Michela Colombo

Efficacy of double intrauterine insemination in controlled ovarian hyperstimulation cycles

OBJECTIVE To investigate the effectiveness of double IUI and to determine the optimal timing of IUI in relation to hCG administration. DESIGN Prospective randomized study. SETTING Infertility Center, Department of Obstetrics and Gynecology, University of Milan. PATIENT(S) Patients with male factor and unexplained infertility undergoing controlled ovarian hyperstimulation (COH) and IUI. INTERVENTION(S) After COH with clomiphene citrate and gonadotropins, patients were randomly assigned to one of the following groups: group A received a single IUI 34 hours after hCG administration, group B received a double IUI 12 hours and 34 hours after hCG administration, and group C received a double IUI 34 hours and 60 hours after hCG administration. MAIN OUTCOME MEASURE(S) Number of follicles > 15 mm in diameter on the day of hCG administration, number of motile spermatozoa inseminated, clinical pregnancy rate. RESULT(S) Two hundred seventy-three patients underwent 449 treatment cycles: 90 patients were treated for 156 cycles in group A, 92 patients for 144 cycles in group B, and 91 patients for 149 cycles in group C. The overall pregnancies rates for groups A, B, and C were 13 (14.4% per patient and 8.3% per cycle), 28 (30.4% per patient and 19.4% per cycle), and 10 (10.9% per patient and 6.7% per cycle), respectively. There was a statistically significant difference between group B and groups A and C. CONCLUSION(S) Our data indicate that two IUIs performed 12 hours and 34 hours after hCG administration is the most cost-effective regimen for women undergoing COH cycles with clomiphene citrate and gonadotropins. Although the second insemination adds up to a slightly higher cost, it significantly increases the chance of pregnancy.

Effectiveness of Sonohysterography in Infertile Patient Work-Up: A Comparison with Transvaginal Ultrasonography and Hysteroscopy

Objective: The aim of this comparative study was to evaluate the accuracy of transvaginal ultrasonography (TVU), sonohysterography (SHG) with sterile saline solution compared to hysteroscopy in the diagnosis of intrauterine pathology in a population of infertile patients before an in vitro fertilization (IVF) program. Patients/Methods: 98 infertile patients with a mean age of 33.9 years (range 27–41) underwent TVU assessment and SHG with sterile saline solution immediately followed by hysteroscopy. Results: The clinical findings obtained by TVU, SHG and hysteroscopy were compared. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TVU and SHG compared to hysteroscopy were calculated. The TVU sensitivity and specificity compared with hysteroscopy were 91 and 83% respectively. Using TVU, a 9.2% false positive rate (9 cases) and a 5.1% false negative rate (5 cases) were detected compared to hysteroscopy. The TVU PPV and NPV were 85.4 and 90% respectively. SHG yielded better results: sensitivity and specificity when compared to hysteroscopy were 98 and 94% respectively. The SHG PPV and NPV were 95 and 98% respectively. Accuracy of SHG was significantly better than TVU considering all intrauterine pathologies and polyps. Conclusion: In experienced hands, SHG is an easy, safe, and well-tolerated alternative to diagnostic hysteroscopy in the initial evaluation of uterine cavity infertile patients.

The Role of Human Papilloma Virus (HPV) Infection in Non-Anogenital Cancer and the Promise of Immunotherapy: A Review

Over the past 30 years, human papilloma virus (HPV) has been shown to play a role in the development of various cancers. Most notably, HPV has been linked to malignant progression in neoplasms of the anogenital region. However, high-risk HPV has also been suggested to play a significant role in the development of cancers in other anatomic locations, such as the head and neck, lung, breast and bladder. In 2006, the first vaccine for HPV, Gardasil, was approved for the prevention of subtypes 6, 11, 16 and 18. A few years later, Cevarix was approved for the prevention of subtypes 16 and 18, the HPV subtypes most frequently implicated in malignant progression. Although increased awareness and vaccination could drastically decrease the incidence of HPV-positive cancers, these approaches do not benefit patients who have already contracted HPV and developed cancer as a result. For this reason, researchers need to continue developing treatment modalities, such as targeted immunotherapies, for HPV-positive lesions. Here, we review the potential evidence linking HPV infection with the development of non-anogenital cancers and the potential role of immunotherapy in the prevention and eradication of HPV infection and its oncogenic sequela.

