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Anna-Lena Klauser,1 Marc Hirschfeld,1– 3 Andrea Ritter,1,2 Gerta Rücker,1,4 Markus Jäger,1,2 Julia Gundarova,1 Daniela Weiss,1,2 Ingolf Juhasz-Böss,1,2 Kai Berner,1,2 Thalia Erbes,1,2 Jasmin Asberger1,2 1Faculty of Medicine, University of Freiburg, Freiburg, Germany; 2Department of Obstetrics and Gynecology, Medical Center – University of Freiburg, Freiburg, Germany; 3Institute of Veterinary Medicine, University of Göttingen, Göttingen, Germany; 4Institute of Medical Biometry and Statistics, Medical Center – University of Freiburg, Freiburg, GermanyCorrespondence: Jasmin AsbergerDepartment of Obstetrics and Gynecology, Faculty of Medicine and Medical Center – University of Freiburg, Hugstetterstr. 55, Freiburg, 79106, GermanyTel +49 761 270 30020Fax +49 761 270 30370Email jasmin.asberger@uniklinik-freiburg.dePurpose: In 2020, breast cancer still represents the most common type of cancer in women worldwide. Depending on the specific molecular subtype, clinical breast cancer management comprises surgery, radiotherapy, chemotherapy and targeted therapy. Furthermore, there are some therapeutic approaches from the field of complementary and alternative medicine. Current research focuses on the elucidation of new therapeutic targets for treatment development. Odorant substances affect apoptosis, proliferation and cell cycle in healthy and cancerous cells. Exact signalling pathways involved are not entirely clear. The present study aims to analyse their therapeutic potential in breast cancer.Methods: This study focuses on the effect of commonly used odorant substances (citral, citrathal R, cyclovertal, para-cymol, hexylacetat, herbavert, dihydromyrcerol and limonen) on the breast cancer cell lines MDA-MB-231, T47-D and BT474. Methodologically, this study applied cell culturing, MTT assay for detection of IC50 of the odorant substance, RNA purification followed by qRT-PCR, protein isolation and Western Blot, as well as immunocytochemistry. Further, this study investigates the role of transient receptor potential channel V1 (TRPV1), involved in the mechanisms of action for some odorant substances. Therefore, capsazepine, a TRPV1 antagonist, was used.Results: The odorant substances citral, citrathal R and cyclovertal have significant pro-apoptotic (p < 0.001), anti-proliferative (p < 0.001) and cell cycle-arresting effects measurable in RNA expression as well as in protein levels and immunocytochemical staining. The combination of citral and capsazepine no longer showed significant pro-apoptotic, antiproliferative, and cell cycle inhibitory effects compared to the compounds alone. This indicates that TRPV1 is necessary for the signal transduction of citral.Conclusion: This present study reveals three odorant substances with effects on cell viability, indicating their potential use in breast cancer therapy.Keywords: citral, citrathal R, cyclovertal, odorant substances, olfactory receptor and breast cancer

Background: The quest to comprehend the real-world efficacy of CDK4/6 inhibitors (CDKis) in breast cancer continues, as patient responses vary significantly., Methods: This single-center retrospective study evaluated CDKi use outside the trial condition from November 2016 to May 2020. Progression-free survival (PFS), time-to-treatment failure (TTF), short-term and prolonged treatment benefit (≥4 and ≥10 months), as well as prognostic and predictive markers were assessed with Kaplan-Meier and multivariate regression analyses., Results: Out of 86 identified patients, 58 (67.4%) had treatment failure of which 40 (46.5%) were due to progression. Median PFS and TTF were 12 and 8.5 months, respectively. A total of 57 (66.3%) and 42 (48.8%) patients experienced short-term and prolonged treatment benefit. Independent, significant predictors for PFS were progesterone receptor expression (HR: 0.88), multiple metastatic sites (HR: 2.56), and hepatic metastasis (HR: 2.01). Significant predictors for TTF were PR expression (HR: 0.86), multiple sites (HR: 3.29), adverse events (HR: 2.35), and diabetes (HR: 2.88). Aside from tumor biology and adverse events, treatment modifications like pausing and switching of CDKi were predictive for short-term (OR: 6.73) and prolonged (OR: 14.27) therapeutic benefit, respectively., Conclusions: These findings emphasize the importance of tailored treatment strategies, highlighting the role of PR expression, metastatic burden, and therapeutic adjustments in optimizing patient outcomes in real-world breast cancer management.

