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Supplementary Figure Legends 1-9 from Epidermal Growth Factor Receptor Signaling Synergizes with Hedgehog/GLI in Oncogenic Transformation via Activation of the MEK/ERK/JUN Pathway

Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC. [Cancer Res 2009;69(4):1284–92]

Supplementary Figures 1-9, Tables 1-2 from Epidermal Growth Factor Receptor Signaling Synergizes with Hedgehog/GLI in Oncogenic Transformation via Activation of the MEK/ERK/JUN Pathway

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Supplementary Figure 2 from Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

PDF file - 245K, Supplementary Figure S1: Activated, autologous T cells induce CLL cell proliferation. Supplementary Figure S2: CD40L activated CLL cells fully retain their chemotactic response towards CCL21. Supplementary Figure S3: PMA/ionomycin and IgM cross-linking induces FL-HA binding. Supplementary Figure S4: CD44 surface expression on CLL patients does not differ between clinical risk groups. Supplementary Figure S5: The CLL cell subpopulation remaining CD44v6 negative after CD40L-activation displays low activation levels. Supplementary Figure S6: The mAbs used for immunoblotting specifically recognize panCD44 and CD44v6

Supplementary Figure 1 from Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

Purpose The main objective of this prospective study was to assess the value of gallium-68 prostate-specific membrane antigen ([68 Ga]Ga-PSMA-11) positron emission tomography/computed tomography (PET/CT) in primary N and M staging of intermediate- and high-risk prostate cancer (PCa) patients before planned curative-intent radical prostatectomy (RPE) and extended pelvic lymph node dissection (ePLND). The second objective was to compare the [68 Ga]Ga-PSMA-11 PET/CT findings with standard of care pelvic multi-parametric magnetic resonance imaging (mpMRI) in the detection of locoregional lymph node metastases and intraprostatic prostate cancer. Procedures A total of 81 patients (mean age: 64.5 years, baseline mean trigger PSA (tPSA) 15.4 ng/ml, ± 15.9) with biopsy proven PCa (24 intermediate- and 57 high risk) scheduled for RPE and ePLND were enrolled in this prospective study. In 52 patients [68 Ga]Ga-PSMA-11 PET/CT, pelvic mpMRI, and RPE with ePLND have been performed. Clinical risk stratification and related biomarkers as well as Gleason score (GS) were recorded. The location of the index lesion (IL) was documented systematically for each modality using a standardized segmentation of the prostate in six segments. Distant bone and lymph node metastasis detected by [68 Ga]Ga-PSMA-11 PET/CT were documented. [68 Ga]Ga-PSMA-11 PET/CT findings were correlated with results of mpMRI and histopathology. A consensus of imaging, clinical and/or follow-up findings were used for determining the distant metastases, which were not verified by histopathology. Results In the patient cohort who underwent RPE, [68 Ga]Ga-PSMA-11 PET/CT and mpMRI detected the IL in 86.5% and 98.1% of the patients, respectively. The median of the maximum standardized uptake value (SUVmax) in the intraprostatic IL was 12 (range, 4.7–67.8). Intraprostatic IL of the high-risk patients showed significantly higher SUVmax than those in patients with intermediate risk for distant metastases (n = 48; median: 17.84 vs. 8.77; p = 0.02). In total 729 LN were removed by ePLND in 48 patients. The histopathology verified 26 pelvic lymph node metastases (pLNM) in 20.8% (10/48) of the patients, which have been correctly identified in 60% of the patients on [68 Ga]Ga-PSMA-11 PET/CT, and in 50% on mpMRI. All but one pLNM had a maximum diameter below 10 mm. Bone metastases (BM) and distant LNM (dLNM) were found in 17.3% of the patients on [68 Ga]Ga-PSMA-11 PET/CT imaging. 39.0% of the [68 Ga]Ga-PSMA-11 PET-positive BM showed no suspicious morphological correlation on CT. Conclusion [68 Ga]Ga-PSMA-11 PET/CT shows high diagnostic performance for N and M staging of patients with intermediate- and high-risk prostate cancer and seems to be superior to pelvic mpMRI in the detection of locoregional lymph node metastases. A significant correlation was found between SUVmax of the intraprostatic index lesion and risk stratification based on tPSA level and GS. The results of this study emphasize again on the role of metabolic molecular imaging using specific tracers in selected patients, leading to tailored therapy approach.

Background: For hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative early breast cancer (EBC), adjuvant chemotherapy (ACT) is recommended in the case of high-risk features only. The MINDACT trial showed that patients with high clinical risk (CR) but low genomic risk (GR) defined by the 70-gene signature (MammaPrint®; 70-GS) did not benefit from ACT. In this registry, we investigated the frequency and feasibility of 70-GS and concurrent 80-gene subtyping (BluePrint®) use in HR-positive, HER2-negative EBC. Furthermore, we recorded the frequency of ACT recommendation and the adherence to it when the “MINDACT strategy” was used. Methods: This prospective registry included patients from 2 Austrian cancer centers. Similar to MINDACT, a modified version of Adjuvant!Online was used to determine CR, and 70-GC was used to determine GR in high-CR patients. ACT was recommended to patients with high CR and high GR. Results: Of 224 enrolled patients, 76 (33.9%) had high CR and 67 (88.2%) received genomic testing. Of those, 43 (64.2%) were classified as low and 24 (35.8%) as high GR, respectively. All 24 patients with high CR and GR (10.7% of all patients) received the recommendation for ACT, but ACT was started in only 15 patients (62.5%). The median time from surgery to the start of ACT was 45 days (range 32–68), and the median time from test decision to the test result was 15 days (range 9–56). Conclusion: We showed that the results of the MINDACT trial are reproducible in an Austrian population. Incorporating 70-GS into the daily clinical routine is feasible and mostly accepted by physicians for the guidance of treatment recommendations.

