The effect of tinzaparin on biomarkers in FIGO stages III-IV ovarian cancer patients undergoing neoadjuvant chemotherapy - the TABANETOC trial: study protocol for a randomized clinical multicenter trial.

BACKGROUND: Tinzaparin, a low-molecular weight heparin (LMWH), has shown anti-neoplastic properties in animal models and in in vitro studies of human cancer cell lines. The reduction of CA-125 levels during neoadjuvant chemotherapy (NACT) in patients with epithelial ovarian cancer (EOC) co-varies with the prognosis; the larger the decrease in CA-125, the better the prognosis. PURPOSE: This study aims to evaluate the potential anti-neoplastic effects of tinzaparin by investigating changes in serum CA-125 levels in advanced EOC patients who receive NACT. MATERIAL AND METHODS: This is an open randomized multicenter pilot trial. Forty patients with EOC selected to receive NACT will be randomized 1:1 to receive daily addition of tinzaparin or no tinzaparin. The processing and treatment of the patients will otherwise follow the recommendations in the Swedish National Guidelines for Ovarian Cancer. Before every cycle of chemotherapy, preoperatively, and 3 weeks after the last cycle of chemotherapy, a panel of biomarkers, including CA-125, will be measured. PATIENTS: Inclusion criteria are women aged 18 years or older, World Health Organization performance status 0-1, histologically confirmed high-grade serous, endometrioid or clear cell EOC, International Federation of Gynecology and Obstetrics (FIGO) stages III-IV. In addition, a CA-125 level of ≥ 250 kIE/L at diagnosis. Exclusion criteria are contraindications to LMWH, ongoing or recent treatment with unfractionated heparin, LMWH, warfarin or non-vitamin K antagonist oral anticoagulants. INTERPRETATION: This study will make an important contribution to the knowledge of the anti-neoplastic effects of tinzaparin in EOC patients and may thus guide the planning of a future study on the impact of tinzaparin on survival in EOC.

Endometriotic Tissue-derived Exosomes Downregulate NKG2D-mediated Cytotoxicity and Promote Apoptosis: Mechanisms for Survival of Ectopic Endometrial Tissue in Endometriosis.

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a "protective shield" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.

Immunoreactivity of LMO7 and other molecular markers as potential prognostic factors in oropharyngeal squamous cell carcinoma.

BACKGROUND: Despite the better prognosis associated with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC), some patients experience relapse and succumb to the disease; thus, there is a need for biomarkers identifying these patients for intensified treatment. Leucine-rich repeats and immunoglobulin-like domain (LRIG) protein 1 is a negative regulator of receptor tyrosine kinase signaling and a positive prognostic factor in OPSCC. Studies indicate that LRIG1 interacts with the LIM domain 7 protein (LMO7), a stabilizer of adherence junctions. Its role in OPSCC has not been studied before. METHODS: A total of 145 patients diagnosed with OPSCC were enrolled. Immunohistochemical LMO7 expression and staining intensity were evaluated in the tumors and correlated with known clinical and pathological prognostic factors, such as HPV status and LRIG1, CD44, Ki67, and p53 expression. RESULTS: Our results show that high LMO7 expression is associated with significantly longer overall survival (OS) (p = 0.044). LMO7 was a positive prognostic factor for OS in univariate analysis (HR 0.515, 95% CI: 0.267-0.994, p = 0.048) but not in multivariate analysis. The LMO7 expression correlated with LRIG1 expression (p = 0.048), consistent with previous findings. Interestingly, strong LRIG1 staining intensity was an independent negative prognostic factor in the HPV-driven group of tumors (HR 2.847, 95% Cl: 1.036-7.825, p = 0.043). CONCLUSIONS: We show for the first time that high LMO7 expression is a positive prognostic factor in OPSCC, and we propose that LMO7 should be further explored as a biomarker. In contrast to previous reports, LRIG1 expression was shown to be an independent negative prognostic factor in HPV-driven OPSCC.

Long-Term Follow-Up of Tamoxifen Treatment and the Use of Imaging in Psammocarcinoma: A Case Report, Review of the Literature and Discussion of Diagnostic and Therapeutic Challenges.