Background: Breast cancer is the most common type of cancer worldwide. Cyclin-dependent kinase inhibition is one of the backbones of metastatic breast cancer therapy. However, there are a significant number of therapy failures. This study evaluates the biomarker potential of microRNAs for the prediction of a therapy response under cyclin-dependent kinase inhibition., Methods: This study comprises the analysis of intracellular and extracellular microRNA-expression-level alterations of 56 microRNAs under palbociclib mono as well as combination therapy with letrozole. Breast cancer cell lines BT-474, MCF-7 and HS-578T were analyzed using qPCR., Results: A palbociclib-induced microRNA signature could be detected intracellularly as well as extracellularly. Intracellular miR-10a, miR-15b, miR-21, miR-23a and miR-23c were constantly regulated in all three cell lines, whereas let-7b, let-7d, miR-15a, miR-17, miR-18a, miR-20a, miR-191 and miR301a&#95;3p were regulated only in hormone-receptor-positive cells. Extracellular miR-100, miR-10b and miR-182 were constantly regulated across all cell lines, whereas miR-17 was regulated only in hormone-receptor-positive cells., Conclusions: Because they are secreted and significantly upregulated in the microenvironment of tumor cells, miRs-100, -10b and -182 are promising circulating biomarkers that can be used to predict or detect therapy responses under CDK inhibition. MiR-10a, miR-15b, miR-21, miR-23a and miR-23c are potential tissue-based biomarkers.

Background: Currently, there are no data from randomized trials on the use of intraoperative radiotherapy (IORT) as a tumor bed boost in women at high risk of local recurrence. The aim of this retrospective analysis was to compare the toxicity and oncological outcome of IORT or simultaneous integrated boost (SIB) with conventional external beam radiotherapy (WBI) after breast conserving surgery (BCS)., Methods: Between 2009 and 2019, patients were treated with a single dose of 20 Gy IORT with 50 kV photons, followed by WBI 50 Gy in 25 or 40.05 in 15 fractions or WBI 50 Gy with SIB up to 58.80-61.60 Gy in 25-28 fractions. Toxicity was compared after propensity score matching. Overall survival (OS) and progression-free survival (PFS) were calculated using the Kaplan-Meier method., Results: A 1:1 propensity-score matching resulted in an IORT + WBI and SIB + WBI cohort of 60 patients, respectively. The median follow-up for IORT + WBI was 43.5 vs. 32 months in the SIB + WBI cohort. Most women had a pT1c tumor: IORT group 33 (55%) vs. 31 (51.7%) SIB group (p = 0.972). The luminal-B immunophenotype was most frequently diagnosed in the IORT group 43 (71.6%) vs. 35 (58.3%) in the SIB group (p = 0.283). The most reported acute adverse event in both groups was radiodermatitis. In the IORT cohort, radiodermatitis was grade 1: 23 (38.3%), grade 2: 26 (43.3%), and grade 3: 6 (10%) vs. SIB cohort grade 1: 3 (5.1%), grade 2: 21 (35%), and grade 3: 7 (11.6%) without a meaningful difference (p = 0.309). Fatigue occurred more frequently in the IORT group (grade 1: 21.7% vs. 6.7%; p = 0.041). In addition, intramammary lymphedema grade 1 occurred significantly more often in the IORT group (11.7% vs. 1.7%; p = 0.026). Both groups showed comparable late toxicity. The 3- and 5-year local control (LC) rates were each 98% in the SIB group vs. 98% and 93% in the IORT group (LS: log rank p = 0.717)., Conclusion: Tumor bed boost using IORT and SIB techniques after BCS shows excellent local control and comparable late toxicity, while IORT application exhibits a moderate increase in acute toxicity. These data should be validated by the expected publication of the prospective randomized TARGIT-B study., Competing Interests: DK has received honoraria from Merck Sharp & Dome, Onkowissen, and Pfizer as well as research funding from Merck KGaA outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stoian, Exner, Gainey, Erbes, Gkika, Popp, Spohn, Krug, Juhasz-Böss, Grosu and Sprave.)