FOLFIRINOX is superior to gemcitabine in patients with pancreatic cancer, but this regimen is associated with toxicity and biomarkers for response are warranted. MicroRNAs can mediate drug resistance and could provide predictive information. Altered expressions of several microRNAs including miR-21-5p, miR-10b-5p and miR-34a-5p have been previously linked to a worse response to gemcitabine. We investigated the influence of expression levels in tumor tissue of those three microRNAs on outcome to FOLFIRINOX. Twenty-nine patients with sufficient formalin-fixed paraffin-embedded tumor tissue were identified. There was no significant association between high and low expression groups for these three microRNA. We conclude that polychemotherapy combination can overcome intrinsic negative prognostic factors., Lay abstract Treatment outcome with the polychemotherapy FOLFIRINOX is superior to gemcitabine in patients with pancreatic cancer, but FOLFIRINOX can cause severe toxicities. Therefore, we investigated biomarkers that may guide treatment decision. Altered expression of certain microRNAs, small noncoding RNAs, are associated with a worse response to gemcitabine. We found that the expression of the microRNAs miR-21-5p, miR-10b-5p and miR-34a-5p have no preditive value in patients treated with FOLFIRINOX. We therefore, conclude that polychemotherapy combination can overcome intrinsic negative prognostic factors.

Background: Biomarkers predicting response to bevacizumab containing therapy in metastatic breast cancer (MBC) are of urgent need. In a retrospective single-institution analysis we have previously shown that a 3-gene methylation signature (MLH1,POLKand TMBIM6) could discriminate between responders and non-responders to a bevacizumab-based therapy in two independent cohorts of patients with MBC with an AUC of 0.94 and 0.86, respectively (Gampenrieder SP et al. Theranostics. 2018. 8(8):2278-2288). Here, we present the validation of these findings within the prospective phase III trial TANIA (Vrdoljak E et al. Ann Oncol. 2016. 27(11):2046-52) randomizing 494 patients with HER2-negative MBC to chemotherapy plus bevacizumab or chemotherapy alone for two consecutive treatment lines (second- and third-line). All patients had already received bevacizumab-containing therapy in the first-line setting. Patients and methods: DNA isolated from archival FFPE tumor samples was available from 200 patients consenting to optional translational research within the TANIA trial. Out of these, 176 samples were collected prior to first-line bevacizumab therapy and were analyzed retrospectively. Sufficient DNA for methylation analysis was available from 124 patients: 64 treated with chemotherapy plus bevacizumab and 60 treated with chemotherapy alone. All samples were isolated from the primary tumor. Quantitative methylation analysis was performed by pyrosequencing on the PyroMark Q24 Advanced System (Qiagen). PFS and OS analyses were performed in both study arms comparing “predicted responders” (PRED_R) versus “predicted non-responders” (PRED_NR) based either on median dichotomization or according to the cutoffs for individual CpG and the combined 3-CpG methylation logistic regression model. Results:Out of the 124 evaluable patients, 32 (25.8%) were classified as PRED_R and 92 as RED_NR by the 3-gene methylation signature. PRED_R did not have a significantly different second-line PFS (HR 0.95, 95%CI 0.57-1.57; P = 0.84) or OS (HR 0.91, 95%CI 0.51-1.60; P = 0.73) when treated in the bevacizumab-containing study arm compared to PRED_NR. In addition, PRED_R did not show a longer PFS when treated with bevacizumab compared to PRED_R treated with chemotherapy alone (HR 0.95, 95%CI 0.59-1.54; P = 0.83). Furthermore, there was no difference in third-line PFS and the combination of second- and third-line PFS between PRED_R and PRED-NR in the bevacizumab arm. In the control arm, PRED_NR showed a statistically significant shorter PFS compared to PRED_R (HR 0.50, 95%CI 0.22-0.77; P = 0.006), but not OS (HR 0.95, 95%CI 0.51-1.77; P = 0.86). Conclusion: Our 3-gene methylation signature was not confirmed as predictive biomarker for bevacizumab efficacy in metastatic breast cancer. (This research project was partially supported by ROCHE Austria GmbH) Citation Format: Gampenrieder SP, Angela R, Rinnerthaler G, Hackl H, Steiner M, Pulverer W, Weinhaeusel A, Klinglmayr E, Karl T, Ilic S, Hufnagl C, Hauser-Kronberger C, Egle A, Greil R. A 3-gene DNA methylation signature fails to predict response to bevacizumab in metastatic breast cancer patients treated within the TANIA phase III trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-07.

The Warburg theory of cancer postulates that an important driver of tumorigenesis is insufficient respiration due to mitochondrial defects, and concomitant enhancement of lactate production due to increased aerobic glycolysis. We analysed 48 melanoma samples by immunohistochemistry and found that 38% of melanomas are characterized by areas of isolated or combined deficiencies of complexes of the oxidative phosphorylation (OXPHOS) system, whereby the incidence of OXPHOS-deficient areas is associated with an increased Breslow index; 62% of melanomas showed high expression of all OXPHOS complexes. Expression of carbonic anhydrase IX was low, indicating that melanomas generally are well-oxygenated. Expression of HIF-1α and MCT4 was high, which might be a consequence of increased lactate dehydrogenase A levels in melanomas. Our data indicate that there are two types of melanomas: one that features a classic Warburg effect, whereas the other one, despite being glycolytic, maintains a high level of OXPHOS complexes.

