Comprehensive cancer predisposition testing within the prospective MASTER trial identifies hereditary cancer patients and supports treatment decisions for rare cancers.

BACKGROUND: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers. PATIENTS AND METHODS: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies. RESULTS: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation. CONCLUSIONS: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research.

Key roles for GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2, prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Clinical validation of the "in silico" prediction of immunogenicity of a human recombinant therapeutic protein.

Antibodies elicited by protein therapeutics can cause serious side effects in humans. We studied immunogenicity of a recombinant fusion protein (FPX) consisting of two identical, biologically active, peptides attached to human Fc fragment. EpiMatrix, an in silico epitope-mapping tool, predicted promiscuous T-cell epitope(s) within the 14-amino-acid carboxy-terminal region of the peptide portion of FPX. On administration of FPX in 76 healthy human subjects, 37% developed antibodies after a single injection. A memory T-cell response against the above carboxy-terminus of the peptide was observed in antibody-positive but not in antibody-negative subjects. Promiscuity of the predicted T-cell epitope(s) was confirmed by representation of all common HLA alleles in antibody-positive subjects. As predicted by EpiMatrix, HLA haplotype DRB1*0701/1501 was associated with the highest T-cell and antibody response. In conclusion, in silico prediction can be successfully used to identify Class II restricted T-cell epitopes within therapeutic proteins and predict immunogenicity thereof in humans.

Downregulation of iNOS expression in rat mesangial cells by special extracts of Harpagophytum procumbens derives from harpagoside-dependent and independent effects.

Special extracts from the roots of Harpagophytum procumbens (Devil's Claw) are used in the supportive treatment of inflammatory diseases, and the iridoid derivative harpagoside is thought to be the active principle. To investigate, whether Harpagophytum extracts may also be useful therapeutics in the treatment of inflammatory kidney diseases, we studied the effects of two different extracts containing 8.9% (extract 1) and 27% harpagoside (extract 2), respectively, on IL-1beta-induced nitric oxide (NO) formation as well as transcriptional regulation of inducible NO synthase (iNOS) in rat renal mesangial cells. We observed a concentration-dependent suppression of nitrite formation by about 80%, which was due to an inhibition of iNOS expression. Moreover, a reduction of iNOS promoter activity and nuclear NF-kappaB translocation was observed, indicating that the extracts interfere with the transcriptional activation of iNOS. Three further Harpagophytum extracts containing about 2% harpagoside did not inhibit NO formation suggesting, that only extracts with a high harpagoside content elicit iNOS inhibition. However, pure harpagoside was only inhibitory at concentrations between 0.3 and 1 mg/ml, which is much higher than the harpagoside content present in an effective concentration of the total extracts. Moreover, a harpagoside-free extract 1 also markedly inhibited iNOS expression, indicating that other extract constituents are involved in this effect. Extract 1 exerted a strong antioxidative effect, whereas no such effect could be demonstrated for harpagoside. Together, these data show that special Harpagophytum extracts may represent potential antiinflammatory drugs in the treatment of glomerular inflammatory diseases.

Why do patients seek treatment in hospitals of complementary medicine?

OBJECTIVE: This exploratory study evaluated patients' reasons for entering a complementary (alternative) medicine hospital by ranking 15 medical and psychosocial factors that were thought to influence this choice. SUBJECTS AND OUTCOME MEASURES: Two hundred patients (200) from two complementary hospitals, one focusing on Traditional Chinese Medicine and one on the Western type of complementary medicine, completed an extensive questionnaire at the beginning of their inpatient treatment. The questionnaire covered personal background; disease parameters; attitude towards conventional medicine; previous experience with, and knowledge of, complementary therapies; expectations concerning the forthcoming treatment; health-related habits; personality traits; and social support. RESULTS: Optimistic attitudes towards treatment and a positive appraisal of alternative doctors were frequently stated reasons (80%), as was the disease severity (long duration: 86%; acute progression or imminent surgery: 70%). Previous successes with complementary therapies, however, ranked relatively low (53%). Negative opinions concerning conventional therapies and conventional doctors' treatments were mentioned by 68% of the patients. Many patients felt themselves to be under considerable psychologic stress (74%). A majority (73%) was well informed about complementary therapies, and 65% were curious about the forthcoming therapies. Sixty-eight percent (68%) indicated good health behaviors. Fewer patients mentioned contemplative and/or religious attitudes (44%) or lack of social support (25%). Age primarily accounted for variations in the ranking weights of the two subgroups. The specific type of complementary medicine was of minor influence. In 14 out of 21 personality dimensions, the current patient group showed significant deviations from the healthy reference, which is in good agreement with findings from conventionally treated patients.