Accurate determination of resection margins in breast specimens is important as complete removal of malignancy is a prerequisite for patients' outcome. Mammography (DM) as 2D-technique provides only limited value in margin assessment. Therefore, we investigated whether cone-beam computed tomography (CBCT) or digital breast tomosynthesis (DBT) has incremental value in assessing margins to microcalcifications. Three independent readers investigated breast specimens for presence of microcalcifications and the smallest distance to margins. Histopathology served as gold standard. Microcalcifications were detected in 15 out of 21 included specimens (71%). Pooled sensitivity for DM, DBT and CBCT for microcalcifications compared to preoperative DM was 0.98 (CI 0.94-0.99), 0.83 (CI 0.73-0.94) and 0.94 (CI 0.87-0.99), pooled specificity was 0.99 (CI 0.99-0.99), 0.73 (CI 0.51-0.96) and 0.60 (CI 0.35-0.85). Mean measurement error for margin determination for DM, DBT and CBCT was 10 mm, 14 mm and 6 mm (p = 0.002) with significant difference between CBCT and the other devices (p < 0.03). Mean reading time required by the readers to analyze DM, DBT and CBCT, was 36, 43 and 54 s (p < 0.001). Although DM allows reliable detection of microcalcifications, measurement of resection margin was significantly more accurate with CBCT. Thus, a combination of methods or improved CBCT might provide a more accurate determination of disease-free margins in breast specimens., (© 2022. The Author(s).)

Background: Ribosomal biogenesis and ribosomal proteins have attracted attention in the context of tumor biology in recent years. Instead of being mere translational machineries, ribosomes might play an active role in tumor initiation and progression. Despite its importance, regulation of ribosomal biogenesis is still not completely understood., Methods: Using Gene Set Enrichment Analysis of RNA sequencing and proteomical mass spectrometry data in breast cancer cells expressing Krüppel-like factor 7 (KLF7), we identified processes altered by this transcription factor. In silico analyses of a cohort of breast cancer patients in The Cancer Genome Atlas confirmed our finding. We further verified the role of KLF7 the identified ribosomal processes in in vitro assays of mammary carcinoma cell lines and analyses of breast cancer patients' tissue slices., Results: We identified the transcription factor Krüppel-like factor 7 (KLF7) as a regulator of ribosomal biogenesis and translation in breast cancer cells and tissue. Highly significant overlapping processes related to ribosomal biogenesis were identified in proteomics and transcriptomics data and confirmed in patients' breast cancer RNA Seq data. Further, nucleoli, the sites of ribosomal biogenesis, were morphologically altered and quantitatively increased in KLF7-expressing cells. Pre-rRNA processing was identified as one potential process affected by KLF7. In addition, an increase in global translation independent from proliferation and transcription was observed upon exogenous KLF7 expression in vitro. Importantly, in a cohort of breast cancer patients, KLF7-expression levels correlated with aggressiveness of the intrinsic breast cancer subtype and tumor grading. Moreover, KLF7 correlated with nucleolar characteristics in human breast tumor tissue, indicating a role for KLF7 in ribosomal biogenesis., Conclusion: In mammary carcinoma, KLF7 is involved in ribosomal biogenesis. Alterations of ribosomal biogenesis has far reaching quantitative and qualitative implications for the proteome of the cancer cells. This might influence the aggressiveness of cancer cells., (© 2022. The Author(s).)

