Staged neurosurgical approach for giant and progressive neonatal arachnoid cysts: a case series and review of the literature.

OBJECTIVES: Prenatally diagnosed complex arachnoid cysts are very rare. While the true prenatal incidence is still unknown, they account for approximately 1% of intracranial masses in newborns. They rarely exhibit rapid growth or cause obstructive hydrocephalus, but if they increase to such a dimension during pregnancy, the ideal management is not well established. We present our detailed perinatal experience, covering prenatal diagnosis, a compassionate delivery process, and neonatal stabilization. Finally, a thorough postnatal neurosurgical intervention was performed. Initially, our focus was on the gradual reduction of cyst size as a primary effort, followed by subsequent definitive surgical treatment. METHODS: This case series shows the treatment course of three fetuses with antenatally diagnosed large arachnoid cysts. We present pre- and postnatal management and imaging, as well as the surgical treatment plan and the available clinical course during follow-up. RESULTS: Two girls and one boy were included in the current review. All three cases presented with prenatally diagnosed complex arachnoid cysts that increased in size during pregnancy. The mean gestational age at delivery was 35 weeks (range 32 to 37 weeks), and all patients were delivered by a caesarian section. Increasing head circumference and compression of brain structures were indications for delivery, as they are associated with a high risk of excess intracranial pressures and CSF diapedesis, as well as traumatic delivery and maternal complications. All cysts were supratentorial in location; one expanded into the posterior fossa, and one was a multicompartment cyst. All children underwent an initial surgical procedure within the first days of life. To relieve cyst pressure and achieve a reduction in head circumference, an ultrasound-guided or endoscopic-assisted internal shunt with drainage of the cyst to the ventricles or subdural/subarachnoid space was inserted. Definite surgical therapy consisted of cyst marsupialization and/or cysto-peritoneal shunt implantation. All children survived without severe neurodevelopmental impairments. CONCLUSION: With the cases presented, we demonstrate that the slow reduction of immense cyst size as an initial procedure until optimal requirements for final surgical treatment were achieved has proven to be optimal for neurological outcome. Special emphasis has to be taken on the delicate nature of premature newborn babies, and surgical steps have to be thoroughly considered within the interdisciplinary team.

Pentaerythrityl tetranitrate improves the outcome of children born to mothers with compromised uterine perfusion-12-months follow-up and safety data of the double-blind randomized PETN trial.

BACKGROUND: This is a follow-up study to the pentaerythrityl tetranitrate randomized controlled multicenter trial that reports neonatal outcome data of newborns admitted to neonatal intensive care units and outcome data of the offspring at 12 months of age. OBJECTIVE: We present data on adverse events reported during the study to document the safety of pentaerythrityl tetranitrate treatment during pregnancy. To further evaluate the effects of pentaerythrityl tetranitrate on neonatal and long-term outcomes, we present follow up data from of 240 children at 12 months of age, including information on height, weight, head circumference, developmental milestones, and the presence of chronic disease and of 144 newborns admitted to the neonatal intensive care unit during the trial. STUDY DESIGN: The pentaerythrityl tetranitrate trial was a randomized, double-blind, placebo-controlled study designed to assess the efficacy and safety of the nitric oxide-donor pentaerythrityl tetranitrate in the prevention of fetal growth restriction and perinatal death in pregnancies complicated by abnormal placental perfusion. RESULTS: Results at 12 months demonstrated that significantly more children were age appropriately developed without impairments in the pentaerythrityl tetranitrate group (P=.018). In addition, the presence of chronic disease was lower in the pentaerythrityl tetranitrate group (P=.041). Outcome data of the 144 newborns admitted to the neonatal intensive care unit did not reveal differences between the treatment and placebo groups. There were no differences in the number or nature of reported adverse events between the study groups. CONCLUSION: The analysis shows that study children born in the pentaerythrityl tetranitrate cohort have a clear advantage compared with the placebo group at the age of 12 months, as evidenced by the increased incidence of normal development without the presence of chronic disease. Although safety has been proven, further follow-up studies are necessary to justify pentaerythrityl tetranitrate treatment during pregnancies complicated by impaired uterine perfusion.