Psammocarcinoma (PsC) represents a rare form of low-grade serous tumor of the ovary or peritoneum. Although ovarian cancer generally has a poor prognosis in its late stages, PsC seems to have a more indolent course. We present a patient with a history of unspecific abdominal pain for more than a year, with sudden acute onset of severe inguinal pain. On admission to the hospital, a computed tomography (CT) revealed a pelvic mass of suspected ovarian origin. Radical surgery was attempted but not achieved due to widespread tumor growth. Histopathological evaluation revealed estrogen receptor-positive stage III PsC. Tamoxifen treatment was thus initiated, still maintaining stable disease 10 years later. The patient has undergone extensive radiological work-up, including CT, chest X-ray, 18F-fluoro-deoxy-glucose positron emission tomography (PET)/CT, 99mTc- hydroxymethylene diphosphonate (HDP) bone scintigraphy, 18F-fluoro-thymidine (FLT) PET/CT, Tc-99m depreotide scintigraphy and magnetic resonance imaging. In conclusion, we demonstrate that PsC has characteristic radiological features and different imaging modalities can be suitable in different clinical situations. In contrast to most other ovarian cancers, PsC does not always warrant adjuvant chemotherapy, even in advanced stages. This emphasizes the need for a deeper knowledge of the biological behavior of this rare tumor, to select the optimal treatment strategy.

NKG2D-mediated cytotoxicity improves after primary surgery for high-grade serous ovarian cancer.

PROBLEM: Tumors compromise the patients' immune system to promote their own survival. We have previously reported that HGSC exosomes play a central role, downregulating NKG2D cytotoxicity. Primary surgery's effect on tumor exosomes and NKG2D cytotoxicity in HGSC patients has not been studied before. The overall objective of this study was to explore the effect of surgery on the exosome-induced impairment of NKG2D cytotoxicity in HGSC. METHOD OF STUDY: Paired pre- and post-operative blood samples were subjected to cell and exosome analyses regarding the NKG2D receptor and ligands, and NKG2D-mediated cytotoxicity. Lymphocytes were phenotyped by immunoflow cytometry. Exosomes, isolated by ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission and immune electron microscopy and western blot were used in functional cytotoxic experiments. HGSC explant culture-derived exosomes, previously studied by us, were used for comparison. RESULTS: HGSC exosomes from patients' sera downregulated NKG2D-mediated cytotoxicity in NK cells of healthy donors. In a subgroup of subjects, NKG2D expression on CTLs and NK cells was upregulated after surgery, correlating to a decrease in the concentration of exosomes in postoperative sera. An overall significantly improved NKG2D-mediated cytotoxic response of the HGSC patients' own NK cells in postoperative compared to preoperative samples was noted. CONCLUSIONS: Surgical removal of the primary tumor has a beneficial effect, relieving the exosome-mediated suppression of NKG2D cytotoxicity in HGSC patients, thus boostering their ability to combat cancer.

Combined treatment with radiotherapy, chemotherapy and avelumab results in regression of metastatic Merkel cell carcinoma and improvement of associated Lambert-Eaton myasthenic syndrome: A case report.

Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroendocrine malignancy arising from mechanoreceptors in the basal epidermis. Due to a pronounced risk of spread and a high propensity for recurrence after treatment, immediate treatment is of utmost importance. Lambert-Eaton myasthenic syndrome (LEMS) is a paraneoplastic phenomenon affecting the muscles with autoimmune pathophysiology, and >50% of known cases are associated with an underlying malignancy. In the present report, the case of a 67-year-old man presenting with progressive proximal muscle weakness, autonomic dysfunction and involuntary weight loss is described. Symptoms and detection of voltage-gated calcium channel antibodies were consistent with LEMS. Distant metastases were found in the inguinal and iliac lymph nodes, and these were immunohistochemically confirmed to be of epithelial and neuroendocrine origin, consistent with MCC. Local radiotherapy and chemotherapy improved the symptoms; however, a change of treatment was required due to the side effects of the chemotherapy. Avelumab, an immune checkpoint inhibitor, was therefore introduced, and within a year the patient did not only experience tumor remission but also exhibited marked improvements in muscle strength and mobility. At present, 2 years later, the MCC is still in remission. To the best of our knowledge, the present report is the first to describe MCC with associated LEMS, which was successfully treated with avelumab after previous radiotherapy and chemotherapy, with both improved functional motor recovery and tumor reduction. In conclusion, the present case report demonstrated that the present treatment strategy is a potential treatment option and could thus be considered in similar cases.