Notch pathway promotes ovarian cancer growth and migration via CXCR4/SDF1α chemokine system

Ovarian cancer is the most deadly gynecological malignancy. Understanding the molecular pathogenesis of ovarian cancer is critical to provide new targeted therapeutic strategies. Recent evidence supports a role for Notch in ovarian cancer progression and associates its dysregulation to poor overall survival. Similarly, CXCR4/SDF1α signalling correlates with ovarian cancer progression and metastasis. Recent findings indicate that Notch promotes CXCR4/SDF1α signalling and its effect on cell growth and migration; nonetheless, up to now, the association between Notch and CXCR4/SDFα in ovarian cancer has not been reported. Thereby, the aim of this study was to investigate if Notch and CXCR4/SDF1α cooperate in determining ovarian cancer growth, survival and migration. To address this issue, Notch signalling was inhibited by using γ-secretase inhibitors, or upregulated by forcing of Notch1 expression in ovarian cancer cell lines. Our results indicated that Notch activity influenced tumour cell growth and survival and positively regulated CXCR4 and SDF1α expression. CXCR4/SDF1α signalling mediated the effect of Notch pathway on ovarian cancer cell growth and SDF1α-driven migration. Additionally, for the first time, we demonstrated that Notch signalling activation can be detected in ovarian cancer specimens by immunohistochemistry analysis of the Notch transcriptional target, HES6 and is positively correlated with high expression levels of CXCR4 and SDF1α. Our results demonstrate that Notch affects ovarian cancer cell biology through the modulation of CXCR4/SDF1α signalling and suggest that Notch inhibition may be a rationale therapeutic approach to hamper ovarian cancer progression mediated by the CXCR4/SDF1α axis.

Notch signaling deregulation in multiple myeloma: A rational molecular target

Despite recent therapeutic advances, multiple myeloma (MM) is still an incurable neoplasia due to intrinsic or acquired resistance to therapy. Myeloma cell localization in the bone marrow milieu allows direct interactions between tumor cells and non-tumor bone marrow cells which promote neoplastic cell growth, survival, bone disease, acquisition of drug resistance and consequent relapse. Twenty percent of MM patients are at high-risk of treatment failure as defined by tumor markers or presentation as plasma cell leukemia. Cumulative evidences indicate a key role of Notch signaling in multiple myeloma onset and progression. Unlike other Notch-related malignancies, where the majority of patients carry gain-of-function mutations in Notch pathway members, in MM cell Notch signaling is aberrantly activated due to an increased expression of Notch receptors and ligands; notably, this also results in the activation of Notch signaling in surrounding stromal cells which contributes to myeloma cell proliferation, survival and migration, as well as to bone disease and intrinsic and acquired pharmacological resistance. Here we review the last findings on the mechanisms and the effects of Notch signaling dysregulation in MM and provide a rationale for a therapeutic strategy aiming at inhibiting Notch signaling, along with a complete overview on the currently available Notch-directed approaches.

Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche. We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease. This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche. This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

PI3K/AKT signaling inhibits NOTCH1 lysosome‐mediated degradation

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T‐cell acute lymphoblastic leukemia (T‐ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T‐ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co‐immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c‐Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T‐ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T‐ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling.