Effect of pentaerythritol tetranitrate (PETN) on the development of fetal growth restriction in pregnancies with impaired uteroplacental perfusion at midgestation-a randomized trial.

BACKGROUND: Fetal growth restriction is strongly associated with impaired placentation and abnormal uteroplacental blood flow. Nitric oxide donors such as pentaerythritol tetranitrate are strong vasodilators and protect the endothelium. Recently, we demonstrated in a randomized controlled pilot study a 38% relative risk reduction for the development of fetal growth restriction or perinatal death following administration of pentaerythritol tetranitrate to pregnant women at risk, identified by impaired uterine perfusion at midgestation. Results of this monocenter study prompted the hypothesis that pentaerythritol tetranitrate might have an effect in pregnancies with compromised placental function as a secondary prophylaxis. OBJECTIVE: This study aimed to test the hypothesis that the nitric oxide donor pentaerythritol tetranitrate reduces fetal growth restriction and perinatal death in pregnant women with impaired placental perfusion at midgestation in a multicenter trial. STUDY DESIGN: In this multicenter, randomized, double-blind, placebo-controlled trial, 2 parallel groups of pregnant women presenting with a mean uterine artery pulsatility index >95th percentile at 19+0 to 22+6 weeks of gestation were randomized to 50-mg Pentalong or placebo twice daily. Participants were assigned to high- or low-risk groups according to their medical history before randomization was performed block-wise with a fixed block length stratified by center and risk group. The primary efficacy endpoint was the composite outcome of perinatal death or development of fetal growth restriction. Secondary endpoints were neonatal and maternal outcome parameters. RESULTS: Between August 2017 and March 2020, 317 participants were included in the study and 307 were analyzed. The cumulative incidence of the primary outcome was 41.1% in the pentaerythritol tetranitrate group and 45.5% in the placebo group (unadjusted relative risk, 0.90; 95% confidence interval, 0.69-1.17; adjusted relative risk, 0.90; 95% confidence interval, 0.69-1.17; P=.43). Secondary outcomes such as preterm birth (unadjusted relative risk, 0.73; 95% confidence interval, 0.56-0.94; adjusted relative risk, 0.73; 95% confidence interval, 0.56-0.94; P=.01) and pregnancy-induced hypertension (unadjusted relative risk, 0.65; 95% confidence interval, 0.46-0.93; adjusted relative risk, 0.65; 95% confidence interval, 0.46-0.92; P=0.01) were reduced. CONCLUSION: Our study failed to show an impact of pentaerythritol tetranitrate on the development of fetal growth restriction and perinatal death in pregnant women with impaired uterine perfusion at midgestation. Pentaerythritol tetranitrate significantly reduced secondary outcome parameters such as the incidence of preterm birth and pregnancy-induced hypertension in these pregnancies.

Novel Method for Three-Dimensional Facial Expression Recognition Using Self-Normalizing Neural Networks and Mobile Devices.

Introduction To date, most ways to perform facial expression recognition rely on two-dimensional images, advanced approaches with three-dimensional data exist. These however demand stationary apparatuses and thus lack portability and possibilities to scale deployment. As human emotions, intent and even diseases may condense in distinct facial expressions or changes therein, the need for a portable yet capable solution is signified. Due to the superior informative value of three-dimensional data on facial morphology and because certain syndromes find expression in specific facial dysmorphisms, a solution should allow portable acquisition of true three-dimensional facial scans in real time. In this study we present a novel solution for the three-dimensional acquisition of facial geometry data and the recognition of facial expressions from it. The new technology presented here only requires the use of a smartphone or tablet with an integrated TrueDepth camera and enables real-time acquisition of the geometry and its categorization into distinct facial expressions. Material and Methods Our approach consisted of two parts: First, training data was acquired by asking a collective of 226 medical students to adopt defined facial expressions while their current facial morphology was captured by our specially developed app running on iPads, placed in front of the students. In total, the list of the facial expressions to be shown by the participants consisted of "disappointed", "stressed", "happy", "sad" and "surprised". Second, the data were used to train a self-normalizing neural network. A set of all factors describing the current facial expression at a time is referred to as "snapshot". Results In total, over half a million snapshots were recorded in the study. Ultimately, the network achieved an overall accuracy of 80.54% after 400 epochs of training. In test, an overall accuracy of 81.15% was determined. Recall values differed by the category of a snapshot and ranged from 74.79% for "stressed" to 87.61% for "happy". Precision showed similar results, whereas "sad" achieved the lowest value at 77.48% and "surprised" the highest at 86.87%. Conclusions With the present work it can be demonstrated that respectable results can be achieved even when using data sets with some challenges. Through various measures, already incorporated into an optimized version of our app, it is to be expected that the training results can be significantly improved and made more precise in the future. Currently a follow-up study with the new version of our app that encompasses the suggested alterations and adaptions, is being conducted. We aim to build a large and open database of facial scans not only for facial expression recognition but to perform disease recognition and to monitor diseases' treatment progresses.