Summary Objective The aim of our investigation was to evaluate the clinicopathological characteristics and mutation patterns in newly diagnosed cases of thyroid cancer in the federal state of Salzburg, Austria, in the year 2013. Methods The medical records of all patients newly diagnosed with thyroid cancer in 2013 in the federal state of Salzburg were retrospectively reviewed. The clinicopathological characteristics and mutations of thyroid cancers were analyzed. Results 63 patients (mean age: 51.0 years, range: 21–81 years; female 75%, male 25%) were identified. 53 patients had papillary (12 follicular variant), 4 patients follicular (1 oxyphilic variant), 3 patients medullary, and 3 patients anaplastic thyroid cancer. T1 tumors were found in 34 patients (pT1a, 20 patients; pT1b, 14 patients), T2 tumors in 10 patients, T3 tumors in 16 patients, and T4 tumors in 3 patients. Lymph node involvement was seen in 15 patients and metastatic disease in 1 patient. Mutations of BRAF (B-type Raf kinase) were detected in 23 and mutation of NRAS (Neuroblastoma RAS Viral Oncogene Homolog) in 2 papillary thyroid cancers. No concomitant mutations of BRAF and NRAS were found. Conclusion Females accounted for 75% of the patients with newly diagnosed thyroid cancer and the incidence peaked at a younger age than in males. Papillary thyroid cancer was the most frequent tumor type, accounting for 84% of the cases. A high frequency of T1 tumors and cancers with no lymph node involvement was found. Males had a higher proportion of large tumors and more aggressive forms of thyroid cancer than females. Mutations (mostly of BRAF) were found in 47% of the cases. Neither mutations of KRAS (Kirsten rat sarcoma viral oncogene homologue) nor concomitant mutations of BRAF and NRAS were found.

Aquaporins (AQPs) are important for ocular homeostasis and function. While AQP expression has been investigated in ocular tissues of human, mouse, rat and dog, comprehensive data in rabbits are missing. As rabbits are frequently used model organisms in ophthalmic research, the aim of this study was to analyze mRNA expression and to localize AQPs in the rabbit eye. The results were compared with the data published for other species. In cross sections of New Zealand White rabbit eyes AQP0 to AQP5 were labeled by immunohistology and analyzed by confocal microscopy. Immunohistological findings were compared to mRNA expression levels, which were analyzed by quantitative reverse transcription real time polymerase chain reaction (qRT-PCR). The primers used were homologous against conserved regions of AQPs. In the rabbit eye, AQP0 protein expression was restricted to the lens, while AQP1 was present in the cornea, the chamber angle, the iris, the ciliary body, the retina and, to a lower extent, in optic nerve vessels. AQP3 and AQP5 showed immunopositivity in the cornea. AQP3 was also present in the conjunctiva, which could not be confirmed for AQP5. However, at a low level AQP5 was also traceable in the lens. AQP4 protein was detected in the ciliary non-pigmented epithelium (NPE), the retina, optic nerve astrocytes and extraocular muscle fibers. For most tissues the qRT-PCR data confirmed the immunohistology results and vice versa. Although species differences exist, the AQP protein expression pattern in the rabbit eye shows that, especially in the anterior section, the AQP distribution is very similar to human, mouse, rat and dog. Depending on the ocular regions investigated in rabbit, different protein and mRNA expression results were obtained. This might be caused by complex gene regulatory mechanisms, post-translational protein modifications or technical limitations. However, in conclusion the data suggest that the rabbit is a useful in-vivo model to study AQP function and the effects of direct and indirect intervention strategies to investigate e. g. mechanisms for intraocular pressure modulation or cornea transparency regulation.

Biomarkers for bevacizumab efficacy in metastatic breast cancer (MBC) are of urgent need. The genetic variability of genes involved in angiogenesis could explain the interpatient variability of drug effects. For this biomarker study DNA was extracted from tumor blocks or blood samples of patients with human epidermal growth factor receptor 2 (HER2)-negative MBC treated with bevacizumab in combination with chemotherapy (bevacizumab cohort, 163 patients) or chemotherapy only (control cohort, 105 patients). We assessed the correlation of 10 single-nucleotide polymorphisms (SNPs) in genes modulating angiogenesis (vascular endothelial growth factor-A (VEGF-A), VEGF receptor 1 (VEGFR-1), serine threonine kinase 39 (STK39)) or hypertension (endothelin-1 and uromodulin) with outcome and toxicity. In the bevacizumab cohort, the SNP rs5370-TT in endothelin-1 (EDN1) showed a significantly shorter median overall survival (OS, 6.3 vs 21.3 months; hazard ratio (HR) 2.89, 95% confidence interval (CI) 1.34-6.26; log-rank P=0.0069) and a trend toward worse median progression-free survival (3.5 vs 7.9 months; HR 2.05, 95% CI 0.96-4.39; log-rank P=0.065) compared with the alternate genotypes combined. Similarly, patients harboring the VEGF-936 (rs3025039) TT alleles showed a significantly shorter median OS than patients with VEGF-936 CC or CT (14.9 vs 21.3 months; HR 2.37, 95% CI 1.09-5.13; P=0.0286). In multivariate analysis including important clinical parameters like disease-free survival (DFS), adjuvant chemotherapy, ECOG (Eastern Cooperative Oncology Group) performance score, histologic subtype, grade, hormone receptor status, visceral metastases and treatment line, only the association of rs5370 (EDN1) with OS was still statistically significant (P=0.012). In the control cohort, no association of the EDN1 genotype with outcome was seen, suggesting a predictive value for bevacizumab. In conclusion, the SNP rs5370 in endothelin-1 could help identifying patients who unlikely gain any benefit from bevacizumab.