The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

[O2 utilization during hyperthermic extremity perfusion with rhTNF alpha and melphalan]. / O2-Utilisation während und nach hyperthermer Extremitätenperfusion mit rhTNF alpha und Melphalan.

During isolated limb perfusion (ILP) severe metabolic impairment with a subsequent alteration in oxygen consumption can be observed. The mechanisms responsible for this may be extracorporeal circulation, hyperthermia, and application of cytostatic drugs and cytokines. Thirty-three patients underwent ILP with rhTNF alpha and melphalan for melanoma or soft-tissue sarcoma. Cardiopulmonary monitoring consisted of arterial and mixed venous blood-gas analysis and a Swan-Ganz catheter was inserted after induction of general anesthesia prior to any surgical intervention. Arterial (SaO2) and mixed venous (SvO2) oxygen saturation, serum lactate and end-expiratory CO2 concentration were determined peri- and postoperatively for 72 h. Oxygen supply and consumption rates were measured systemically (DO2I, VO2I) and in the extracorporeal circuit ('DO2I, 'VO2I). For statistical analysis we used the t-test. During extracorporal circulation an increase of DO2I and VO2I was observed. A slight increase of lactate values began during the wash-out phase. Immediately after reperfusion. DO2I, VO2I and lactate increased significantly with normalization until the 2nd postoperative day. SaO2 and SvO2 remained unchanged. A significant correlation between regional toxicity and the postoperative maximum of serum lactate values was found. The increase of DO2I and VO2I in the tissues during ILP and after reperfusion was achieved by a significant increase in cardiac output while the oxygen extraction rate was not altered. Elevation of lactate values after reperfusion and the increase in oxygen utilization might be due to oxygen depletion in the perfused limb. This could contribute to the development of lactacidosis or rhabdomyolysis. Therefore, to minimize toxicity it seems to be mandatory to measure adequate tissue oxygen supply during ILP.

[Advanced age--a risk factor in autologous blood donation?]. / Fortgeschrittenes Lebensalter--ein Risiko zur Eigenblutspende?

Tolerance of autologous blood donation was investigated in a patient group aged between 66 and 75 years (median = 70 years). Autologous blood donors between 18 and 65 years (median = 51 years) served as a control. A total of 38 patients were examined. Only blood donors which did not exceed ASA criteria II were accepted. Blood donation was performed weekly with a daily ferrum intake of 200 mg. Patients were divided into two groups with and without volume replacement, respectively. The parameters investigated were: blood pressure and pulse before and after the first and second autologous blood donation, circulatory response during 24 h after blood donation, and hemoglobin concentration before the first, second, and third donation. Regarding pulse and blood pressure, there was no statistical difference between the elder and younger patient group. Hemoglobin reduction from the first to the second donation was 1.1 g/dl in both groups and from the second to the third donation 1.1 g/dl in the younger group and 1.2 g/dl in the elder patient group. Again, no significant difference between both groups could be shown. None of the 38 patients showed negative side effects regarding the circulatory response during a time period of 24 h after blood donation.

Retrograde transport of nerve growth factor from hippocampus and amygdala to trkA messenger RNA expressing neurons in paraventricular and reuniens nuclei of the thalamus.

We previously reported the presence of trkA messenger RNA expressing non-cholinergic neurons in the paraventricular anterior and reuniens nuclei, which are located in the thalamic midline. In the present study, retrograde labeling with iodinated (125I) nerve growth factor was used to identify the innervation target of these cells. Neurons in the paraventricular anterior and reuniens nuclei were labeled following injection of iodinated nerve growth factor into amygdala and hippocampus, but not into nucleus accumbens and entorhinal cortex, the two other main areas receiving strong innervation from the thalamic midline. Target ablation of hippocampus or amygdala failed to down-regulate trkA messenger RNA expression in the two thalamic nuclei, thus suggesting a role for nerve growth factor different from a critical survival factor. The thalamic paraventricular anterior and reuniens nuclei are part of the reticular formation which plays a role in general cortical activation, behavioral arousal and control of awareness. Retrograde transport of nerve growth factor by trkA messenger RNA expressing neurons in these nuclei suggests a physiological role of this trophic factor in the function of these cells.

Thoracotomy in adult dairy cattle: 14 cases (1979-1991).