The femur too short? 1373 fetuses with short femur during second-trimester screening.

PURPOSE: A short fetal femur in prenatal diagnosis might be an indicator for intrauterine growth retardation (IUGR), a genetically determined small child (SGA) with or without associated fetal malformations and/or an adverse fetal outcome. METHODS: 1373 singleton pregnancies with a femoral length < 5th percentile detected between 1999 and 2015 during second-trimester screening in a tertiary prenatal diagnostic center were subjected to a descriptive retrospective analysis with regard to fetal characteristics as well as pregnancy outcome. RESULTS: 685 (49.9%) fetuses presented an isolated short femur, while 688 (50.1%) showed additional abnormalities. 293 (42.6%) of those were SGA babies without any malformation, while 395 (57.4%) had one or more severe anomaly of the following organ systems: 157 (11.5%) cardiovascular, 101 (7.4%) musculoskeletal, 82 (6.0%) urogenital, 72 (5.2%) cerebrocephalic, 50 (3.6%) gastrointestinal, and 5 (0.4%) thoracic. 75 (5.5%) of the fetuses showed chromosomal aberrations of which Trisomy 13, 18 and 21 were found in 2, 13 and 27 of the cases, respectively. Fetuses with associated malformations had a significantly lower live birth rate than those without (64.2% vs. 98.1%, p < 0.001); in addition, a higher rate of preterm births 36.6% vs. 11.3%, p < 0.001) and SGA babies (51.4% vs. 30.4%, p < 0.001) were observed in the first collective. CONCLUSION: Diagnosis of a short fetal femur should lead to an extended organ screening; in the case of associated abnormalities, additional genetic testing has to be offered, as well as intensified pregnancy monitoring in pregnancies at risk for IUGR and/or preterm birth.

Novel and flexible ultrasound simulation with smartphones and tablets in fetal echocardiography.

PURPOSE: Evaluation of a novel ultrasound-simulation-app for training fetal echocardiography as a possible useful addition for students, residents and specialist doctors. Furthermore, comparison to a conventional learning-method with special attention on orientation and recognition of physiological structures. METHODS: Prospective two-arm study with the participation of 226 clinical students. 108 students were given an extract from a textbook on fetal echocardiography (PDF-group, n = 108) for 30 min to study. 118 students were able to use the new ultrasound-simulator-app (Simulator-group, n = 118) to learn for 30 min. The knowledge of the students was examined both before and after the learning-period by having them identify sonographic structures in videos using single-choice selection. RESULTS: There were no significant differences between the two groups regarding age (p = 0.87), gender (p = 0.28), and the number of previously performed ultrasound-examinations (p = 0.45). In the Simulator-group, there was a significantly higher learning effect regarding the proportion of students with an increase of correct answers in the video test examination (p = 0.005). At the end of learning, the students in the Simulator-group needed significantly less time to display the structures in the app's simulation (median initially 10.9 s vs. 6.8 s at the end; p < 0.001). CONCLUSIONS: The novel ultrasound-simulation-app seems to be a useful addition and improvement to ultrasound training. Previous difficulties such as simultaneously having patients, ultrasound-machines, and professors at disposal can thus be avoided. This means that another important step towards remote learning can be taken, which has been proven increasingly essential lately, due to the COVID-19 pandemic.

Prenatally Diagnosed Single Umbilical Artery (SUA) - Retrospective Analysis of 1169 Fetuses.