Background: In metastatic breast cancer (MBC) patients, no biomarker predicting benefit to a bevacizumab-containing therapy has been established yet. MicroRNAs (miRNAs) are involved in angiogenesis and treatment resistance and therefore could be of predictive value. Methods: Profiling of 754 miRNAs was performed in tumor samples of 58 MBC patients treated with a bevacizumab-containing first-line regimen (learning set). Based on progression-free survival (PFS), patients were divided into responders (R) and non-responders (NR). Differentially expressed miRNAs between R and NR were analyzed in a cohort of 57 patients treated with first-line chemotherapy without bevacizumab (control set), to exclude miRNAs providing prognostic information. MiRNA candidates significantly associated with PFS in multivariate analysis were further validated in tumor samples of 203 patients treated within the phase III trial TANIA randomizing between chemotherapy either alone or with bevacizumab after progression on first-line bevacizumab. Results: Low expression of miR-20a-5p (multivariate p = 0.035) and miR-21-5p (multivariate p = 0.004) were significantly associated with longer PFS in the learning set, but not in the control set. In samples from the TANIA trial, low expression of miR-20a-5p was also significantly associated with longer PFS (hazard ration (HR) 0.60, 95%-CI 0.37&ndash, 0.89, p = 0.012) and longer overall survival (OS, HR 0.54, 95%-CI 0.32&ndash, 0.83, p = 0.007) in the bevacizumab arm but not in the chemotherapy-only arm (PFS: HR 0.73, p = 0.119, OS: HR 1.01, p = 0.964). For miR-21-5p no significant association with PFS or OS in both treatment arms was observed. Conclusion: MiR-20a-5p expression in breast cancer tissue was predictive for a greater benefit from bevacizumab-containing therapy in two independent cohorts.

// Raphael Johannes Morscher 1, 4 , Sepideh Aminzadeh-Gohari 1 , Cornelia Hauser-Kronberger 2 , Rene Gunther Feichtinger 1 , Wolfgang Sperl 3 , Barbara Kofler 1 1 Laura Bassi Centre of Expertise-THERAPEP, Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria 2 Department of Pathology, Paracelsus Medical University, 5020 Salzburg, Austria 3 Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria 4 Division of Medical Genetics, Medical University Innsbruck, 6020 Innsbruck, Austria Correspondence to: Raphael Johannes Morscher, e-mail: raphael.morscher@pmu.ac.at Keywords: neuroblastoma, ketogenic diet, glucose, metronomic cyclophosphamide, anti-angiogenic Received: September 15, 2015 Accepted: February 05, 2016 Published: March 05, 2016 ABSTRACT Background: MYCN -amplification in high-grade Neuroblastoma (NB) tumors correlates with increased vascularization and therapy resistance. This study combines an anti-angiogenic approach with targeting NB metabolism for treatment. Methods and Results: Metronomic cyclophosphamide (MCP) monotherapy significantly inhibited NB growth and prolonged host survival. Growth inhibition was more pronounced in MYCN -amplified xenografts. Immunohistochemical evaluation of this subtype showed significant decrease in blood vessel density and intratumoral hemorrhage accompanied by blood vessel maturation and perivascular fibrosis. Up-regulation of VEGFA was not sufficient to compensate for the effects of the MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were evident. In contrast non MYCN -amplified tumors developed resistance, which was accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or calorie-restriction significantly enhanced the anti-tumor effect. Calorie-restricted ketogenic diet in combination with MCP resulted in tumor regression in all cases. Conclusions: Our data show efficacy of combining an anti-angiogenic cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model. These findings might open a new front in NB treatment.

Background: Biomarkers predicting response to bevacizumab containing therapy in breast cancer are of urgent need for a personalized treatment approach. DNA methylation is involved in regulation of angiogenesis and development of treatment resistance and could therefore provide predictive information for bevacizumab efficacy. Patients and methods: A genome-wide methylation profiling using the Illumina Infinium HumanMethylation450 BeadChip was performed in FFPE specimen of 36 patients with HER2-negative metastatic breast cancer treated with chemotherapy in combination with bevacizumab as first-line therapy (discovery set). Twenty-eight (78%) samples came from primary tumor and 8 (22%) from metastasis (2 lung mets., 1 pleural met., 1 liver met., 2 soft tissue mets., 1 ovarial met, 1 bone marrow infiltration). Based on progression-free survival (PFS) and breast cancer subtype (ER+ vs. triple-negative) patients were divided in responder (R) and non-responder (NR). By biostatistical methods differences in the methylation pattern between R and NR were detected. The 48 most interesting genes (e.g. because of there involvement in angiogenesis or carcinogenesis) showing a differential methylation status between R and NR are currently validated in two further metastatic breast cancer cohorts treated with (main set) and without bevacizumab (validation set), respectively. These validated results will be presented at the meeting. Citation Format: Gampenrieder SP, Rinnerthaler G, Weinhäusel A, Pulverer W, Hugnagl C, Hackl H, Romeder F, Monzo Fuentes C, Hauser-Kronberger C, Mlineritsch B, Greil R. DNA methylation patterns correlating with outcome in patients treated with first-line bevacizumab for metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-43.

The purpose of this work was to retrospectively evaluate survival and local control rates of triple-negative breast cancer subtypes classified as five marker negative (5NP) and core basal (CB), respectively, after breast-conserving surgery and intraoperative boost radiotherapy with electrons (IOERT) followed by whole breast irradiation. A total of 71 patients with triple-negative breast cancer were enrolled, who were treated with lumpectomy, axillary lymph node dissection, and IOERT with 9.6 Gy (median Dmax) followed by normofractionated whole breast irradiation to median total doses of 54 Gy. Chemotherapy was applied in a neoadjuvant (12 %), adjuvant (75 %), or combinational setting (7 %). After a median follow-up of 97 months (range 4–170 months), 5 in-breast recurrences were detected (7.0 %). For all patients, 8-year actuarial rates for local control, metastases-free survival, disease-specific survival, and overall survival amounted to 89, 75, 80, and 69 %, respectively. All local recurrences occurred in grade 3 (G3) tumors irrespective of their specific immunohistochemical phenotype; thus, the local control rate for grades 1/2 (G1/2) was 100 % for both 5NP and CB, while for G3 it was 88 % for 5NP and 90 % for CB (p = 0.65 and 0.82, respectively, n.s.). For disease-specific survival, only the difference of the best-prognosis group 5-NP/G3 vs. the worst-prognosis cohort CB/G1/2 was statistically significant: 90 % vs. 54 % (p = 0.03). Boost-IOERT provides acceptable long-term in-breast control in triple negative breast cancer. The best subgroup in terms of disease-specific survival was represented by 5NP in combination with tumor grading G3.