Background: Radiotherapy using the deep inspiration breath-hold (DIBH) technique compared with free breathing (FB) can achieve substantial reduction of heart and lung doses in left-sided breast cancer cases. The anatomical organ movement in deep inspiration also cause unintended exposure of locoregional lymph nodes to the irradiation field., Methods: From 2017-2020, 148 patients with left-sided breast cancer underwent breast conserving surgery (BCS) or mastectomy (ME) with axillary lymph node staging, followed by adjuvant irradiation in DIBH technique. Neoadjuvant or adjuvant systemic therapy was administered depending on hormone receptor and HER2-status. CT scans in FB and DIBH position with individual coaching and determination of the breathing amplitude during the radiation planning CT were performed for all patients. Intrafractional 3D position monitoring of the patient surface in deep inspiration and gating was performed using Sentinel and Catalyst HD 3D surface scanning systems (C-RAD, Catalyst, C-RAD AB, Uppsala, Sweden). Three-dimensional treatment planning was performed using standard tangential treatment portals (6 or 18 MV). The delineation of ipsilateral locoregional lymph nodes was done on the FB and the DIBH CT-scan according to the RTOG recommendations., Results: The mean doses (D mean ) in axillary lymph node (AL) level I, II and III in DIBH were 32.28 Gy (range 2.87-51.7), 20.1 Gy (range 0.44-53.84) and 3.84 Gy (range 0.25-39.23) vs. 34.93 Gy (range 10.52-50.40), 16.40 Gy (range 0.38-52.40) and 3.06 Gy (range 0.21-40.48) in FB (p < 0.0001). Accordingly, in DIBH the D mean for AL level I were reduced by 7.59%, whereas for AL level II and III increased by 22.56% and 25.49%, respectively. The D mean for the supraclavicular lymph nodes (SC) in DIBH was 0.82 Gy (range 0.23-4.11), as compared to 0.84 Gy (range 0.22-10.80) with FB (p = 0.002). This results in a mean dose reduction of 2.38% in DIBH. The D mean for internal mammary lymph nodes (IM) was 12.77 Gy (range 1.45-39.09) in DIBH vs. 11.17 Gy (range 1.34-44.24) in FB (p = 0.005). This yields a mean dose increase of 14.32% in DIBH., Conclusions: The DIBH technique may result in changes in the incidental dose exposure of regional lymph node areas., (© 2022. The Author(s).)

The stage and molecular pathology-dependent prognosis of breast cancer, the limited treatment options for triple-negative carcinomas, as well as the development of resistance to therapies illustrate the need for improved early diagnosis and the development of new therapeutic approaches. Increasing data suggests that some answers to these challenges could be found in the area of epigenetics. In this study, we focus on the current research of the epigenetics of breast cancer, especially on the potential of epigenetics for clinical application in diagnostics, risk stratification and therapy. The differential DNA methylation status of specific gene regions has been used in the past to differentiate breast cancer cells from normal tissue. New technologies as detection of circulating nucleic acids including microRNAs to early detect breast cancer are emerging. Pattern of DNA methylation and expression of histone-modifying enzymes have been successfully used for risk stratification. However, all these epigenetic biomarkers should be validated in larger clinical studies. Recent preclinical and clinical studies show a therapeutic benefit of epigenetically active drugs for breast cancer entities that are still difficult to treat (triple negative, UICC stage IV). Remarkably, epigenetic therapies combined with chemotherapies or hormone-based therapies represent the most promising strategy. At the current stage, the integration of epigenetic substances into established breast cancer therapy protocols seems to hold the greatest potential for a clinical application of epigenetic research.

Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 10 5 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.

In about 20-30% of all women with breast cancer, an increased number of cases of breast cancer can be observed in their family history. However, currently, only 5-10% of all breast cancer cases can be attributed to a pathogenic gene alteration. Molecular genetic diagnostics underwent enormous development within the last 10 years. Next-generation sequencing approaches allow increasingly extensive analyses resulting in the identification of additional candidate genes. In the present work, the germline molecular diagnostic analysis of a cohort of 228 patients with suspected hereditary breast and ovarian cancer syndrome (HBOC) was evaluated. The 27 pathogenic gene variants initially detected are listed, and their distribution in the high-risk BRCA1 and BRCA2 genes is presented in this study. In ten high-risk patients, in whom, to date, no pathogenic variant could be detected, an extended genetic analysis of previously not considered risk genes was performed. Three variants of uncertain significance and one pathogenic variant could be described. This proves the importance of extended analysis using current molecular genetic methods.