PURPOSE: The incidence of a fetal single umbilical artery (SUA) is about 0.5 % and has been associated with an increased risk of congenital malformations, fetal aneuploidy and intrauterine growth restriction (IUGR). MATERIALS AND METHODS: A retrospective analysis of 1169 women with singleton pregnancies diagnosed with fetal SUA between 1997 and 2014 in a specialized practice for prenatal diagnostics has been performed. Data was obtained on maternal and fetal findings as well as pregnancy outcome. RESULTS: 989 (84.6 %) fetuses showed an isolated SUA (iSUA) while 180 (15.4 %) presented with SUA and additional structural and/or chromosomal abnormalities. Structural malformations were distributed as follows: 9.0 % cardiovascular, 3.5 % urogenital, 2.9 % musculoskeletal, 3.0 % gastrointestinal and 2.1 % cerebral. 2.1 % of the fetuses had chromosomal aberrations. 50.8 % (49.2 %) of the fetuses were female (male) and right vs. left SUA was found in 64.2 % (35.8 %) of the cases. Fetuses with SUA and additional abnormalities showed lower rates of live births (85.0 % vs. 98.5 %, p < 0.001), a lower median birth weight (2825 g vs. 3220 g, p < 0.001), higher rates of preterm delivery before week 34 + 0 (13.7 % vs. 3.8 %, p < 0.001) and weighed less than the 5th growth percentile in 21.6 % vs. 9.3 % (p < 0.001) of the fetuses with iSUA. In 5.1 % (60) of the children, chromosomal or structural abnormalities were detected post-partum. CONCLUSION: Once fetal SUA is diagnosed, intense sonoanatomy of the fetus is required and, if associated malformations are found, genetic testing must be offered. In iSUA intermittent biometry is recommended for the early detection of IUGR but additional genetic testing is not necessarily recommended.

Foetal Doppler Parameters as a Prognostic Marker Before Induction of Labour.

Introduction The value of foetal Doppler ultrasonography before induction of labour for prognostic assessment of the duration of labour and foetal outcome is presented. Patients and Methods Doppler ultrasound of the foetal middle cerebral artery (MCA) and of the umbilical artery (UA) was performed in addition to evaluation of the Bishop score in 49 women around the expected date of confinement (38 + 0 - 42 + 0 weeks of gestation) prior to planned pharmacological induction of labour. These parameters were studied using non-parametric statistical methods for associations with the duration of induction until delivery, the mode of delivery and foetal outcome. Results The resistance index (RI) of the MCA (rs = 0.547, p < 0.001), but not the RI of the UA (rs = - 0.055, p = 0.707) correlated positively with the duration of induction. Moreover, a negative correlation was found between the RI of the UA and the baby's arterial cord pH at birth (rs = - 0.287, p = 0.046). No differences in the RI of MCA or UA were found between babies born vaginally and those delivered by secondary section. Conclusion The present data show that Doppler measurement of the foetal MCA and UA before pharmacological induction of labour at term can be a further parameter for prognostic estimation of the duration and success of induction and of foetal outcome in addition to the established Bishop score.

Value of endometrial thickness assessed by transvaginal ultrasound for the prediction of endometrial cancer in patients with postmenopausal bleeding.

PURPOSE: Histological confirmation of endometrial cancer by dilatation/curettage (D/C) in women with postmenopausal bleeding (PMB) can be challenging due to anesthesiological and/or surgical risks. Thus, less invasive methods for diagnostics are required to identify patients with minimal risk for endometrial cancer (EC) to avoid unnecessary surgical intervention. The objective of this single-center cohort study was to assess the diagnostic validity of transvaginal ultrasound (TVUS) measurements of endometrial thickness (ET) in patients with PMB for the detection of EC. METHODS: A retrospective analysis of data from patients presenting between January 2005 and August 2014 at the Department of Obstetrics and Gynecology, University Hospital Ulm, Germany, with PMB and subsequent D/C was performed. Complete data with TVUS documentation of ET and histological results of tissue samples were available from 254 patients. In addition, data on age, body mass index (BMI), ASA-score, diabetes, hypertension, and hematological laboratory values (for a smaller subsample) were recorded. To identify independent risk factors, a multivariate logistic regression with endometrial cancer as binary response variable (yes/no) was performed. Diagnostic efficacy data for different ET cutoff points (≤1 to ≤26 mm) were obtained by a receiver operator characteristic (ROC) curve analysis. RESULTS: The multivariate logistic regression revealed a significant independent predictive value for age and ET. However, none of the analyzed ET cutoff points showed optimal diagnostic validity, as all cutoff points with sensitivity rates above 90% (≤1 to ≤5 mm) had false positive rates of 70% and higher. CONCLUSIONS: There is no ET cutoff point that provides good diagnostic accuracy and/or reliably excludes the presence of endometrial cancer in patients with PMB. Thus, our data analysis supports the actual German approach of histological evaluation of any PMB to confirm or exclude EC.

