First report on successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe.

RESEARCH QUESTION: Cryopreservation of ovarian tissue is one feasible option to preserve female fertility prior to cancer treatment. The slow freezing protocol represents the current standard approach, while vitrification has been suggested as a promising alternative. This paper reports the follow-up and first successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe. DESIGN: After the patient received a diagnosis of breast cancer, ovarian tissue was removed laparoscopically and sent via overnight transportation to University Hospital Bonn for vitrification on site. The patient was treated with chemotherapy, leading to ovarian failure. After 2 years, retransplantation of the vitrified, rapid warmed tissue was conducted on site. RESULTS: Two months after grafting, the patient reported regular menstrual cycles. After 1 further month a clinical pregnancy occurred, which ended in a spontaneous abortion at the 8th week of pregnancy. Six months after grafting, another naturally conceived pregnancy was determined, resulting in the birth of a healthy boy 14 months after retransplantation of the ovarian tissue. CONCLUSIONS: Complementing the successful deliveries reported by the groups of Suzuki (Japan) and Silber (USA) regarding vitrified tissue, the current results confirm the high potential of this cryopreservation method in a clinical routine setting as an alternative approach to the widespread slow freezing method.

Evaluation of the Gonadotoxicity of Cancer Therapies to Improve Counseling of Patients About Fertility and Fertility Preservation Measures: Protocol for a Retrospective Systematic Data Analysis and a Prospective Cohort Study.

BACKGROUND: Cytotoxic treatments such as chemo- and radiotherapy and immune therapies are required in cancer diseases. These therapies have the potential to cure patients but may also have an impact on gonadal function and, therefore, on fertility. Consequently, fertility preservation treatments such as freezing of gametes and gonadal tissue might be required. However, as detailed data about the necessity to perform fertility preservation treatment are very limited, this study was designed to fill this data gap. OBJECTIVE: Primary objective of this study is to analyze the impact of cancer therapies and chemotherapies on the ovarian reserve and sperm quality. Secondary objectives are to analyze the (1) impact of cancer therapies and chemotherapies on other fertility parameters and (2) probability of undergoing fertility preservation treatments in relation to specific cancer diseases and treatment protocols and the probability to use the frozen gametes and gonadal tissue to achieve pregnancies. METHODS: First, previously published studies on the gonadotoxicity of chemo- and radiotherapies among patients with cancer will be systematically analyzed. Second, a prospective cohort study set up by approximately 70 centers in Germany, Switzerland, and Austria will collect the following data: ovarian function by analyzing anti-Müllerian hormone (AMH) concentrations and testicular function by analyzing sperm parameters and total testosterone immediately before and around 1 year after gonadotoxic therapies (short-term fertility). A follow-up of these fertility parameters, including history of conceptions, will be performed 5 and 10 years after gonadotoxic therapies (long-term fertility). Additionally, the proportion of patients undergoing fertility-preserving procedures, their satisfaction with these procedures, and the amount of gametes and gonadal tissue and the children achieved by using the frozen material will be analyzed. Third, the data will be merged to create the internet-based data platform FertiTOX. The platform will be structured in accordance with the ICD (International Classification of Diseases) classification of cancer diseases and will be easily be accessible using a specific App. RESULTS: Several funding bodies have funded this study. Ten systematic reviews are in progress and the first one has been accepted for publication. All Swiss and many German and Austrian ethics committees have provided their approval for the prospective cohort study. The study registry has been set up, and a study website has been created. In total, 50 infertility centers have already been prepared for data collection, which started on December 1, 2023. CONCLUSIONS: The study can be expected to bridge the data gap regarding the gonadotoxicity of cancer therapies to better counsel patients about their infertility risk and their need to undergo fertility preservation procedures. Initial data are expected to be uploaded on the FertiTOX platform in 2026. TRIAL REGISTRATION: ClinicalTrials.gov NCT05885048; https://clinicaltrials.gov/study/NCT05885048. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51145.

