Kirsten Kübler

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Targeted activation of RNA helicase retinoic acid-inducible gene-I induces proimmunogenic apoptosis of human ovarian cancer cells.

Most malignant cells are poorly immunogenic and fail to elicit an effective antitumor immune response. In contrast, viral infections of cells are promptly detected and eliminated by the immune system. Viral recognition critically hinges on cytosolic nucleic acid receptors that include the proinflammatory RNA helicase retinoic acid-inducible gene-I (RIG-I). Here, we show that targeted delivery of RIG-I agonists induced ovarian cancer cells to upregulate HLA class I and to secrete the proinflammatory cytokines CXCL10, CCL5, interleukin-6, tumor necrosis factor-alpha, and IFN-beta. Ovarian cancer cells stimulated via RIG-I became apoptotic and were readily phagocytosed by monocytes and monocyte-derived dendritic cells, which in turn upregulated HLA class I/II and costimulatory molecules and released CXCL10 and IFN-alpha. Our findings offer proof of principle that mimicking viral infection in ovarian cancer cells triggers an immunogenic form of tumor cell apoptosis that may enhance immunotherapy of ovarian cancer.

Immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(I:C) leads to myeloid cell maturation and activates NK cells

Owing to high rates of tumor relapse, ovarian cancer remains a fatal disease for which new therapeutic approaches are urgently needed. Accumulating evidence indicates that immune stimulation may delay or even prevent disease recurrence in ovarian cancer. In order to elicit proinflammatory signals that induce or amplify antitumor immune reactivity, we mimicked viral infection in ascites‐derived ovarian cancer cells. By transfection or electroporation we targeted the synthetic double‐stranded RNA poly(I:C) intracellularly in order to activate melanoma differentiation‐associated gene‐5 (MDA‐5), a sensor of viral RNA in the cytosol of somatic cells. Cancer cells reacted with enhanced expression of HLA‐class I, release of CXCL10, IL‐6, and type I IFN as well as tumor cell apoptosis. Monocytes and monocyte‐derived DCs (MoDCs) engulfed MDA‐5‐activated cancer cells, and subsequently upregulated HLA‐class I/II and costimulatory molecules, and secreted CXCL10 and IFN‐α. Further, this proinflammatory milieu promoted cytolytic activity and IFN‐γ secretion of NK cells. Thus, our data suggest that the engagement of MDA‐5 in a whole tumor cell vaccine is a promising approach for the immunotherapy of ovarian cancer.

Prognostic significance of tumor-associated macrophages in endometrial adenocarcinoma

OBJECTIVE Endometrial adenocarcinoma is one of the most common gynecologic malignancies worldwide and in stages confined to the uterus considered to have an excellent prognosis. However, in advanced or recurrent cases when surgery fails to achieve disease control other treatment options are less effective. Thus, new therapeutic avenues are needed. METHODS To provide the rationale for the use of novel agents that target immune checkpoints 163 type I endometrial cancer samples were immunohistochemically screened for the presence of CD163(+) tumor-associated macrophages and Foxp3(+) regulatory T cells. Further, a D2-40-based evaluation of lymph vessel density and lymphovascular space invasion was carried out. Correlation analysis with clinicopathological parameters was performed; Kaplan-Meier curves were generated; multivariate analysis was undertaken as appropriate. RESULTS A substantial amount of tumor-associated macrophages and regulatory T cells was detected in all specimens characterizing endometrial cancer as an immunogenic tumor. However, only the increased infiltration of tumor-associated macrophages was proportionally associated with advanced FIGO stages, high tumor grade, increased lymph vessel density, lymphovascular space invasion and lymph node metastasis. Thus, the presence of tumor-associated macrophages indicates aggressive tumor behavior and appeared to be an independent prognostic factor for recurrence-free survival. CONCLUSIONS Our results make future therapeutic approaches that target tumor-associated macrophages reasonable to improve the outcome of women with advanced or recurrent endometrial adenocarcinoma.

HLA-class II haplotype associations with ovarian cancer

The development of cancer is a multistep process that is characterized by the accumulation of genetic alterations in cells and changed cellular interactions with the surrounding healthy tissues. The human immune system is believed to be intrinsically involved in this process. The correlation of certain human leukocyte antigen (HLA)‐class I and II haplotypes with tumorigenesis is documented in a variety of tumors. However, few data exist on the possible association of specific HLA‐class II alleles or haplotypes with ovarian cancer. In our sample of 52 Caucasian patients with primary ovarian carcinoma and 239 female healthy local controls, we observed a significantly increased incidence of the HLA‐class II haplotypes DRB1*0301 – DQA1*0501 – DQB1*0201 (p < 0.001) and DRB1*1001 – DQA1*0101 – DQB1*0501 (p < 0.001) in the patients. Our data suggest that HLA‐class II loci or individual HLA‐class II haplotypes may be involved in the pathogenesis of ovarian cancer.