Cancer testis antigen Sperm Protein 17 as a new target for triple negative breast cancer immunotherapy

Breast carcinoma is a major health issue for millions of women. Current therapies have serious side effects, and are only partially effective in patients with metastatic tumors. Thus, the need for novel and less toxic therapies is urgent. Moreover, hormonal and antibody therapies effective in other subtypes are not effective in Triple Negative Breast Cancer (TNBC). Immunotherapeutic strategies directed against specific tumor-associated antigens (TAAs) and mediated by specific cytotoxic T lymphocytes (CTL) have been largely underexplored in this disease. Cancer-testis antigens (CTA) are a group of TAAs displaying the ideal characteristics of promising vaccine targets, i.e. strong immunogenicity and cancer specificity. The CTA, Sperm Protein 17 (SP17), has been found to be aberrantly expressed in different neoplasms, including ovarian and esophageal cancers, nervous system tumors and multiple myeloma, and has been suggested as a candidate target for immunotherapy. Here, we evaluated SP17 expression levels in breast cancer cell lines, invasive ductal breast carcinoma, including patients with TNBC, and adjacent non-neoplastic breast tissue, and determined whether SP17 was capable of generating SP17-specific cytotoxic T lymphocytes in vitro. We showed that SP17 is expressed in breast cancer cell lines and primary breast tumors and importantly in TNBC subtype, but not in adjacent non-tumoral breast tissue or unaffected tissues, except in male germinal cells. Furthermore, we detected specific anti-SP17 antibodies in patients’ sera and we generated SP17-specific, HLA class I-restricted, cytotoxic T lymphocytes capable of efficiently killing breast cancer cells.

Targeting Notch as a Therapeutic Approach for Human Malignancies

BACKGROUND Notch is a multifaceted protein that plays a fundamental role in fetal development and tissue homeostasis by directing many cellular functions, including cell growth and differentiation, cell fate determination and regulation of stem cells maintenance. The Notch family consists of four receptors (Notch 1-4) and five ligands (Jagged1-2 and Delta-like 1-3-4) widely expressed in human tissues. Given the crucial contribution of Notch signaling in many physiological processes, it is not surprising that a variety of human malignancies is characterized by a dysregulation of one or more components of this pathway. METHODS In this review, we are going to provide a broad overview on the role of Notch pathway in solid and hematological malignancies and a survey on possible Notch-directed therapeutic strategies. RESULTS We present the most recent findings indicating that Notch signaling dysregulation in human cancers may be due to genetic and epigenetic alterations or to the interactions with other oncogenic pathways. Furthermore, Notch activity may have an oncogenic or a tumor suppressor effect. Finally, we describe the latest preclinical and clinical studies concerning the different pharmacological approaches targeting Notch. CONCLUSION The provided evidence confirms the importance of Notch pathway in human malignancies indicating that a strong rationale exists for the development of a Notch-tailored therapy.

Identification of small molecules uncoupling the Notch::Jagged interaction through an integrated high-throughput screening

Notch signaling plays an important role in several cellular functions including growth, differentiation, cell fate determination and stemness. Increased Notch activity has been linked to several types of cancers. Activation of Notch signaling is triggered by the interaction of Notch receptors (Notch1-4) with 5 different ligands (Jagged1-2 and Dll1-3-4) expressed on the neighbouring cells. Currently, indirect approaches to inhibit Notch signalling are based on the inhibition of the key step of Notch activation catalyzed by the γ-Secretase and thereby affect several different γ-Secretase substrates; conversely direct strategies get advantage of antibody-based drugs. The evidence that Jagged-mediated Notch activation plays a key role in cancer cell biology and the interplay with the surrounding microenvironment prompted us to develop a strategy to directly inhibit Notch activation by uncoupling its interaction with the Jagged, using an unprecedented approach based on small molecules. We set-up a screening strategy based on: protein::protein docking of crystallographic structures of Notch1 with Jagged1; comparative modelling of the Notch2:Jagged2 complex, based on the Notch1::Jagged1 complex; in silico high-throughput screening directed to Notch2 interaction surface of a virtual chemical library containing a large variety of molecules commercially available. The predicted pharmacological activity of the selected compounds was validated in vitro by a gene reporter and a viability assay. This approach led to the successful identification of two candidates with different anti-proliferative potency and efficacy. This represents the first step towards the rational identification of candidate molecules for the development of entirely novel drugs directed to inhibit Notch signaling in cancer.