High FSH levels impair VEGF secretion of human, frozen-thawed ovarian cortical tissue in vitro.

Cryopreservation and reimplantation of human ovarian tissue restore the ovarian hormonal function and fertility due to the preservation of follicles. As the success depends on proper angiogenesis, different approaches aim to support this process. In mice, pretreatment of ovarian tissue with FSH shows increased follicular numbers probably due to the supported angiogenesis by an increased vascular endothelial factor (VEGF) expression. However, in human tissue it remains completely unclear, which effect the hormonal status of the patient has at the time point of reimplantation. Frozen-thawed human ovarian cortical tissue was cultured for 48 h with 0, 1 or 10 ng/mL recombinant human FSH. VEGF-A expression was assessed by ELISA and immunohistofluorescence (IHF) analysis. By IHF, HIF-1α and FSHR expression dependency on culture and FSH concentration was analyzed. Follicles at all stages expressed VEGF-A, which increases during folliculogenesis. Frozen-thawed human ovarian cortical tissue secreted a not statistically different amount of VEGF-A, when cultured in presence of 1 ng/mL FSH (17.5 mIU/mL). However, the presence of 10 ng/mL FSH (175 mIU/mL) significantly decreased VEGF-A expression and secretion. The high FSH concentration increased especially the VEGF-A expression of already growing follicles. The presence of pre-menopausal concentrations of FSH had no significant effect on VEGF-A expression, whereas the presence of elevated FSH levels decreased cortical VEGF-A expression. A hormonal pre-treatment of women with elevated FSH concentrations prior to reimplantation might be considered to support angiogenesis. Here, we show that VEGF-A expression by follicles is affected by FSH dependent on the concentration.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue.

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

Cryostorage of human ovarian tissue: evaluating the storage and disposal pattern over a 22-year period in 2475 patients.

RESEARCH QUESTION: What are the parameters of age, indications for ovarian tissue cryopreservation, storage characteristics and reasons for tissue disposal in a large cohort of individuals undertaking cryopreservation? DESIGN: The relevant parameters in a single university centre were revised and digitalized in the period from 2019 to 2021. To assess patients' motivation at the end of storage, patients were contacted by letter, e-mails and telephone calls. RESULTS: A group of 2475 patients with stored ovarian tissue were analysed in the time period between 2000 and 2021; the response rate for contact calls and letters was 28.8% (224/777). Where storage had ended (n = 1155), patients had on average stored for 3.8 years and begun storing at age 30 years; the main indications were breast cancer (53%) and lymphoma (17.5%). Of these participants, 2.5% had a transplantation on site, 10.3% transferred their tissue to another cryobank and 11.5% were deceased. The majority of the group (75.7%) ended their storage due to pregnancy (49.1%), a lack of desire to have children (25.9%), storage fees that were too expensive (8.9%), death (8.5%), recurrence of cancer (8.5%), lack of a partner (4%) and fear of surgery in the future (3.1%); 6.7% retrospectively regretted ending storage. CONCLUSIONS: The pregnancy rate of 49.1%, resulting from ovarian tissue that was not removed during surgery for scheduled ovarian tissue cryopreservation supports the clinical approach of removing and cryopreserving only 25-50% of one ovary. It is proposed that interdisciplinary counselling should be implemented not only prior to fertility preservation, but also when intending to end storage.

Fertility preservation counselling for women with endometriosis: a European online survey.