Fourteen cows were subjected to thoracotomy as an aid in the treatment of either septic pericarditis (n = 7) or unilateral pleuritis (fibrous or purulent; n = 7). Thoracic lesions were primary in 4 cows, secondary to traumatic reticuloperitonitis in 9 cows, and secondary to extension of a liver abscess in 1 cow. Thoracotomy was performed on 9 cows under local anesthesia. Surgery was performed on 5 cows under general anesthesia; 2 died during anesthesia, and 2 others were euthanatized. Of the 10 cows allowed to recover from surgery, 4 had pericarditis and 6 had pleuritis. Four cows with pleuritis had thoracic abscesses. All but 1 cow with pericarditis died or were euthanatized, and 5 of the 6 cows with pleuritis were discharged from the hospital. A year after surgery, 1 cow was culled because of infertility, and the other 5 cows were returned to production.

Serum trace element levels in HIV-infected subjects.

The absolute number of T4 cells and the serum concentrations of Ca, Cu, Fe, K, Mg, P, Se, and Zn were determined in 59 Walter-Reed staged, HIV-infected men, compared to healthy controls, serum levels of Ca, Cu, and Fe were significantly higher, those of P and Se significantly lower in the HIV-infected subjects. In the HIV-infected cases, but not in the controls, the concentrations of Se and Zn, of Ca with Cu and Fe, and of Fe with P, were directly correlated. In the controls, the correlation between the levels of K and Mg was direct, and inverse between those of Zn and P. Trace element levels did not significantly correlate with WR-stage. However, the absolute number of T4 cells was directly correlated with the serum Mg concentration.

[Trace element concentrations in HIV infected patients]. / Spurenelementkonzentrationen bei HIV-Infizierten.

The serum concentrations of Ca, Cu, Fe, K, Mg, P, Se and Zn and the absolute number of T4 cells were determined in 59 HIV-infected men. Serum levels of Ca, Cu and Fe were significantly higher, those of P and Se significantly lower in the infected, compared to healthy controls. In the HIV-infected cases the concentrations of Se and Zn, of Ca with Cu and Fe, and of Fe with P, were directly correlated. In the controls, the correlation between the levels of K and Mg was direct, and inverse between those of Zn and P. WR-stage and trace element levels did not correlate. The absolute number of T4-lymphocytes was directly correlated with the serum Mg concentration.

Carbon monoxide diffusing capacity of the lungs determined by single-breath and steady-state exercise methods.

We measured carbon monoxide diffusing capacity of the lungs (DL,CO) by both the resting single-breath (SB) and steady-state (SS) exercise methods in 95 patients referred for pulmonary function testing. A 10-second breath-holding method was used for the SB test. DL,CO (SS) was measured during the last minute of a 3-minute exercise test on a 9-inch step. Results of the two methods showed good agreement, the SB-SS difference averaging -0.70 (SD, 3.39) ml/min per mm Hg. The difference between the two methods was not correlated with other measurements of pulmonary function except minute ventilation during the exercise performed in the DL,CO (SS) procedure. In a separate study of laboratory personnel, the day-to-day variabilities of the two tests were similar (SD, 1.4 ml/min per mm Hg). Alveolar volume obtained by helium dilution during the SB test was comparable to total lung capacity (TLC) estimated by multiple-breath nitrogen washout in patients without severe airway obstruction. In severe airway obstruction, the mean SB alveolar volume was 13.8% less than the TLC by nitrogen washout, a difference that may be useful as an indicator of inefficiency of gas mixing in the lungs. We conclude that the SB and SS exercise methods provide similar estimates of DL,CO in patients referred to a pulmonary function laboratory.

Actions of enflurane, isoflurane, vecuronium, atracurium, and pancuronium on pulmonary resistance in dogs.

The effects of enflurane, isoflurane, vecuronium, atracurium, and pancuronium on pulmonary resistance and heart rate were studied in 30 vagotomized dogs lying supine and anesthetized with chloralose-urethane. None of the five drugs affected pulmonary resistance when the airway was unstimulated. Enflurane and isoflurane significantly attenuated the increase in pulmonary resistance induced by electrical stimulation of the vagus nerves. This effect was dose-dependent and similar for both anesthetics at equivalent multiples of their minimum alveolar concentration. Atracurium significantly (P less than 0.05) enhanced the increase in pulmonary resistance induced by vagus nerve stimulation; vecuronium had no significant effect. Pancuronium, up to a cumulative dose of 0.14 mg/kg, also significantly (P less than 0.05) enhanced the increase in pulmonary resistance induced by vagus nerve stimulation; but this effect was reversed by further increasing the dose. Pancuronium also attenuated the cardiodecelerator response to vagus nerve stimulation in a dose-dependent fashion. The underlying mechanisms for the attenuation of responses to vagus nerve stimulation by enflurane or isoflurane or for the increase in response with atracurium are unknown. Pancuronium at lower doses increases the response most likely by blocking prejunctional muscarinic receptors (M2) that physiologically inhibit vagally mediated increases in pulmonary resistance.