Molecular precision oncology faces two major challenges: first, to identify relevant and actionable molecular variants in a rapidly changing field and second, to provide access to a broad patient population. Here, we report a four-year experience of the Molecular Tumor Board (MTB) of the Comprehensive Cancer Center Freiburg (Germany) including workflows and process optimizations. This retrospective single-center study includes data on 488 patients enrolled in the MTB from February 2015 through December 2018. Recommendations include individual molecular diagnostics, molecular stratified therapies, assessment of treatment adherence and patient outcomes including overall survival. The majority of MTB patients presented with stage IV oncologic malignancies (90.6%) and underwent an average of 2.1 previous lines of therapy. Individual diagnostic recommendations were given to 487 patients (99.8%). A treatment recommendation was given in 264 of all cases (54.1%) which included a molecularly matched treatment in 212 patients (43.4%). The 264 treatment recommendations were implemented in 76 patients (28.8%). Stable disease was observed in 19 patients (25.0%), 17 had partial response (22.4%) and five showed a complete remission (6.6%). An objective response was achieved in 28.9% of cases with implemented recommendations and for 4.5% of the total population (22 of 488 patients). By optimizing the MTB workflow, case-discussions per session increased significantly while treatment adherence and outcome remained stable over time. Our data demonstrate the feasibility and effectiveness of molecular-guided personalized therapy for cancer patients in a clinical routine setting showing a low but robust and durable disease control rate over time.

Breast cancer (BC) represents the most common type of cancer in females worldwide. Endocrine therapy evolved as one of the main concepts in treatment of hormone-receptor positive BC. Current research focuses on the elucidation of tumour resistance mechanisms against endocrine therapy. In a translational in vitro approach, potential regulatory effects of clinically implemented BC anti-oestrogens on ERα, its coactivators DDX5, DDX17 and other DEADbox proteins as well as on the proliferation markers cyclin D1 and Ki67 were investigated on both the RNA and protein level. BC in vitro models for hormone-receptor positive (MCF-7, T-47D) and hormone-receptor negative cells (BT-20) were subjected to endocrine therapy. Anti-oestrogen-dependent expression regulation of target genes on the transcriptional and translational level was quantified and statistically assessed. Endocrine therapy decreases the expression levels of Ki67, cyclin D1 and ERα in hormone-receptor positive cells. In the hormone-receptor negative cells, the three parameters remained stable after endocrine therapy. Endoxifen triggers a downregulation of DDX5 and DDX23 in MCF-7 cells. Fulvestrant treatment downregulates the expression levels of all investigated DEADbox proteins in MCF-7 cells. In T-47D cells, endoxifen and fulvestrant lead to a decrease of all target gene expression levels. Interestingly, endocrine therapy affects DEADbox RNA expression levels in BT-20 cells, too. However, this result could only be confirmed for DDX1, immunocytologically. The investigated DEADbox proteins appear to correlate with the oestrogen-dependent tumourigenesis in hormone-receptor positive BC and show expression alterations after endocrine treatment.

Breast cancer tumor draining lymph nodes (TDLNs) display distinct morphologic changes depending on the breast cancer subtype. For triple-negative breast cancers (TNBC), draining LNs display a higher amount of secondary lymphoid follicles, which can be regarded as a surrogate marker for an activated humoral immune response. In the present study, we focus on PD1 + T-follicular helper cells (Tfh) in TDLNs of TNBC, since PD1 + Tfh are drivers of the germinal center (GC) reaction. We quantified PD1 + Tfh in 22 sentinel LNs with 853 GCs and interfollicular areas from 19 patients with TNBC by morphometry from digitalized immunostained tissue sections. Overall survival was significantly worse for patients with a higher number and area density of PD1 + Tfh within GCs of TDLNs. Further, we performed T-cell receptor gamma chain (TRG) analysis from microdissected tissue in the primary tumor and TDLNs. Eleven patients showed the same TRG clones in the tumor and the LN. Five patients shared the same TRG clones in the tumor and the GCs. In two patients, those clones were highly enriched inside the GCs. Enrichment of identical TRG clones at the tumor site vs. the TDLN was associated with improved overall survival. TDLNs are important relays of cancer immunity and enable surrogate approaches to predict the outcome of TNBC itself.

Ion mobility coupling to mass spectrometry facilitates enhanced identification certitude. Further coupling to liquid chromatography results in multi-dimensional analytical methods, especially suitable for complex matrices with structurally similar compounds. Modified nucleosides represent a large group of very similar members linked to aberrant proliferation. Besides basal production under physiological conditions, they are increasingly excreted by transformed cells and subsequently discussed as putative biomarkers for various cancer types. Here, we report a method for modified nucleosides covering 37 species. We determined collisional cross-sections with high reproducibility from pure analytical standards. For sample purification, we applied an optimized phenylboronic acid solid-phase extraction on media obtained from four different pancreatic cancer cell lines. Our analysis could discriminate different subtypes of pancreatic cancer cell lines. Importantly, they could clearly be separated from a pancreatic control cell line as well as blank medium. m1A, m27G, and Asm were the most important features discriminating cancer cell lines derived from well-differentiated and poorly differentiated cancers. Eventually, we suggest the analytical method reported here for future tumor-marker identification studies. Graphical abstract.