Background: Biomarkers predicting response to bevacizumab in breast cancer are still missing. Since epigenetic modifications can contribute to an aberrant regulation of angiogenesis and treatment resistance, we investigated the influence of DNA methylation patterns on bevacizumab efficacy. Methods: Genome-wide methylation profiling using the Illumina Infinium HumanMethylation450 BeadChip was performed in archival FFPE specimens of 36 patients with HER2-negative metastatic breast cancer treated with chemotherapy in combination with bevacizumab as first-line therapy (learning set). Based on objective response and progression-free survival (PFS) and considering ER expression, patients were divided in responders (R) and non-responders (NR). Significantly differentially methylated gene loci (CpGs) with a strong change in methylation levels (Δβ>0.15 or Δβ

Background: Estrogen-receptor (ER) positive breast cancer co-expresses androgen receptor (AR) in 75% to 95% of cases. The Austrian Breast & Colorectal Cancer Study Group (ABCSG) trial 12 compared anastrozole plus goserelin with tamoxifen plus goserelin in premenopausal patients with hormone receptor positive early breast cancer. In addition, patients were randomized to receive zoledronic acid or not. The aim of this analysis was to investigate the effect of AR expression on outcome in dependency of the treatment arm. Patients and methods: AR expression was analyzed by immunohistochemistry using a mouse monoclonal antibody (Novocastra) in formalin-fixed paraffin-embedded specimens from 194 early breast cancer patients treated within the ABCSG 12 trial at our institution. As there is no generally accepted cut-off level defining AR positivity, we used two definitions for this analysis: A) positive staining of ≥ 10% of tumor cells B) an immune reactive score (IRS; intensity of staining X percentage of stained cells) ≥ 3 according to the Remmele score used for ER/PR evaluation. Results: A total of 194 patients were included in this analysis. In tissue samples collected before 2001 AR staining was very weak, interpreted as a loss of antigenicity due to archival time and conditions. Therefore, 40 patients with tissue samples achieved earlier than 2001 were excluded from further analysis. Seventy-nine percent (122 of 154) and 63% (97 of 154) of tumors were AR positive by definition A and B, respectively. All except one of the AR positive tumors according to definition B, were also positive according to definition A. Disease free survival (DFS) and overall survival (OS) data N (%)DFSOSDefinition A (≥ 10%) AR positive122 (79)HR 0.61 (0.21-1.72) P=0.3428HR 0.17 (0.03-0.99) P=0.025AR negative32 (21) Definition B (IRS ≥ 3) AR positive97 (63)HR 0.88 (0.33-2.35) P=0.7994HR 0.39 (0.07-2.34) P=0.2846AR negative57 (37) DFS and OS did not differ between both endocrine treatment arms in a Cox regression model tested for interaction between AR expression and endocrine treatment. Conclusion: In this pilot study patients with AR positive disease had a numerically better DFS and OS compared to AR negative patients, but only prolongation of OS in patients with ≥ 10% AR positive tumor cells was statistically significant. AR expression did not influence outcome between tamoxifen and anastrozole treated patients, but based on the small number of events, this results have to be interpreted with caution. This data will be confirmed in a larger proportion of patients treated within the ABCSG-12 trial. Citation Format: Gabriel Rinnerthaler, Anna M Knopp, Cornelia Hauser-Kronberger, Simon P Gampenrieder, Patrick Morre, Brigitte Mlineritsch, Christian Fesl, Michael Gnant, Richard Greil. Androgen receptor expression in pre-menopausal early breast cancer patients treated with endocrine therapy within the ABCSG-12 trial - a single center pilot analysis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-47.

FOLFIRINOX is a highly active regimen for the treatment of patients with unresectable pancreatic cancer. However, treatment with FOLFIRINOX is associated with relevant toxicity and predictors for response to therapy are warranted. We retrospectively analyzed 49 patients with unresectable pancreatic cancer treated with FOLFIRINOX in order to evaluate a possible predictive role of clinical parameters and tumor characteristics for response to chemotherapy. Tumor samples were characterized histopathologically before treatment and expression of p53 and Ki67 was analyzed using automated immunohistochemistry. Overall survival (OS) and progression-free survival (PFS) were estimated by the Kaplan-Meier method. The overall objective response rate was 55.1%, the disease control rate was 70.6%. Female gender was associated with a significantly higher disease control rate of 91.7 compared to 48.0% in male patients (p=0.001) which reached 100% in female patients when primarily treated compared to treatment after surgical resection and relapse (77.8%, p=0.057). For all patients median PFS was 3.5 months (95% CI, 2.7-4.3 months) and median OS was 13 months (95% CI, 9.4-16.6 months). Female patients showed a tendency towards a longer median PFS (5.0 months, 95% CI, 3.6-6.4 months) compared to males (3.0 months, 95% CI, 2.4-3.6 months) (p=0.099). Serum levels of CA19.9 and CEA were significantly higher in female patients compared to male patients (p=0.037, p=0.05). Tumors of patients with response to FOLFIRINOX showed a higher expression level of p53 and Ki67 as well as higher serum levels of CA19.9 compared to non-responders, which was statistically not significant. Our study indicates that female gender is a positive predictor for therapy response to FOLFIRINOX in patients with unresectable pancreatic cancer. Female gender in turn was associated with increased levels of tumor markers CEA and CA19.9 and patients with higher serum levels of CA19.9 were more responsive to FOLFIRINOX.