Cytokine mRNA and protein expression by cell cultures of epithelial ovarian cancer-Methodological considerations on the choice of analytical method for cytokine analyses.

PROBLEM: To get a comprehensive picture of cytokine expression in health and disease is difficult, cytokines are transiently and locally expressed, and protein analyses are burdened by biological modifications, technical issues, and sensitivity to handling of samples. Thus, alternative methods, based on molecular techniques for cytokine mRNA analyses, are often used. We compared cytokine mRNA and protein expression to evaluate whether cytokine mRNA profiles can be used instead of protein analyses. METHOD OF STUDY: In kinetic experiments, cytokine mRNA and protein expression of IL-1ß, IL-6, IL-8, TNF-α, and TNF-ß/LTA were studied using real-time RT-qPCR and Luminex® microarrays in the ovarian cancer cell lines OVCAR-3, SKOV-3 and the T-cell line Jurkat, after activation of transcription by thermal stress. In addition, we analyzed IL-6 and IL-8 mRNA and protein in a small number of ovarian cancer patients. RESULTS: Ovarian cancer cells can express cytokines on both mRNA and protein level, with 1-4 hours' time delay between the mRNA and protein peak and a negative Spearman correlation. The mRNA and protein expression in patient samples was poorly correlated, reflecting previous studies. CONCLUSION: Cytokine mRNA and protein expression levels show diverging results, depending on the material analyzed and the method used. Considering the high sensitivity and reproducibility of real-time RT-qPCR, we suggest that cytokine mRNA profiles could be used as a proxy for protein expression for some specific purposes, such as comparisons between different patient groups, and in defining mechanistic pathways involved in the pathogenesis of cancer and other pathological conditions.

Differential expression of ligands for NKG2D and DNAM-1 receptors by epithelial ovarian cancer-derived exosomes and its influence on NK cell cytotoxicity.

Cancers constitutively produce and secrete into the blood and other biofluids 30-150 nm-sized endosomal vehicles called exosomes. Cancer-derived exosomes exhibit powerful influence on a variety of biological mechanisms to the benefit of the tumors that produce them. We studied the immunosuppressive ability of epithelial ovarian cancer (EOC) exosomes on two cytotoxic pathways of importance for anticancer immunity-the NKG2D receptor-ligand pathway and the DNAM-1-PVR/nectin-2 pathway. Using exosomes, isolated from EOC tumor explant and EOC cell-line culture supernatants, and ascitic fluid from EOC patients, we studied the expression of NKG2D and DNAM-1 ligands on EOC exosomes and their ability to downregulate the cognate receptors. Our results show that EOC exosomes differentially and constitutively express NKG2D ligands from both MICA/B and ULBP families on their surface, while DNAM-1 ligands are more seldom expressed and not associated with the exosomal membrane surface. Consequently, the NKG2D ligand-bearing EOC exosomes significantly downregulated the NKG2D receptor expression on peripheral blood mononuclear cells (PBMC) while the DNAM-1 receptor was unaffected. The downregulation of NKG2D receptor expression was coupled to inhibition of NKG2D receptor-ligand-mediated degranulation and cytotoxicity measured in vitro with OVCAR-3 and K562 cells as targets. The EOC exosomes acted as a decoy impairing the NKG2D mediated cytotoxicity while the DNAM-1 receptor-ligand system remained unchanged. Taken together, our results support and explain the mechanism behind the recently reported finding that in EOC, NK-cell recognition and killing of tumor cells was mainly dependent on DNAM-1 signaling while the contribution of the NKG2D receptor-ligand pathway was complementary and uncertain.