Cancer research of immune-modulating mechanisms mainly addresses the role of tumor-infiltrating immune cells. Mechanisms modulating the adaptive immune system at the primary activation site - the draining lymph node (LN) - are less investigated. Here we present tumor-caused histomorphological changes in tumor draining LNs of breast cancer patients, dependent on the localization (sentinel LN vs. non-sentinel LN), the tumor size, the intrinsic subtype and nodal metastatic status. The quantitative morphological study was conducted in breast cancer patients with at least one sentinel LN and no neoadjuvant therapy. All LNs were annotated considering to their topographical location, stained for IgD/H&E, digitized and quantitatively analyzed. In 206 patients, 394 sentinels and 940 non-sentinel LNs were categorized, comprising 40758 follicles and 7074 germinal centers. Subtype specific immunomorphological patterns were detectable: Follicular density was higher in LNs of Her2 enriched hormone receptor positive and triple-negative breast cancers whereas hormone receptor positive breast cancers showed more macrophage infiltrations in the LN cortex. Follicles are rounder in metastatic LNs and non-sentinel LNs. The identified immunomorphological changes reflect different underlying immunomodulations taking place in the tumor-draining LNs and should therefore be considered as possible prognostic and predictive markers for LN metastasis and therapy associated immunomodulation.

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

Background/aim: The pro-angiogenic Cyr61 protein has been associated with tumorigenesis and cancer progression in different gynecological carcinomas. In this study, we evaluated the potential impact and clinical relevance of Cyr61 expression in patients with primary non-metastatic cervical cancer (CC)., Patients and Methods: Cyr61 expression was assessed in tissue specimen of 48 patients with primary CC by immunohistochemical analysis. Expression levels were scored and correlated to clinico-pathological factors and outcome data., Results: High Cyr61 expression levels were present in 54.2% of CC tissues. Associations with histological grade (p=0.030), depth of tumor invasion (p=0.007) and GOG score (p=0.027) were observed. Patients who overexpressed Cyr61 displayed an increased death rate (30.8% vs. 18.2%) and a decreased 5-year-survival (76.9% vs. 86.4%)., Conclusion: Our data indicate a potential functional impact of Cyr61 in development and the progression of CC. The definite tumor-relevant function (suppressive/promoting) of Cyr61 in CC and the prognostic relevance of Cyr61 overexpression has to be evaluated in larger cohorts., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Introduction: The gastrin-releasing peptide receptor (GRPR) is overexpressed in breast cancer. The present study evaluates GRPR imaging as a novel imaging modality in breast cancer by employing positron emission tomography (PET) and the GRPR antagonist (68)Ga-RM2., Methods: Fifteen female patients with biopsy confirmed primary breast carcinoma (3 bilateral tumors; median clinical stage IIB) underwent (68)Ga-RM2-PET/CT for pretreatment staging. In vivo tumor uptake of (68)Ga-RM2 was correlated with estrogen (ER) and progesterone (PR) receptor expression, HER2/neu status and MIB-1 proliferation index in breast core biopsy specimens., Results: 13/18 tumors demonstrated strongly increased (68)Ga-RM2 uptake compared to normal breast tissue (defined as PET-positive). All PET-positive primary tumors were ER- and PR-positive (13/13) in contrast to only 1/5 PET-negative tumors. Mean SUVMAX of ER-positive tumors was 10.6±6.0 compared to 2.3±1.0 in ER-negative tumors (p=0.016). In a multivariate analysis including ER, PR, HER2/neu and MIB-1, only ER expression predicted (68)Ga-RM2 uptake (model: r(2) =0.55, p=0.025). Normal breast tissue showed inter- and intraindividually variable, moderate GRPR binding (SUVMAX 2.3±1.0), while physiological uptake of other organs was considerably less except pancreas. Of note, (68)Ga-RM2-PET/CT detected internal mammary lymph nodes with high (68)Ga-RM2 uptake (n=8), a contralateral axillary lymph node metastasis (verified by biopsy) and bone metastases (n=1; not detected by bone scan and CT)., Conclusion: Our study demonstrates that (68)Ga-RM2-PET/CT is a promising imaging method in ER-positive breast cancer. In vivo GRPR binding assessed by (68)Ga-RM2-PET/CT correlated with ER expression in primary tumors of untreated patients.

