells, which expressed the specific receptors produced an increase in cell invasiveness only in those tumor cells possessing the specific receptors. This effect was also dependent on PKA activity. We conclude that LH/hCG can regulate EC cells invasiveness, and this result provides a rationale for the use of inhibitors of LH secretion such as GnRH analogues in the treatment of EC.

Analysis of estrogen receptor (ERalpha and ERbeta) and progesterone receptor (PR) polymorphisms in uterine leiomyomas.

BACKGROUND: Leiomyomas are the most common tumors in women, found in up to 30% of women in active reproductive life. These tumors are estrogen- and progestin-responsive. In fact, they do not occur before menarche, undergoing rapid increase in size during pregnancy with consequent fetal wastage. Conversely, they can regress or even calcify after menopause or castration. Given the central role of sex steroids in uterus trophism, estrogen and progesterone receptors are important mediators of hormonal bioeffects, and the genes encoding these receptors become easy candidates for uterine proliferative disorders. MATERIAL/METHODS: We examined the distribution of common polymorphisms of ERalpha, ERbeta and PR genes in 413 Caucasian females: 225 post-menopausal healthy controls and 188 premenopausal women affected by uterine leiomyomas. The polymorphisms were evaluated both in constitutive and tissue DNA by PCR amplification, specific endonuclease digestion, and then detection with agarose or polyacrylamide gel electrophoresis. RESULTS: The observed allele frequencies did not deviate from the expected Hardy-Weinberg distribution in our population. In case-control analysis, no variant frequency of the studied genes differed significantly between control subjects and patients. CONCLUSIONS: Polymorphisms in the genes encoding for ERalpha, ERbeta and PR did not correlate with the occurrence of uterine leiomyomas in our Caucasian population.