Peripheral Immunological Cells in Pregnant Women and their Change during Diabetes.

During the last decades the incidence of diabetes has dramatically increased as well as the number of pregnant diabetic women. There is still missing data regarding patterns and shifts of immune cell populations due to pregnancy with or without diabetes. The study aimed to investigate the impact of pregnancy, type 1 diabetes (T1D) and gestational diabetes mellitus (GDM) on different immune cells in female. The number and proportion of CD3-, CD4-, CD8- and γδ T-cells as well as B-, NK-, NKT- and dendritic cells (DC) incl. rate of apoptosis was analyzed in peripheral blood samples from 24 non-pregnant women, 24 pregnant controls, 25 non-pregnant T1D, 18 women with GDM and 15 pregnant T1D (PT1D) women. Compared to healthy controls, healthy pregnant women had reduced numbers of lymphoid DC and γδ T-cells, while women with gestational diabetes presented with increased numbers of γδ T-cells. Pregnant women with T1D showed increased NKT cells and a decrease of NK cells compared to healthy pregnant or non-pregnant T1D women. Apoptosis of γδ T-cells in healthy pregnant women was found to be decreased in comparison to their non-pregnant controls while apoptosis of myeloid and lymphoid DC was increased in pregnant T1D in comparison to non-pregnant T1D. Those results may indicate that increased complication rates during diabetic pregnancies might be due to an impaired adaptation of the immune system.

Surgery of Anterior Skull Base Lesions in Children.

INTRODUCTION: Lesions affecting the anterior skull base represent a rare group of craniofacial pathologies. A tumor of the facial midline, meningitis, or rhinoliquorrhea may be indicative of malformations like dermoid cysts, gliomas, encephaloceles, or nasal fistulas. METHODS: We present a case series of 13 children (4 months to 12 years; 8 males, 5 females) with lesions involving the anterior skull base, which were treated surgically in an interdisciplinary setting. This case series includes cases of nasal fistulae (n = 5), nasal cysts (n = 5), aneurysmal bone cyst, nasal glioma, and meningoencephalocele (n = 1). RESULTS: All lesions were resected with a transnasal, transcutaneous, and/or transcranial approach with reconstruction of the anterior skull base if intracranial/intradural extension was detected. In 5 cases, a dura leakage was visible, which was sealed via Onlay-technique in 3 cases, whereas in 2 cases involving a greater dural defect, the GAP-CAS technique was performed. No complications occurred, and no recurrence was visible in a long-term follow-up. An algorithm for a systematic approach to these various pathologies is provided. CONCLUSION: Congenital pathologies of the anterior skull base are rare, challenging to diagnose, and present as clinical emergencies. An interdisciplinary surgical approach is needed for best functional and aesthetic results.

A possible ambivalent role for relaxin in human myometrial and decidual cells in vitro.

PURPOSE: Based on the reported tocolytic action of the hormone relaxin (RLX) in rodents, locally produced in reproductive tissues and the corpus luteum in mammals, the present study aimed to evaluate the influence of RLX on contraction-mediating cyclooxygenases-1 and -2 (COX) and the contractile prostaglandin PGE(2) in human myometrial and decidual cells. Primary cultured cells were obtained from uteri and placentas of term and preterm women undergoing elective caesarean section. METHODS: In vitro culture of primary myometrial and decidual cells, immunocytochemistry, reverse transcription and real-time PCR, Western blot, ELISA. RESULTS: We demonstrate for the first time an activating effect of RLX for human COX-1 and COX-2 in primary myometrial and decidual cells in vitro. CONCLUSIONS: These effects might potentially contribute to birth-associated induction of contractions in vivo.