Synuclein gamma (SNCG) is under consideration as a potential biomarker in cancer biology. Up to date four different SNCG variants are described. Due to growing evidence suggesting correlations between aberrant alternative splicing processes and cancer progression, we investigated the effects of peritumoural conditions on expression pattern of SNCG in endometrial cancer (EC) in vitro. Compared to breast cancer cell lines, mRNA expression levels of all known SNCG isoforms 1-4 are significantly reduced in EC cell lines. We identified a novel alternatively spliced variant of isoform 2 (isoform 2 short) which is found highly expressed in EC cell lines. Hypoxia and acidosis trigger an up-regulation of isoform 2 short. EC cell lines are characterized by low SNCG protein levels under control conditions, but exhibit a significant increase triggered by hypoxia and acidosis. In addition we analysed the potential association between SNCG protein expression and clinico-pathological parameters in human EC samples. Our findings indicate a grade-dependent induction of SNCG protein expression in endometrial cancer. We identified for the first time a novel isoform of SNCG that is found specifically expressed in EC. Our results also strongly indicate the existence of a corresponding protein of isoform 2 short that potentially plays a critical role in EC cancer progression.

Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

Background: Since recent studies revealed the feasibility to detect blood-based microRNAs (miRNAs, miRs) in breast cancer (BC) patients a new field has been opened for circulating miRNAs as potential biomarkers in BC. In this pilot study, we evaluated to our knowledge for the first time whether distinct pattern of urinary miRNAs might be also applicable as innovative biomarkers for BC detection., Methods: Urinary miRNA expression levels of nine BC-related miRNAs (miR-21, miR-34a, miR-125b, miR-155, miR-195, miR-200b, miR-200c, miR-375, miR-451) from 24 untreated, primary BC patients and 24 healthy controls were quantified by realtime-PCR. The receiver operating characteristic analyses (ROC) and logistic regression were calculated to assess discriminatory accuracy., Results: Significant differences were found in the expression of four BC-associated miRNAs quantified as median miRNA expression levels. Urinary miR-155 levels were significantly higher in BC patients compared to healthy controls (1.49vs.0.25; p < 0.001). In contrast, compared to healthy controls, BC patients exhibited significantly lower urinary expression levels of miR-21 (2.27vs.5.07; p < 0.001), miR-125b (0.71vs.1.62; p < 0.001), and miR-451 (0.02vs.0.59 p = 0.004), respectively. The ROC including all miRNAs as well as the group of the four significant deregulated miRNAs separated BC patients from healthy controls with a very high (area under the receiver operating characteristic curve [AUC] = 0.932) and high accuracy (AUC = 0.887), respectively., Conclusions: We were able to demonstrate for the first time the feasibility to detect distinct BC-dependent urinary miRNA profiles. The expression levels of four urinary miRNAs were specifically altered in our cohort of BC patients compared to healthy controls. This distinct pattern offers the possibility for a specific discrimination between healthy women and primary BC patients. This sustains the potential role of urinary miRNAs as non-invasive innovative urine-based biomarkers for BC detection.