Objectives/Hypothesis Congenital granular cell tumor (CGCT), also known as congenital epulis, is a rare benign mesenchymal tumor of the oral cavity. We report of a 3 years and 7 months old female patient undergoing surgical excision of an oral tumor. Subsequent histological and immunohistological investigations within the clinical course led to the diagnosis of CGCT. However, clinical findings in this case, such as primary onset and an untypical location within the oral cavity, clearly stand in contrast to those usually found in CGCT, resulting in an exceptional case not previously described in the literature. Laryngoscope, 123:2459–2462, 2013

Background Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser-generated micropores to subcutaneous injection. Methods BALB/c mice were sensitized by intraperitoneal injection of recombinant grass pollen allergen Phl p 5 together with alum. Subsequently, lung inflammation was induced by repeated intranasal challenge. During the treatment phase, adjuvant-free Phl p 5 was applied in solution to microporated skin or was subcutaneously injected. Lung function and cellular infiltration; Phl p 5–specific serum levels of IgG1, IgG2a, and IgE; and cytokine levels in bronchoalveolar lavage fluids as well as in supernatants of splenocyte cultures were assessed. Results Both therapeutic approaches reduced airway hyperresponsiveness and leukocyte infiltration into the lungs. Whereas subcutaneous immunotherapy induced a systemic increase in Th2-associated cytokine secretion, transcutaneous application revealed a general downregulation of Th1/Th2/Th17 responses. Successful therapy was associated with induction of IgG2a and an increase in FOXP3+ CD4+ T cells. Conclusions Transcutaneous immunotherapy via laser microporation is equally efficient compared with conventional subcutaneous treatment but avoids therapy-associated boosting of systemic Th2 immunity. Immunotherapy via laser-microporated skin combines a painless application route with the high efficacy known from subcutaneous injections and therefore represents a promising alternative to established forms of immunotherapy.

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

Inhibition of Hedgehog (HH)/GLI signalling in cancer is a promising therapeutic approach. Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI. Cooperation of HH/GLI with epidermal growth factor receptor (EGFR) signalling promotes transformation and cancer cell proliferation in vitro. However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined. In this report we show that genetic and pharmacologic inhibition of EGFR signalling reduces tumour growth in mouse models of HH/GLI driven basal cell carcinoma (BCC). We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells. The data validate EGFR signalling as drug target in HH/GLI driven cancers and shed light on the molecular processes controlled by HH-EGFR signal cooperation, providing new therapeutic strategies based on combined targeting of HH-EGFR signalling and selected downstream target genes.

Summary Aims Activation of β-catenin has been identified as a possible mechanism for the development of nephroblastomas. In our study we investigated whether this activation occurs already in precursor lesions of nephroblastomas, called nephrogenic rests (NRs). Inactivation of the adenomatous polyposis coli (APC) protein is an important regulatory mechanism of activating p-catenin. We clarified the role of APC by assessing loss of heterozygosity (LOH) and possible mutations within the genomic region. Methods Activation of β-catenin was examined by immunohistochemistry identifying nuclear translocation. Two polymorphic loci of the APC gene were investigated for LOH and sequence analysis was performed for the mutation cluster region of the APC gene on formalin fixed, paraffin embedded samples. Results Four of the 18 nephroblastomas available for immunohistochemistry exhibited nuclear staining of β-catenin, but none of the NRs. Analysis of LOH revealed 14 homozygous samples, 10 heterozygous tumours and six tumours exhibiting LOH of the APC gene. One blastematype nephroblastoma showed nuclear localisation of β-catenin in conjunction with LOH of the APC gene. Analysis of 12 nephroblastomas revealed no sequence aberration. Conclusion Our results indicate that nuclear activation of p-catenin is a late event in the tumorigenesis of nephroblastomas coinciding in some tumours with LOH of the APC gene.

Altered cellular proton handling and cell volume regulation are hallmarks of tumorigenesis. To investigate a possible involvement of the non-gastric H + /K + ATPase ATP12A (ATP1AL1) in prostate cancer, we performed immunohistochemistry in formalin-fixed, paraffin-embedded histological sections from benign and malignant human prostate lesions. Normal prostate tissue displayed a membrane-bound ATP12A staining with focal accumulated pattern, whereas in the benign prostate hyperplasia (BPH) and cancerous prostate tissue (tumor grade I–III) the protein appears to be displaced in the luminal cells of the glandular epithelium. Hence, the expression pattern of ATP12A is markedly altered in BPH and prostate cancer. To test for altered gene expression of ATP12A we performed quantitative reverse transcriptase PCR (QRT-PCR) in normal (tumor-free) prostate tissue, BPH and tumor stages I-III using a prostate cancer cDNA array. However, no significantly different expression levels could be detected in the various disease states compared to normal tissue, which contrasts the findings from immunohistochemistry and points to the possibility of altered post-translational processing and/or sorting of the protein. We further show that ATP12A mRNA is expressed at different levels in PC-3 and LNCaP prostate cancer cells, with a significant ~26-fold higher expression in the latter cell type. Protein expression in these tumor cell lines was verified by Western blot.

We previously demonstrated that methylation of neugogenic differentiation 1 (NEUROD1) gene, a polycomb group target (PCGT) gene is a predictor of response to neoadjuvant chemotherapy in breast cancer. Here, we address the question whether NEUROD1 methylation provides clinical information independent from its expression level, and whether PCGT methylation is homogeneous in breast cancer. We examined: (1) NEUROD1 methylation and mRNA expression in 9 breast cancer cell lines and 63 tumour specimens, (2) DNA methylation in a training set of 55 PCGT genes taken from the centre (TUC) and periphery (TUP) of 15 breast cancer specimens, and compared this with 22 non neoplastic controls, and finally, (3) validated statistically significant genes in an independent set of 20 cases versus 18 controls. 8/9 cell lines demonstrated NEUROD1 methylation, whereas, there was only one cell-line that showed NEUROD1 expression. There was no association between methylation and expression in breast tumour specimens, with only 14% exhibiting NEUROD1 expression. Of the 55 PCGT genes analysed, 24% (13/55) were shown to be cancer specific (p0.05) with a receiver-operating-characteristic (ROC) area-under-the-curve (AUC) of0.7 (range 0.71-0.95). DNA methylation accurately predicted the presence of cancer in both TUC and TUP. DNA methylation of PCGT genes predicts the presence of breast cancer and is not subject to tumour heterogeneity. Further work will reveal if methylation of PCGT genes will serve as a robust means for the clinical detection and assessment of breast cancer.