Circulating N-terminal pro-B-type natriuretic peptide in fetal anemia before and after treatment

Background:N-terminal pro-B-type natriuretic peptide (nt-proBNP) is an established marker of heart failure in adult cardiology. We analyzed nt-proBNP in the circulation of fetuses with increased volume load secondary to anemia and investigated the effect of treatment on nt-proBNP concentration.Methods:Fetuses undergoing intrauterine transfusion (IUT) were examined. nt-proBNP was measured before IUT and correlated with hemoglobin concentrations, ultrasonographic findings, and Doppler measurements of the peak systolic velocity of the middle cerebral artery (MCA-PSV).Results:A total of 27 patients (7 with hydrops) and 78 controls were examined. nt-proBNP was markedly elevated in anemia (P < 0.001). Concentrations were highest in hydropic fetuses (P < 0.03); no differences were present in hemoglobin and MCA-PSV values between hydropic and nonhydropic cases. In fetuses undergoing multiple IUTs nt-proBNP normalized after the third IUT, whereas hemoglobin and MCA-PSV remained abnormal.Conclusion:Levels of circulating nt-proBNP correlate well with the degree of myocardial workload in the hyperdynamic state of fetal anemia. We hypothesize that normalization of nt-proBNP after serial transfusions is an indicator of myocardial adjustment to chronic anemia. nt-proBNP measurement may be useful in the management of fetal anemia, particularly in cases at risk of hydrops and fetuses requiring multiple transfusions.

Reference values for N-terminal pro-B-type natriuretic peptide in fetal circulation between 20 and 34 weeks of gestation.

OBJECTIVE We aimed to investigate the range of fetal NT-proBNP values in normal pregnancy between 20 and 34 weeks of gestation. METHOD NT-proBNP was measured in 56 fetal blood samples. RESULTS Mean (+ or - 2 SD) NT-proBNP concentration was 1998 (242-3754) ng/L; a significant decline occurred with advancing gestational age (p=0.012). CONCLUSIONS Gestational age has to be taken in to consideration in the assessment of NT-proBNP. Our data may be used as reference values in fetal and neonatal medicine.

Genetic alterations of HLA-class II in ovarian cancer

The immune system controls tumor formation through identification and elimination of cellular alterations. Consequently, cancer development in immune competent hosts depends on strategies to evade the immune system. Modulation of tumor antigen‐specific immune responses by aberrant expression of HLA‐class I and II molecules is well documented in a variety of carcinomas including ovarian cancer. To date, little data are available about molecular mechanisms responsible for altered HLA‐class II phenotypes in tumors. In our sample of 10 Caucasian patients with ovarian carcinoma, a semiquantitative analysis was performed for HLA‐class II loci DRB1 and DQB1 in malignant and normal ovarian tissue. Gene amplifications were identified in 62.5% of analyzed alleles and deletions in 17.5%, demonstrating that genomic aberrations of 6p21.3 are common and that copy number gain is more frequent than loss. Moreover, amplifications are most pronounced in advanced‐stage tumors. To evaluate genotype‐phenotype relation, immunohistochemical analyses were performed and revealed de novo expression of HLA‐class II in 30% of tumors with an inverse association between antigen level and HLA copy number. It remains to be elucidated whether the profound changes of the latter quantities are the result of the hosts immunological self‐defense, indicate the presence of an oncogene located within the MHC‐complex or merely reflect the increasing loss of differentiation of the tumor tissue.

High-risk pregnancy management in women with hypopituitarism

Pregnancy after complete loss of pituitary function is uncommon. However, advances in fertility treatment have led to increased pregnancy rates in hypopituitary women. We hereby present a literature review of pregnancies affected by hypopituitarism, including a comparison with published controls; further, we add one case report of severe hypopituitarism where third-trimester oxytocin supplementation was performed. As only limited information is available on management and outcome, our purpose was to determine obstetric complications associated with deficiency of pituitary hormones. The analysis of 31 pregnancies in 27 women revealed that hypopituitary women are at increased risk: postpartum hemorrhage occurred in 8.7%, transverse lie in 16%; 42.4% of the newborns were small for gestational age. These findings are supposedly the result of uterine dysfunction caused by hormone deficiency. Oxytocin supplementation was performed with the aim to establish physiologic conditions and to prevent postpartum uterine inertia. In this case substitution may have contributed to correct fetal presentation but did not prevent postpartum hemorrhage. Further investigations into both oxytocin-dependent and -independent mechanisms regulating uterine contractions and contractility are necessary to develop strategies for prevention of uterine inertia in oxytocin-deficient pregnancies.

Interval Debulking Surgery in Patients with Federation of Gynecology and Obstetrics (FIGO) Stage IIIC and IV Ovarian Cancer

Background: The feasibility of neoadjuvant chemotherapy (NAC) and the outcome in patients with Federation of Gynecology and Obstetrics (FIGO) IIIC and IV ovarian cancer were assessed. Patients and Methods: 67 patients undergoing interval debulking surgery (IDS) and ≥ 4 courses of platinum-based NAC were analyzed for survival, perioperative morbidity and mortality. Results: The median follow-up was 30 months. The median progression-free survival (PFS) was 17 months, the overall survival (OS) 34 months. The PFS of patients without residual disease (n = 23; 34.3%) was 31 months (p = 0.003), the OS 65 months (p = 0.001). PFS and OS were significantly longer in patients with no residual disease than in patients with 1-10 mm (n = 34; 47.9%) (p = 0.005 and p = 0.0001, respectively) residual disease. No survival benefit was seen for patients with 1-10 mm compared to > 1 cm (n = 12; 16.9%) residual disease (PFS p = 0.518; OS p = 0.077). 1 patient (1.4%) died; 12 patients needed interventional treatment or operation (16.9%) within the first 30 days postoperatively. Out of these, 5 patients (7.0%) had residual or lasting disability. Conclusions: NAC and IDS are safe and feasible in this series of patients with unfavorable prognosis. IDS does not change the goal of complete cytoreduction and therefore does not compensate for a less radical surgical approach.