A novel mechanism for anti-EGFR antibody action involves chemokine-mediated leukocyte infiltration.

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are currently used for therapy of recurrent or metastatic disease; however, their mode of action is not completely understood. To investigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leukocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab')2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltration, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemokines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immunospot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo.

Alterations in the p53 pathway and their association with radio- and chemosensitivity in head and neck squamous cell carcinoma.

Chemotherapy and/or radiotherapy are established measures in treatment protocols of head and neck squamous cell carcinoma (HNSCC). However, we still lack reliable predictive markers for the response to radio- and chemotherapy. The p53 pathway is involved in stress response and thus might influence chemo-/radiosensitivity. Using 29 HNSCC cell lines previously characterized for p53 mutations, we simultaneously analyzed several key players in the p53 pathway by RT-PCR, transcript sequencing and immunohistochemistry, and investigated their association with chemosensitivity and radiosensitivity. Cell lines with p53 mutations were slightly more sensitive to cisplatin than those with wild-type p53. The type of mutation did not influence radio- or chemosensitivity. p14(ARF), an activator of p53, was lost or mutated in all cell lines. Three cell lines showed overexpression of HDM-2, a major negative regulator of p53; however, HDM-2 levels did not correlate with radio- or chemosensitivity. HPV-16 oncoproteins were detected in one highly chemoresistant cell line. Our findings suggest that molecular events resulting in the inactivation of the p53 pathway occur in all HNSCC cell lines. However, single alterations in the p53 pathway are not reliable predictors for the response to radio- or chemotherapy in HNSCC.

The effect of relaxin on the oxytocin receptor in human uterine smooth muscle cells.

EXPERIMENTAL OBJECTIVES: Activation of the oxytocin receptor (OTR) induces phospholipase C induced PIP(2) turnover in the human uterus. Relaxin (RLX), a polypeptide hormone produced in the corpus luteum of pregnancy as well as in the placenta and decidua inhibits PIP(2) turnover and subsequent signaling in human myometrium. The purpose of this study was to evaluate a possible effect of RLX on OTR regulation in human uterine smooth muscle cells. Primary cultures of myometrium from term pregnant women undergoing elective caesarean section were incubated for different time periods (0-96 h) and with different concentrations of RLX [10 pg/ml-20 microg/ml]. The effects on OTR binding, mRNA and protein expression were evaluated by means of (125)I-OVT binding assay, RT-PCR and flow cytometry. RESULTS: Prolonged RLX incubation was able to inhibit 30-40% of OTR binding while binding affinity remained unchanged. Oxytocin receptor mRNA and protein expression were down regulated by RLX about 50% and 35% respectively. CONCLUSION: We report for the first time an effect of RLX on OTR regulation in human uterine myometrial cells. The above results indicate that high local uterine RLX concentrations may be involved in uterine quiescence during human pregnancy by down regulating the OTR.

Gamma-delta T-cells in patients with squamous cell carcinoma of the head and neck.

In our attempt to characterize a general immune-suppression found in patients with squamous cell carcinoma of the head and neck (SCCHN) we now focused on a subset of CD3 lymphocytes described as gamma/delta-T-cells, a cell type with potential relevance in non-MHC restricted anti-tumor immune responses. Peripheral blood of 33 SCCHN patients and 33 age-matched controls (CON) was evaluated for the frequency of gamma/delta-T-cells among CD3+ T-cells and their onset of apoptosis (Annexin V binding) by multicolor flow cytometry. Results were correlated with clinical parameters. Patients with SCCHN had a significantly higher proportion of gamma/delta-T-cells compared to healthy controls (4.4+/-0.4% for SCCHN vs. 3.0+/-0.3% for CON, p=0.01). However, this increase was not paralleled with a difference in the onset of apoptosis if compared to CON. There was also no correlation between the proportion of gamma/delta-T-cells and tumor stage. However, a significantly higher proportion of gamma/delta-T-cells was found in patients with recurrent or metachronous second primary SCCHN (6.0+/-1.0%) if compared to the other SCCHN (3.8+/-0.4%, p=0.02). In a follow up 3-6 months post-treatment patients showed a decrease of gamma/delta-T-cells among CD3+cells (2.7+/-0.4%, n=4) if they were operated only and an increase if primary radio-chemotherapy (6.7+/-1.7%, n=8) or a combination of operation plus radio-chemotherapy (6.8+/-2.3%, n=3) was applied. Furthermore, patients receiving palliative treatment including radio-chemotherapy had highest values of gamma/delta-T-cells (9.1+/-2.7%, n=4) overall implicating that the treatment modality significantly influences the proportion of gamma/delta-T-cells. Since patients with SCCHN, particularly those with recurrent or second primary disease after treatment, had a higher proportion of gamma/delta-T-cells without signs of a reduced onset of apoptosis this could be due to an increased de novo generation. The current study implies that increased frequencies of gamma/delta-T-cells in patients with SCCHN may not only be the result of tumor-host interactions but the consequence of applied treatment modalities.