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.

Hedgehog (HH) signaling in the epidermis is primarily mediated by the zinc finger transcription factors GLI1 and GLI2. Exquisite regulation of HH/GLI signaling is crucial for proper specification of the epidermal lineage and development of its derivatives, whereas dysregulation of HH/GLI signaling disrupts tissue homeostasis and causes basal cell carcinoma (BCC). Similarly, bone morphogenetic proteins (BMPs) and activins have been described as key signaling factors in the complex regulation of epidermal fate decisions, although their precise interplay with HH/GLI is largely elusive. Here we show that, in human epidermal cells, expression of the activin/BMP antagonist follistatin (FST) is predominantly up-regulated by the HH effector GLI2. Consistently, we found strong FST expression in the outer root sheath of human hair follicles and BCC. Detailed promoter analysis showed that two sequences with homology to the GLI consensus binding site are required for GLI2-mediated activation. Interestingly, activation of the FST promoter is highly GLI2-specific, because neither GLI1 nor GLI3 can significantly increase FST transcription. GLI2 specificity requires the presence of a 518-bp fragment in the proximal FST promoter region. On the protein level, sequences C-terminal to the zinc finger are responsible for GLI2-specific activation of FST transcription, pointing to the existence of GLI-interacting cofactors that modulate GLI target specificity. Our results reveal a key role of GLI2 in activation of the activin/BMP antagonist FST in response to HH signaling and provide new evidence for a regulatory interaction between HH and activin/BMP signaling in hair follicle development and BCC.

Background A long-term analysis by the Early Breast Cancer Trialist Group (EBCTG) revealed a strong correlation between local control and cancer-specific mortality. MicroRNAs (miRs), short (20–25 nucleotides) non-coding RNAs, have been described as prognosticators and predictors for breast cancer in recent years. The aim of the current study was to identify miRs that can predict local control after breast conserving therapy (BCT) in early stage breast cancer. Results Clinical data of 46 early stage breast cancer patients with local relapse after BCT were selected from the institutional database. These patients were matched to 101 control patients showing identical clinical features but without local relapse. The study was conducted in two steps. (1) In the pilot study, 32 patients (16 relapses versus 16 controls) were screened for the most de-regulated microRNAs (= candidate microRNAs) in a panel of 1250 miRs by microarray technology. Eight miRs were found to be significantly de-regulated. (2) In the validation study, the candidate microRNAs were analyzed in an independent cohort of 115 patients (30 relapses versus 85 controls) with reverse transcription quantitative polymerase chain reaction (RT-qPCR). From these eight candidates, hsa-miR-375 could be validated. Its median fold change was 2.28 (Mann-Whitney U test, corrected p value = 0.008). In the log-rank analysis, high expression levels of hsa-miR-375 correlated with a significantly higher risk of local relapse (p = 0.003). In a multivariate analysis (forward stepwise regression) including established predictors and prognosticators, hsa-miR-375 was the only variable that was able to distinguish the statistical significance between relapse and control groups (raw p value = 0.000195 HR = 0.76, 95 % CI 0.66–0.88; corrected p value = 0.005). Conclusions Hsa-miR-375 predicts local control in patient with early stage breast cancer, especially in estrogen receptor α (ER-α)-positive patients. It can therefore serve as an additional molecular marker for treatment choice independently from known predictors and prognosticators. Validation in larger prospective studies is warranted. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0198-1) contains supplementary material, which is available to authorized users.

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)® microarrays from Agilent® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.

Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a T(H)1-like immune reaction on the cellular level, i.e. generation of IFN-gamma secreting CD8(+) T-cells, yet a T(H)2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule beta-galactosidase has been argued to prevent IgE induction and to promote T(H)1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing beta-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule beta-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a T(H)2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This T(H)2 effect was influenced by the nature of the antigen, with a more pronounced T(H)2-bias for the allergens Bet v 1 and Phl p 5 compared to beta-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly T(H)1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy.

Fulvestrant (Faslodex) is an oestrogen receptor (ER) antagonist with demonstrated efficacy in patients with advanced and pretreated breast cancer. We present a single-centre experience with fulvestrant administered under the compassionate use programme (CUP) to a total of 54 postmenopausal women with metastatic breast cancer progressing on multiple endocrine and cytotoxic therapies. Patients received 250 mg fulvestrant i.m. once monthly as second- (n = 8), third- (n = 30), fourth- (n = 14) and fifth-line (n = 2) hormonal treatment. The median number of previous endocrine therapies was 2 (range 1–4). Most of the patients also had multiple palliative chemotherapies with a median of 1.7 (range 0–6) prior therapies. The median duration of fulvestrant treatment was 6.3 months (range 1–39 months) and the median duration of follow-up was 19.4 months (range 1–63 months). Objective response was achieved by five patients (9.3%): one complete remission (CR) (1.9%) and four partial remissions (PR) (7.4%). Stable disease (SD) lasting ≥6 months was achieved by 16 patients (29.6%). Thus in all, fulvestrant conferred clinical benefit (CB) on 21 women (38.9%). The median time to progression (TTP) was 6.4 months. In all patients with CR and PR, tumour cells were positive for both ER and progesterone receptor (PgR), but lacked HER2/neu overexpression; one patient with PR had an unknown HER2/neu status. Overall, the drug was well tolerated. No grade 3/4 toxicities were reported. Fulvestrant appears to be an efficient and well-tolerated drug even in women with advanced breast cancer progressing after multiple endocrine and/or cytotoxic treatments.

