Short-course subcutaneous treatment with PQ Grass strongly improves symptom and medication scores in grass allergy.

BACKGROUND: A modified grass allergen subcutaneous immunotherapy (SCIT) product with MicroCrystalline Tyrosine and monophosphoryl lipid-A as an adjuvant system (Grass MATA MPL [PQ Grass]) is being developed as short-course treatment of grass-pollen allergic rhinitis (SAR) and/or rhinoconjunctivitis. We sought to evaluate the combined symptom and medication score (CSMS) of the optimized cumulative dose of 27,600 standardized units (SU) PQ Grass in a field setting prior to embarking on a pivotal Phase III trial. METHODS: In this exploratory, randomized, double-blind, placebo-controlled trial subjects were enrolled across 14 sites (Germany and the United States of America). Six pre-seasonal subcutaneous injections of PQ Grass (using conventional or extended regimens) or placebo were administered to 119 subjects (aged 18-65 years) with moderate-to-severe SAR with or without asthma that was well-controlled. The primary efficacy endpoint was CSMS during peak grass pollen season (GPS). Secondary endpoints included Rhinoconjunctivitis Quality of Life Questionnaire standardized (RQLQ-S) and allergen-specific IgG4 response. RESULTS: The mean CSMS compared to placebo was 33.1% (p = .0325) and 39.5% (p = .0112) for the conventional and extended regimens, respectively. An increase in IgG4 was shown for both regimens (p < .01) as well as an improvement in total RQLQ-S for the extended regimen (mean change -0.72, p = .02). Both regimens were well-tolerated. CONCLUSIONS: This trial demonstrated a clinically relevant and statistically significant efficacy response to PQ Grass. Unprecedented effect sizes were reached for grass allergy of up to ≈40% compared to placebo for CSMS after only six PQ Grass injections. Both PQ Grass regimens were considered equally safe and well-tolerated. Based on enhanced efficacy profile extended regime will be progressed to the pivotal Phase III trial.

Quantitative proteomic analysis of formalin-fixed, paraffin-embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. RESULTS: In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non-malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins. CONCLUSION: Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC.

[Update on Barrett esophagus and Barrett carcinoma]. / Update zum Barrett-Ösophagus und Barrett-Karzinom.

The definition of Barrett esophagus is currently under discussion. It is now suggested that a distal esophagus coated with cylinder epithelium with cardia-fundus mucosa should also be classified as Barrett esophagus because the risk of cancer is significantly increased even without histological evidence of intestinal metaplasia with goblet cells. The results of recent epidemiological investigations imply that the cancer risk of cylinder cell metaplasia and low grade intraepithelial neoplasia in Barrett esophagus has previously been overestimated. The histological detection of dysplasia still remains the best biomarker for estimation of the risk of cancer of Barrett esophagus. Exact determination of invasion depth in the mucosa, respective submucosa is now established as prognostic marker for overall survival in Patients with early carcinomas and this classification is useful for therapy decisions (endoscopic versus surgical removal). In advanced Barrett carcinoma following neoadjuvant therapy the lymph node status (ypN) is a better prognostic factor than the ypT category. In metastasized tumors therapies targeting HER2/new, EGFR or c-Met have been investigated explicitly in Barrett carcinoma only in phase I/II studies, whereby the predictive value of appropriate molecular pathology investigations is not yet reliably established.

[Intraepithelial neoplasia of Barrett's esophagus: prognosis of potential malignancy]. / Intraepitheliale Neoplasie des Barrett-Ösophagus: Vorhersage des malignen Potenzials.

Adenocarcinomas of the distal esophagus mainly develop from intestinal metaplasia (Barrett's esophagus) through intermediate steps of low-grade and high-grade intraepithelial neoplasia. Histopathological examination of endoscopic biopsies constitutes the gold standard for estimating the cancer risk of a patient with Barrett's esophagus. Several prospective biomarker phase IV studies have demonstrated the predictive value of e.g. allelic loss of TP53, tetraploidy and aneuploidy as well as cyclin D1 expression. Among the relevant biomarkers from retrospective phase III studies are polysomy and specific DNA gains and losses, markers of proliferation (Mib-1) and methylation markers. As there are conflicting results in the literature and these analyses are costly, their use in routine patient care cannot yet be recommended. However, immunostaining for several markers may assist in the classification of intraepithelial neoplasia in individual difficult cases.

CpG-island methylation of the ER promoter in colorectal cancer: analysis of micrometastases in lymph nodes from UICC stage I and II patients.