BACKGROUND: Endometriosis is a common cause for infertility. Decreased ovarian reserve due to pathology or surgical management can reduce the chances of natural pregnancy and limit the effectiveness of controlled ovarian stimulation during fertility treatment. Cryopreservation of oocytes or ovarian cortex prior to surgery or before loss of follicular capital is a strategy to preserve fecundity. METHODS: An online survey was sent to reproductive specialists and gynecological surgeons representing major centers of reproductive medicine in Europe to investigate current fertility preservation practices for endometriosis patients. RESULTS: Of 58 responses, 45 (77.6%) in 11/13 countries reported the existence of endometriosis management guidelines, of which 37/45 (82.2%) included treatment recommendations for infertile patients. Most centers (51.7%) reserved fertility counselling for severe endometriosis (large endometriomas with or without deep endometriosis) while 15.5% of centers did not offer fertility preservation for endometriosis. CONCLUSIONS: To address non-uniformity in available guidelines and the diversity in fertility preservation practices, we propose an algorithm for managing patients with severe endometriosis most likely to be impacted by reduced ovarian reserve. Improved awareness about the possibilities of fertility preservation and clear communication between gynaecological surgeons and reproductive medicine specialists is mandatory to address the unmet clinical need of preventing infertility in women with endometriosis.

Metabolic and secretory recovery of slow frozen-thawed human ovarian tissue in vitro.

Within the options available for fertility preservation, cryopreservation of ovarian cortical tissue has become an important technique. Freezing and thawing procedures have been optimized to preserve tissue integrity and viability. However, the improvement of the tissue retransplantation is currently of great interest. Rapid angiogenesis is needed at the retransplantation site to accomplish sufficient blood supply to provide oxygen and nutrients. Many studies address this issue. However, we need to understand the physiology of the thawed tissue to gain further understanding of the complexities of the procedure. As freezing and thawing generally impairs cellular metabolism, we aimed to characterize the changes in metabolic activity and secretion of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) of frozen-thawed ovarian cortical tissue over time. Biopsy punches of ovarian cortical tissue from patients undergoing fertility preservation were maintained in culture without freezing or after a slow-freezing and thawing procedure. VEGF-A secretion was measured after 48 h by ELISA. To examine temporary changes, metabolic activity was assessed for both fresh and frozen-thawed tissue of the same patient. Metabolic activity and VEGF-A secretion were measured at 0, 24 and 48 h in culture. Thawed ovarian cortical tissue secreted significantly less VEGF-A compared to fresh ovarian cortical tissue within 48 h of culture. After thawing, metabolic activity was significantly reduced compared to fresh ovarian cortex but over the course of 48 h, the metabolic activity recovered. Similarly, VEGF-A secretion of thawed tissue increased significantly over 48 h. Here, we have shown that it takes 48 h for ovarian cortical tissue to recover metabolically after thawing, including VEGF-A secretion.

In vitro growth (IVG) of human ovarian follicles in frozen thawed ovarian cortex tissue culture supplemented with follicular fluid under hypoxic conditions.

BACKGROUND: Despite its clinical success rates, transplantation after ovarian tissue cryopreservation (OTC) remains a matter of concern. Certain cancer subtypes may lead to the transfer of malignant cells when transplantation of affected ovarian tissue is conducted. IVG and subsequent isolation of vital follicles obtained from frozen thawed ovarian tissue for further in vitro maturation (IVM) would expand current fertility protection techniques while reducing the risk of retransplanting malignant cells. METHODS: A total of 216 cortical biopsies from 3 patients were included in this study in 4 treatment groups. After freezing, thawing and 8 days of hypoxic tissue culture supplemented with different concentrations of human follicular fluid (HuFF) and follicle-stimulating hormone (FSH), follicles were isolated enzymatically and stained with calcein to determine follicular viability. Numbers and size of vital follicles were assessed by fluorescence microscopy (Ti2, Nikon) and specified by computer assisted, semi-automated measurement (NIS software, Nikon). To estimate the effect of in vitro culture on apoptosis, tissue sections were stained for nicked DNA (TUNEL) prior and after tissue culture. RESULTS: Analysing 3025 vital follicles, we observed significant differences [P < 0.01] regarding follicle size when hypoxic tissue culture was supplemented with HuFF compared with the control group on day 1, individual follicles reached sizes > 100 µm. CONCLUSIONS: The results implicate that HuFF contains valuable factors contributing to significant IVG of follicles in human ovarian tissue and could be regarded as an additional tool in personalized fertility restoration prior to retransplantation of ovarian tissue.