Background Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. Methods We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. Results The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. Conclusions Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0717-4) contains supplementary material, which is available to authorized users.

Background Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T H 1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. Objective A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. Methods Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG 1 , IgG 2a , and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-γ and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. Results Prevaccination with ubiquitinated Bet v 1–stimulated T H 1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. Conclusion The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. Clinical implications Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.

Objective To test if a high degree of apoptosis in cumulus cells could indicate diminished oocyte quality and developmental competence. Design Prospective analysis. Setting Public hospital and university. Patient(s) Thirty seven women who gave written consent to participate in this study. Intervention(s) Cumulus–oocyte complexes were denuded separately and the resulting cumulus cell suspensions were analyzed for presence of apoptosis using a terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay detection kit. Main Outcome Measure(s) Percentage of apoptotic cumulus cells and blastocyst formation rate. Result(s) Younger patients (≤35 years) showed significantly fewer apoptotic cumulus cells than older ones. In all patients gamete maturity was shown to be highly correlated to the rate of apoptosis in cumulus cells. At zygote and cleavage stages (days 1 to 4) no morphologic features were related to the degree of programmed cell death. However, blastocyst development was predictable taking into account the percentage of apoptosis in associated somatic cells. In addition, there was a trend toward better quality blastocysts from follicles with a lower rate of apoptotic cells. No influence on pregnancy and implantation rate was observed. Conclusion(s) Apoptotic processes within follicles seem to impair oocyte maturation. Though not manifested in the morphologic appearance, gametes and embryos derived from cumulus complexes with no or minor apoptosis have an increased chance of giving rise to optimal blastocysts.

Background: Biomarkers predicting response to bevacizumab containing therapy in breast cancer are still missing. Since epigenetic modifications can contribute to an aberrant regulation of angiogenesis and to treatment resistance, we investigated the influence of DNA methylation on bevacizumab efficacy. Patients and methods: A genome-wide methylation profiling using the Illumina Infinium HumanMethylation450 BeadChip was performed in archival FFPE specimen of 36 patients with HER2-negative metastatic breast cancer treated with chemotherapy in combination with bevacizumab as first-line therapy (learning set). Based on objective response and progression-free survival (PFS) and considering ER expression, patients were divided in responders (R) and non-responders (NR). Differentially methylated gene loci (methylation variable position = MVP) and differentially methylated regions (DMR) between R and NR were identified. Only significant sites with a strong change in methylation levels (Δβ>0.15 or Δβ Results: In the learning set 435 MVPs (p Conclusion: A 24-gene methylation signature can reproducible discriminate between responders and non-responders to a bevacizumab-based therapy in breast cancer and could help to identify patients deriving greater benefit from anti-VEGF agents. Citation Format: Gampenrieder SP, Rinnerthaler G, Pulverer W, Weinhäusel A, Hufnagl C, Hackl H, Hauser-Kronberger C, Mlineritsch B, Greil R. DNA methylation signature predicting bevacizumab efficacy in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-10.

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

The high incidence of human epidermal growth factor receptor (HER)2 overexpression on breast and various other cancer cells and the prognostic and potentially predictive value of HER2 render this growth receptor a novel and important therapeutic target. Out of a wide range of assays that have been used in research for the detection of HER2 status, only two techniques are now predominant and readily applicable in the routine clinical pathology laboratory: determination of HER2 overexpression by immunohistochemistry (IHC) and HER2 gene amplification by fluorescence in situ hybridisation (FISH). In a retrospective study on a cohort of 173 archival invasive breast carcinomas a chromogenic in situ hybridisation (CISH) assay for the detection of HER2 amplification was established. Results were compared to HercepTest, which is the most frequently used method for detecting HER2 alteration. Additionally, HER2 gene copy number was investigated using differential PCR (dPCR) as a testing system. Discrepant cases between CISH and HercepTest and all IHC positive cases (2 + and 3 +), a total of 42 cases, were analysed with FISH Path Vysion(Vysis) assay. HER2 overexpression was found by IHC in 24.3%, HER2 amplification by CISH in 19.1% and by dPCR in 9.2% of the tumours. The overall concordance rate between CISH and IHC was 95.9%, between dPCR and IHC 85% and between CISH and FISH 100%, respectively. Among 25 HercepTest positive cases (score 3+) two showed no gene amplification and four out of 13 tumours with score 2 + were negative with CISH and FISH. The current study showed that CISH offers an ideal approach that allows detection of HER2 amplification in the context of morphology, whereas the major drawback of dPCR is the impracticability of tissue differentiation of invasive and non-invasive carcinoma.

To elucidate the relationship between tumor genesis and the mitochondrial energy metabolism in renal neoplasms, we studied three individual enzyme activities of the oxidative phosphorylation, two components of the Krebs cycle and the mitochondrial DNA content of renal carcinomas including 29 conventional, five papillary, two unclassified carcinomas with sarcomatoid features and one collecting duct carcinoma. A significant reduction of all mitochondrial enzyme activities including complex V, as well as of the mitochondrial DNA content was detected in 34 of 37 renal carcinoma tissues as compared with control kidney. Mitochondrial enzyme activities and mitochondrial DNA levels were not statistically different between the conventional, papillary and unclassified sarcomatoid type of renal carcinoma and did not correlate with tumour grade, metastasis, ploidy and proliferative activity as determined by Ki-67 staining. Taken together, our data indicate that a co-ordinated down-regulation of all components necessary for mitochondrial energy metabolism occurs in most renal carcinomas as an early event in carcinoma formation, which does not change with progression of the disease.