Patients with UICC stage II colorectal cancer (CRC) have a risk of approximately 20% to develop disease recurrence after tumour resection. The presence and significance of micrometastases for locoregional recurrence in these patients lacking histopathological lymph node involvement on routine stained HE sections is undefined. Oestrogen receptor (ER) promoter methylation has earlier been identified in CRC. Therefore, we evaluated the methylation status of the ER promoter in lymph nodes from 49 patients with CRC UICC stage I and II as a molecular marker of micrometastases and predictor of local recurrence. DNA from 574 paraffin-embedded lymph nodes was isolated and treated with bisulphite. For the detection of methylated ER promoter sequences, quantitative real-time methylation-specific PCR was used. Of the 49 patients tested, 15 (31%) had ER methylation-positive lymph nodes. Thirteen of those (86%) remained disease free and two (14%) developed local recurrence. In the resected lymph nodes of 34 of the 49 patients (69%), no ER promoter methylation could be detected and none of these patients experienced a local relapse. The methylation status of the ER promoter in lymph nodes of UICC stage I and II CRC patients may be a useful marker for the identification of patients at a high risk for local recurrence.

Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes.

BACKGROUND: The aim of this study was to investigate the prognostic effect of tumour-infiltrating lymphocytes (TILs) in serous stage III ovarian carcinoma to determine TIL clonality and to correlate this to Her2/neu expression. METHODS: Formalin-fixed and paraffin-embedded ovarian carcinomas were examined for CD20-, CD3-, CD4- and CD8-positive lymphocytes (n=100), and for Her2/neu-positive tumour cells (n=55/100) by immunohistochemistry. Clonality analysis was carried out by T-cell receptor gamma (TCRgamma) gene rearrangements (n=93/100). Statistical analyses included experimental and clinico-pathological variables, as well as disease-free (DFS) and overall (OS) survival. RESULTS: CD20-positive B lymphocytes were present in 57.7% (stromal)/33.0% (intraepithelial) and CD3-positive T lymphocytes in 99.0% (stromal)/90.2% (intraepithelial) of ovarian carcinomas. Intraepithelial CD3-positive T lymphocytes were correlated with improved DFS in optimally debulked patients (P=0.0402). Intraepithelial CD8-positive T lymphocytes were correlated with improved OS in all optimally debulked patients (P=0.0201) and in those undergoing paclitaxel/carboplatin therapy (P=0.0092). Finally, rarified and clonal TCRgamma gene rearrangements were detected in 37 out of 93 (39.8%) and 15 out of 93 (16.1%) cases, respectively. This was marginally associated with improved DFS (P=0.0873). Despite a significant correlation of HER2/neu status and intraepithelial CD8-positive lymphocytes (P=0.0264), this was non-directional (R=-0.257; P=0.0626). CONCLUSION: Improved survival of ovarian cancer patients is related to the infiltration, clonal selection and intraepithelial persistence of T lymphocytes.

Strong dose response after immunotherapy with PQ grass using conjunctival provocation testing.

BACKGROUND: Pollinex Quattro Grass (PQ Grass) is an effective, well-tolerated, short pre-seasonal subcutaneous immunotherapy to treat seasonal allergic rhinoconjunctivitis (SAR) due to grass pollen. In this Phase II study, 4 cumulative doses of PQ Grass and placebo were evaluated to determine its optimal cumulative dose. METHODS: Patients with grass pollen-induced SAR were randomised to either a cumulative dose of PQ Grass (5100, 14400, 27600 and 35600 SU) or placebo, administered as 6 weekly subcutaneous injections over 31-41 days (EudraCT number 2017-000333-31). Standardized conjunctival provocation tests (CPT) using grass pollen allergen extract were performed at screening, baseline and post-treatment to determine the total symptom score (TSS) assessed approximately 4 weeks after dosing. Three models were pre-defined (Emax, logistic, and linear in log-dose model) to evaluate a dose response relationship. RESULTS: In total, 95.5% of the 447 randomized patients received all 6 injections. A highly statistically significant (p < 0.0001), monotonic dose response was observed for all three pre-specified models. All treatment groups showed a statistically significant decrease from baseline in TSS compared to placebo, with the largest decrease observed after 27600 SU (p < 0.0001). The full course of 6 injections was completed by 95.5% of patients. Treatment-emergent adverse events were similar across PQ Grass groups, and mostly mild and transient in nature. CONCLUSIONS: PQ Grass demonstrated a strong curvilinear dose response in TSS following CPT without compromising its safety profile.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Targeted delivery of an ADP-ribosylating bacterial toxin into cancer cells.

The actin cytoskeleton is an attractive target for bacterial toxins. The ADP-ribosyltransferase TccC3 from the insect bacterial pathogen Photorhabdus luminescence modifies actin to force its aggregation. We intended to transport the catalytic part of this toxin preferentially into cancer cells using a toxin transporter (Protective antigen, PA) which was redirected to Epidermal Growth Factor Receptors (EGFR) or to human EGF receptors 2 (HER2), which are overexpressed in several cancer cells. Protective antigen of anthrax toxin forms a pore through which the two catalytic parts (lethal factor and edema factor) or other proteins can be transported into mammalian cells. Here, we used PA as a double mutant (N682A, D683A; mPA) which cannot bind to the two natural anthrax receptors. Each mutated monomer is fused either to EGF or to an affibody directed against the human EGF receptor 2 (HER2). We established a cellular model system composed of two cell lines representing HER2 overexpressing esophageal adenocarcinomas (EACs) and EGFR overexpressing esophageal squamous cell carcinomas (ESCCs). We studied the specificity and efficiency of the re-directed anthrax pore for transport of TccC3 toxin and established Photorhabdus luminescence TccC3 as a toxin suitable for the development of a targeted toxin selectively killing cancer cells.

Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations.

Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) ß, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFß. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAPα.

Role of S-100beta as potential autoantigen in an autoimmune disease of the inner ear.

In Lewis rats, adoptive transfer of T cells specific for the calcium-binding protein S-100beta mediates experimental autoimmune encephalomyelitis, but surprisingly also induced a marked labyrinthitis associated with impairment of hearing. This suggests that transfer of S-100beta-specific T cells into susceptible animals could be a novel model to study autoimmunity of inner ear diseases. The investigation demonstrated in detail an inner ear involving, central nervous system (CNS)-specific autoimmune disease in order to identify a putatively shared autoantigen(s) in these pathologies. In fact, the model will be a useful tool to investigate in detail, the pathological mechanisms of the human inner ear disease associated with Vogt-Koyanagi-Harada disease.

Radioprotection of salivary glands by amifostine in high-dose radioiodine therapy.

UNLABELLED: Salivary gland impairment after high-dose radioiodine treatment is well recognized. Because differentiated thyroid cancer has a good prognosis, reduction of long-term side effects is important. This study investigated the radioprotective effects of amifostine in animals and humans receiving high-dose radioiodine therapy. METHODS: Quantitative salivary gland scintigraphy was performed in five rabbits before and up to 3 mo after high-dose radioiodine therapy applying 1 GBq 131I. Three animals received 200 mg/kg amifostine before high-dose radioiodine therapy, and two served as controls. All animals were examined histopathologically. Quantitative salivary gland scintigraphy also was performed in 17 patients with differentiated thyroid cancer before and 3 mo after high-dose radioiodine therapy with 6 GBq 131I. Eight patients were treated with 500 mg/m2 amifostine before high-dose radioiodine therapy, and nine served as controls. RESULTS: In two control rabbits, high-dose radioiodine therapy significantly reduced parenchymal function by 63% and 46% in parotid and submandibular glands, respectively. In contrast, there was no significant decrease in parenchymal function in amifostine-treated animals. Histopathologically, lipomatosis was observed in control animals but was negligible in amifostine-treated animals. Similar findings were observed in differentiated thyroid cancer patients. In nine control patients, high-dose radioiodine therapy significantly (p < 0.01) reduced parenchymal function by 37% and 31% in parotid and submandibular glands, respectively. Three patients exhibited Grade I (World Health Organization) xerostomia. In contrast, there was no significant decrease in parenchymal function in amifostine-treated patients and no incidence of xerostomia. CONCLUSION: Parenchymal damage in salivary glands induced by high-dose radioiodine therapy can be reduced significantly by amifostine. This may increase the quality of life of patients with differentiated thyroid cancer.

[Quantitative salivary gland scintigraphy--a recommended examination prior to and after radioiodine therapy]. / Die quantitative Sialoszintigraphie--eine sinnvolle Untersuchung im Vorfeld und in der Nachsorge der Radiojodtherapie.

AIM: The aim of this study was to evaluate possible deterioration of salivary gland function due to radioiodine therapy with low activities using standardized quantitative salivary gland scintigraphy (qSZ). In addition, the prevalence of sialopathies was estimated in patients with thyroid dysfunction. METHODS: Prior to routine thyroid scintigraphy qSZ was performed after i.v. injection of 36-126 MBq 99mTc-pertechnetate, and both uptake and excretion fraction were calculated as a measure of parenchymal function and saliva excretion, respectively 312 healthy patients served as reference for a normal data base. 144 patients underwent qSZ prior to and 3 months after radioiodine therapy. Results of qSZ in another 674 thyroid patients were evaluated for determining the prevalence of salivary gland dysfunction. RESULTS: Normal uptake was 0.45 +/- 0.14% and 0.39 +/- 0.12%, and normal excretion fraction amounted to 49.5 +/- 10.6% and 39.1 +/- 9.2% in parotid and submandibular glands, respectively. Despite salivary gland stimulation with ascorbic acid during radioiodine therapy a significant activity-related functional impairment of 14-90% could be measured after application of 0.4-24 GBq of 131I. Prevalence of pretreatment sialopathies was 77/674 = 11.4% in single glands, and there was a global salivary gland functional impairment in 52/674 = 7.7%. CONCLUSION: Together with thyroid scintigraphy qSZ is an easy to perform examination without additional radiation burden. It can be recommended in all patients prior to and after radioiodine therapy both in order to quantify and to document possible parenchymal impairment induced even by low activities of 131I.