How can fertility counseling be implemented for every newly diagnosed pediatric patient facing gonadotoxic treatment?-A single-center experience.

Since the survival rates of pediatric patients undergoing cancer treatment or hematopoietic stem cell transplantation (HSCT) have increased rapidly in recent decades, the late effects of treatment are now an important focus of patient care. Access to fertility preservation (FP) procedures as well as their financing differs considerably across Europe. However, some countries in Europe have recently changed the legal basis for financing FP procedures; therefore, the implementation of structures is mandatory to give patients access to FP. In this prospective cohort study, we characterized the process for establishing pediatric fertility counseling, including the development of an in-house standard procedure for recommendations regarding FP with potentially gonadotoxic treatment and valuating data from all FP counseling sessions. All data concerning patient characteristics (pubertal status, disease group) and recommendation of FP measures were prospectively collected and adoption of FP measures analyzed. Prior to the establishment of a structured process for FP in our pediatric oncology and stem cell transplantation center, there was no standardized FP counseling. We demonstrate that with the establishment of an inhouse standard procedure, it is possible to give consistent yet individualized FP counseling to approximately 90% of our patients facing gonadotoxic treatment, counseling over 200 patients between 2017 and 2019. This pilot study could potentially be adapted in other pediatric hematology, oncology, and stem cell transplantation centers to allow a more standardized handling of FP counseling for all patients facing gonadotoxic treatment.

DNA Damaged Induced Cell Death in Oocytes.

The production of haploid gametes through meiosis is central to the principle of sexual reproduction. The genetic diversity is further enhanced by exchange of genetic material between homologous chromosomes by the crossover mechanism. This mechanism not only requires correct pairing of homologous chromosomes but also efficient repair of the induced DNA double-strand breaks. Oocytes have evolved a unique quality control system that eliminates cells if chromosomes do not correctly align or if DNA repair is not possible. Central to this monitoring system that is conserved from nematodes and fruit fly to humans is the p53 protein family, and in vertebrates in particular p63. In mammals, oocytes are stored for a long time in the prophase of meiosis I which, in humans, can last more than 50 years. During the entire time of this arrest phase, the DNA damage checkpoint remains active. The treatment of female cancer patients with DNA damaging irradiation or chemotherapeutics activates this checkpoint and results in elimination of the oocyte pool causing premature menopause and infertility. Here, we review the molecular mechanisms of this quality control system and discuss potential therapeutic intervention for the preservation of the oocyte pool during chemotherapy.

Quantitative analysis of the sHLA-G protein in seminal plasma.

BACKGROUND: Recent studies revealed that maternal and embryonic contributions impact on HLA-G protein expression and might contribute to pregnancy success or failure. The main objective of this study was to examine the paternal levels of the immunoregulatory soluble human leukocyte antigen-G (sHLA-G) protein in seminal plasma and testicular biopsy samples during artificial reproductive technique (ART) treatment and to investigate possible correlations with other semen parameters, age, and pregnancy outcome of the female partner. METHODS: Soluble HLA-G levels of 106 seminal plasma samples and eight testicular biopsy samples were determined using a commercial sHLA-G Enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: We observed a significant negative correlation of male age with total sHLA-G amount (P 0.023, R -0.221) and semen volume (P = 0.047, R -0.193). Testicular biopsy samples were analyzed and tested positively with sHLA-G ELISA. Levels of sHLA-G in seminal plasma samples from men with normozoospermia did not deviate significantly from those with reduced semen quality. No significant difference of sHLA-G levels in seminal plasma and pregnancy outcome of the female partner was detected. Our data showed that age of men with normozoospermia was significantly lower when the female partner conceived after ART treatment (P = 0.016, Mann-Whitney U test). CONCLUSION: High sHLA-G levels in seminal plasma of the male partner appear not to be required for pregnancy but might contribute among other factors to the success of establishing and maintaining pregnancy through long-term priming of the female uterine milieu.