Background: Estrogen receptor alpha (ERa/ESR1) expression is regulated by alternative splicing. Its most frequently detectable exon7 skipping isoform (ERaD7) is a dominant negative variant. Elevated expression of ERaD7 was already detected in endometrial cancer (EC), while its potential prognostic significance has not been characterized so far. Exon7 contains potential binding sites for the two functional splicing regulatory opponents, HNRNPG and HTRA2-BETA1 known to trigger opposite effects on EC outcome. This study served to elucidate the influence of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing regulation and the impact of ERaD7 concentration on type 1 EC outcome., Methods: Functional in vitro experiments for HNRNPG and HTRA2-BETA1 in regard to the regulatory impact on endogenous and exogenous ERaD7 splicing were performed. Additionally, real-time PCR determined mRNA levels of ERaD7, HNRNPG and HTRA2-BETA1 in 116 type 1 EC patients., Results: HNRNPG and HTRA2-BETA1 were found to be specific regulators of ERa exon7 splicing. While HTRA2-BETA1 promoted exon7 inclusion, HNRNPG antagonized this effect by inducing exon7 skipping (p = 0.004). ERaD7 was detected in 71 out of 116 type 1 EC specimens. Statistical analyses revealed an inverse correlation between ERaD7 mRNA levels and tumor grading (p = 0.029), FIGO stage (p = 0.033) as well as lymph node metastases (p = 0.032), respectively. Furthermore, higher ERaD7 expression could be correlated to an improved disease-specific survival (p = 0.034)., Conclusions: Our study demonstrates antagonistic regulatory effects of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing with potential impact on type 1 EC clinical outcome due to the consecutively variable expression levels of the ERa isoform D7.

Background: Neoadjuvant chemotherapy (NC) is an established therapy in breast cancer, able to downstage positive axillary lymph nodes, but might hamper their detectibility. Even if clinical observations suggest lower lymph node yield (LNY) after NC, data are inconclusive and it is unclear whether NC dependent parameters influence detection rates by axillary lymph node dissection (ALND)., Methods: We analyzed retrospectively the LNY in 182 patients with ALND after NC and 351 patients with primary ALND. Impact of surgery or pathological examination and specific histomorphological alterations were evaluated. Outcome analyses regarding recurrence rates, disease free (DFS) and overall survival (OS) were performed., Results: Axillary LNY was significantly lower in the NC in comparison to the primary surgery group (median 13 vs. 16; p < 0.0001). The likelihood of incomplete axillary staging was four times higher in the NC group (14.8% vs. 3.4%, p < 0.0001). Multivariate analyses excluded any influence by surgeon or pathologist. However, the chemotherapy dependent histological feature lymphoid depletion was an independent predictive factor for a lower LNY. Outcome analyses revealed no significant impact of the LNY on local and regional recurrence rates as well as DFS and OS, respectively., Conclusion: NC significantly reduces the LNY by ALND and has profound effects on the histomorphological appearance of lymph nodes. The current recommendations for a minimum removal of 10 lymph nodes by ALND are clearly compromised by the clinically already established concept of NC. The LNY of less than 10 by ALND after NC might not be indicative for an insufficient axillary staging.

Background: This paper describes a randomized prospective study conducted in 308 patients undergoing caesarean section in spinal anaesthesia at a single hospital between 2010 and 2012 to find a suitable anti-emetic strategy for these patients., Material and Methods: Spinal anesthesia was performed in left prone position, at L3/L4 with hyperbaric 0.5% Bupivacaine according to a cc/cm body height ratio. There were no opioids given peri-operatively. The patients received either no prophylaxis (Group I) or tropisetron and metoclopramide (Group II) or dimenhydrinate and dexamethasone (Group III), or tropisetron as a single medication (Group IV). The primary outcome was nausea and/or vomiting (NV) in the intraoperative, early (0-2 h) or late (2-24 h) postoperative period. Multivariate statistical analysis was conducted with a regression analysis and a backward elimination of factors without significant correlation., Results: All prophylactic agents significantly reduced NV incidence intraoperatively. Relative risk reduction for NV by prophylaxis was most effective (59.5%) in Group II (tropisetron and metoclopramide). In Group III (dimenhydrinate and dexamethasone), NV risk was reduced by 29.9% and by 28.7% in Group IV (tropisetron mono-therapy). The incidence of NV in the early (0?2 h) and the late (2?24 h) postoperative period was low all over (7.8%), but the relative risk reduction of NV in the early postoperative period was 54.1% (Group IV), 45.1% (Group III), and 34.8% (Group II), respectively. In the late postoperative period, there was no significant difference between the 4 groups., Conclusions: We recommend a prophylactic medication with tropisetron 2 mg and metoclopramide 20 mg for patients during caesarean section. These agents are safe, reasonably priced, and highly efficient in preventing nausea and vomiting.