Severe immune suppression is frequent in late-stage tumor patients and promotes tumor immune evasion and subsequent tumor progression. Regulatory T cells (Treg) are major suppressors of anti-tumor immune responses. Therefore, targeting of Treg has become a key goal of anti-tumor therapy. Several preclinical and clinical observations suggest that Treg can be depleted by cyclophosphamide. Over a period of 3 months, we investigated the effect of metronomic low-dose cyclophosphamide on Treg numbers, suppressive capacity and proliferation on endogenous anti-tumor T-cell responses and on their correlation to clinical outcome in 12 patients with treatment-refractory metastasized breast cancer who received single-agent 50 mg cyclophosphamide p.o. daily. Cyclophosphamide treatment initially caused a significant reduction in circulating Treg by more than 40% (P = 0.002). However, Treg numbers completely recovered during the treatment due to increased proliferative activity and maintained their suppressive capacity. Treg depletion coincided with a strong increase in breast tumor-reactive T cells (P = 0.03) that remained at high levels during the whole period. Numbers of tumor-reactive T cells but not of Treg correlated with disease stabilization (P = 0.03) and overall survival (P = 0.027). We conclude that metronomic low-dose cyclophosphamide only transiently reduces Treg but induces stable tumor-specific T-cell responses, which correlate with improved clinical outcome in advanced-stage breast cancer patients.

Breast cancer is a leading cause of cancer-related death in women. Small open reading frame (sORF)-encoded proteins or microproteins constitute a new class of molecules often transcribed from presumed long non-coding RNA transcripts (lncRNAs). The translation of some of these sORFs has been confirmed, but their cellular function and importance remains largely unknown. Here, we report the identification and characterization of a novel microprotein of 10 kDa, which we named Cancer-Associated Small Integral Membrane Open reading frame 1 (CASIMO1). CASIMO1 RNA is overexpressed predominantly in hormone receptor-positive breast tumors. Its knockdown leads to decreased proliferation in multiple breast cancer cell lines. Its loss disturbs the organization of the actin cytoskeleton, leads to inhibition of cell motility, and causes a G/G cell cycle arrest. The proliferation phenotype upon overexpression is observed only with CASIMO1 protein expression, but not with a non-translatable mutant attributing the effects to the sORF-derived protein rather than a lncRNA function. CASIMO1 microprotein interacts with squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis and a known oncogene in breast cancer. Overexpression of CASIMO1 leads to SQLE protein accumulation without affecting its RNA levels and increased lipid droplet clustering, while knockdown of CASIMO1 decreased SQLE protein abundance and ERK phosphorylation downstream of SQLE. Importantly, SQLE knockdown mimicked the CASIMO1 knockdown phenotype and in turn SQLE overexpression fully rescued the effect of CASIMO1 knockdown. These findings establish CASIMO1 as the first functional microprotein that plays a role in carcinogenesis and is implicated in the cell lipid homeostasis.

BackgroundBreast cancer represents the most common malignant disease in women worldwide. As currently systematic palliative treatment only has a limited effect on survival rates, the concept of health-related quality of life (HRQoL) is gaining more and more importance in the therapy setting of metastatic breast cancer. One of the major patient-reported outcomes (PROs) for measuring HRQoL in patients with breast cancer is provided by the European Organization for Research and Treatment of Cancer (EORTC). Currently, paper-based surveys still predominate, as only a few reliable and validated electronic-based questionnaires are available. Facing the possibilities associated with evolving digitalization in medicine, validation of electronic versions of well-established PRO is essential in order to contribute to comprehensive and holistic oncological care and to ensure high quality in cancer research.ObjectiveThe aim of this study was to analyze the reliability of a tablet-based measuring application for EORTC QLQ-C30 in German language in patients with adjuvant and (curative) metastatic breast cancer.MethodsPaper- and tablet-based questionnaires were completed by a total of 106 female patients with adjuvant and metastatic breast cancer recruited as part of the e-PROCOM study. All patients were required to complete the electronic- (e-PRO) and paper-based versions of the HRQoL EORTC QLQ-C30 questionnaire. A frequency analysis was performed to determine descriptive sociodemographic characteristics. Both dimensions of reliability (parallel forms reliability [Wilcoxon test] and test of internal consistency [Spearman rho and agreement rates for single items, Pearson correlation and Kendall tau for each scale]) were analyzed.ResultsHigh correlations were shown for both dimensions of reliability (parallel forms reliability and internal consistency) in the patient’s response behavior between paper- and electronic-based questionnaires. Regarding the test of parallel forms reliability, no significant differences were found in 27 of 30 single items and in 14 of 15 scales, whereas a statistically significant correlation in the test of consistency was found in all 30 single items and all 15 scales.ConclusionsThe evaluated e-PRO version of the EORTC QLQ-C30 is reliable for patients with both adjuvant and metastatic breast cancer, showing a high correlation in almost all questions (and in many scales). Thus, we conclude that the validated paper-based PRO assessment and the e-PRO tool are equally valid. However, the reliability should also be analyzed in other prospective trials to ensure that usability is reliable in all patient groups.Trial RegistrationClinicalTrials.gov NCT03132506; https://clinicaltrials.gov/ct2/show/NCT03132506 (Archived by WebCite at http://www.webcitation.org/6tRcgQuou).