T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx.

Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)-A2.1 restricted HPV16 E7 protein-derived epitopes, tetramer analysis was performed using peripheral blood lymphocytes of 20 HLA-A2.1+ patients and 20 HLA-A2.1+ healthy individuals. Tetramers specific for 3 HPV16 peptides (E711-20, E782-90 and E786-93), an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV-specific T-cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was performed to demonstrate proliferation and antitumor activity of the HPV-responsive T cells. Frequencies of CD8+ T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p<0.05) of T cells specific for the E711-20 epitope compared to those with tumors negative for both markers. HPV16 E711-20 and HPV16 E786-93 specific T cells were expandable upon IVS with cognate peptide-pulsed DC and were reactive against peptide-pulsed targets or, in case of the E711-20 epitope-specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16+ SCCO, precursor T cells specific for E711-20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recognize in vitro HPV16 E7+ tumor cells. Further studies have to elucidate why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV-based vaccine in patients with SCCO.

Effects of tamoxifen on human squamous cell carcinoma lines of the head and neck.

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

Frequencies of tetramer+ T cells specific for the wild-type sequence p53(264-272) peptide in the circulation of patients with head and neck cancer.

Immunization with wild-type sequence (wt) p53 epitopes represents a novel therapeutic strategy for cancer patients with tumors accumulating mutant p53. To evaluate usefulness of p53-derived peptides as future cancer vaccines, frequencies of wt p53(264-272) peptide-specific CD8+ T cells were determined in the peripheral circulation of patients with squamous cell carcinoma of the head and neck (SCCHN). T cells of 30 HLA-A2.1+ patients and 31 HLA-A2.1+ healthy individuals were evaluated by multicolor flow cytometry analysis using peptide-HLA-A2.1 complexes (tetramers). T cells specific for an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were studied in parallel. Patients with SCCHN had a significantly higher mean frequency of CD8+ T cells specific for wt p53(264-272) than normal donors (P = 0.0041). Surprisingly, the frequency of epitope-specific T cells in the circulation of patients did not correlate with p53 accumulation in the tumor. In patients whose tumors had normal p53 expression or had p53 gene mutations preventing presentation of this epitope, high frequencies of wt p53(264-272)-specific CD8+ T cells were found, of which many were memory T cells. In contrast, patients whose tumors accumulated p53 had low frequencies of wt p53(264-272)-specific CD8+ T cells, which predominantly had a naive phenotype and were unable to proliferate ex vivo in response to the epitope, as reported by us previously (T. K. Hoffmann, J. Immunol., 165: 5938-5944, 2000). This seemingly contradictory relationship between the high frequency of epitope-specific T cells and wt p53 expression in the tumor suggests that other factors may contribute to the observed anti-p53 responses. Human papillomavirus-16 E6/E7 expression is common in SCCHN, and E6 is known to promote presentation of wt p53 epitopes. Although human papillomavirus-16 E6/E7 expression was detected in 46% of the tumors, it did not correlate with the frequency of wt p53(264-272)-specific CD8+ T cells or with p53 expression in the tumor. These findings emphasize the complexity of interactions between the tumor and the host immune system, and, thus, have particularly important implications for future p53-based immunization strategies.