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Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62-92% of in situ samples (n=13), 72-97% of invasive samples from the first validation set (n=32), and 86-100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.

IntroductionVaginal intraepithelial neoplasia (VAIN) is a pre-malignant lesion, potentially leading to vaginal cancer. It is a rare disease, representing less than 1% of all intraepithelial neoplasia of the female genital tract. Similar to cervical intraepithelial neoplasia (CIN), there are three different grades of VAIN. VAIN 1 is also known as a low-grade squamous intraepithelial lesion (LSIL), whereas VAIN 2 and VAIN 3 both represent high-grade squamous intraepithelial lesions (HSIL). Risk factors for the development of VAIN are similar to those for cervical neoplasia, i.e. promiscuity, starting sexual activity at an early age, tobacco consumption and infection with human papillomavirus (HPV). However, compared to other intraepithelial neoplasia such as CIN or VIN (vulvar intraepithelial neoplasia), there still is little understanding about the natural course of VAIN and its capacity for pro- or regression. Furthermore, there is controversial data about the HPV detection rate in VAIN lesions.Patients and Methods67 patients with histologically confirmed VAIN, who were diagnosed between 2003 and 2011 at the University Women´s Hospital of Heidelberg Germany, were included in this study. The biopsies of all participating patients were subjected to HPV genotyping. GP-E6/E7 Nested Multiplex PCR (NMPCR) was used to identify and genotype HPV. Eighteen pairs of type-specific nested PCR primers were assessed to detect the following "high-risk" HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, as well as the "low-risk" genotypes 6/11, 42, 43 and 44. The data was analyzed with the software SAS (Statistical Analysis System).ResultsAll 67 cases were eligible for DNA analysis. The median age was 53 years. The largest group with 53% (n = 36) was formed by women, who were first diagnosed with VAIN between the age of 41 to 60 years. 50% (n = 37) of the patients presented a VAIN in the upper 1/3 of the vagina. 58 (87%) were diagnosed with HSIL (VAIN). The median age in patients with LSIL (VAIN) was 53 years and in patients with HSIL (VAIN) 53.5 years. 12 women (18%) had an immunosuppression. HPV positivity was confirmed in 37 patients (55%). Except for a single patient, who had a triple infection with HPV types 6/11, 16 and 68, only infections with one single HPV genotype were detected. An infection with the HPV genotypes 31, 39, 45, 51, 58, 59, 66, 42, 43 and 44 couldn’t be found in any of the patients. In 28 patients with diagnosed VAIN, an infection with HPV 16 could be shown, 24 (86%) of them were diagnosed with a HSIL (VAIN). 16 (24%) women presented condylomata and 13 of them (81%) had a positive HPV status. However, only 47% of the women without condylomata presented a positive HPV status, resulting in a significant correlation (p = 0.0164) between condylomata and HPV infection. In 28 of all 67 patients (42%), recurrence of the neoplasia occurred.ConclusionHPV 16 is the main virus-type to be associated with the development of a VAIN. Also, HPV 16 infection, VIN or condylomata acuminata in the past ...

Objectives. To evaluate virtual touch tissue imaging quantification (VTIQ) as a new elastography method concerning its intra- and interexaminer reliability and its ability to differentiate benign from malignant breast lesions in comparison to and in combination with ultrasound (US) B-mode breast imaging reporting and data system (BI-RADS) assessment. Materials and Methods. US and VTIQ were performed by two examiners in 103 women with 104 lesions. Intra- and interexaminer reliability of VTIQ was assessed. The area under the receiver operating curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BIRADS, VTIQ, and combined data were compared. Results. Fifty-four of 104 lesions were malignant. Intraexaminer reliability was consistent, and interexaminer agreement showed a strong positive correlation (r = 0.93). The mean VTIQ values in malignant lesions were significantly higher than those in benign (7.73 m/s ± 1.02 versus 4.46 m/s ± 1.87; P < 0.0001). The combination of US-BIRADS with the optimal cut-off for clinical decision making of 5.18 m/s yielded a sensitivity of 98%, specificity of 82%, PPV of 86%, and NPV of 98%. The combination of BIRADS and VTIQ led to improved test validity. Conclusion. VTIQ is highly reliable and reproducible. There is a significant difference regarding the mean maximum velocity of benign and malignant lesions. Adding VTIQ to BIRADS assessment improves the specificity.

Our results suggest that the German version of the MFAS is a reliable and valid questionnaire to measure the emotional relationship of the mother to the unborn child during pregnancy.