Claudin-1 is linked to presence of implants and micropapillary pattern in serous borderline epithelial tumours of the ovary.

AIMS: Expression of Claudin-1 has been associated with prognosis in several cancers. Here we investigated the expression pattern of Claudin-1 in borderline tumours of the ovary (BOT). METHODS: We analysed a cohort of 114 cases of borderline tumour (BOT). Claudin-1 expression was studied by immunohistochemistry using a polyclonal antibody and was compared with clinical and histopathological characteristics. RESULTS: Strong Claudin-1 expression was found in 30 cases (26.3%) independent of histological subtype. Expression was significantly less frequent in International Federation of Gynecology and Obstetrics (FIGO) stage I (p= 0.045), while the presence of microinvasion did not correlate with Claudin-1 expression. In contrast, we detected a highly significant association of Claudin-1 expression with the presence of peritoneal implants (p=0.003) and micropapillary pattern (p=0.047), which are features exclusively seen in serous BOT. Moreover, when we restricted our analysis to the subtype of serous BOT, the association of Claudin-1 expression with peritoneal implants (p<0.001) and micropapillary pattern (p =0.003) remained highly significant. CONCLUSIONS: In conclusion, Claudin-1 expression is associated with the presence of peritoneal implants and micropapillary pattern, which have been shown to be associated with poor prognosis. We speculate that overexpression of Claudin-1 might be linked to the mitogen-activated protein kinase pathway activation in BOT and suggest further studies to define its prognostic and potential therapeutic value.

[Fertility Preservation in Prepubertal und Pubertal Children and Adolescents]. / Fertilitätserhalt bei präpubertären und pubertären Kindern und Jugendlichen.

BACKGROUND: Due to rising survival rates in cancer and autoimmune diseases fertility preservation before gonadotoxic therapies has become increasingly important. Although fertility can be significantly affected by gonadotoxic therapies, the possibility of fertility preservation during childhood has not been sufficiently considered so far. METHODS: Selective literature research with presentation of fertility preservation methods, their indications, implementations, risks and efficacy. RESULTS: Measures are indicated in all girls and boys at high risk of gonadal damage. The complexity of the techniques requires special expertise in the counseling and implementation, which is offered to girls in counselling Germany especially in the centers of FertiPROTEKT (www.fertiprotekt.com). In girls, mainly cryopreservation of ovary tissue is considered. In postpubertal girls cryopreservation of oocytes is also possible. In postpubertal boys sperm can be preserved. Freezing of testicular tissue is still experimental in prepubertal boys. Success rates are still difficult to quantify; birth rates of about 50% are discussed. All procedures are not covered by health insurance. CONCLUSION: In children and adolescents, measures of fertility preservation should be considered in cases of highly gonadotoxic therapies, and appropriate advice should be given by specialists.

TFF3 Expression as Stratification Marker in Borderline Epithelial Tumors of the Ovary.

Borderline tumors (BOT) of the ovary account for 10% to 20% of ovarian neoplasms. Like ovarian cancer, BOT encompass several different histological subtypes (serous, mucinous, endometrioid, clear cell, transitional cell and mixed) with serous (SBOT) and mucinous (MBOT) the most common. Current hypotheses suggest low-grade serous carcinoma may develop in a stepwise fashion from SBOT whereas the majority of high grade serous carcinomas develop rapidly presumably from inclusion cysts or ovarian surface epithelium. The pathogenesis of mucinous ovarian tumors is still puzzling. Molecular markers could help to better define relationships between such entities. Trefoil factor-3 (TFF3) is an estrogen-regulated gene associated with prognosis in different types of cancer. It has also been included in a recent marker panel predicting subtypes of ovarian carcinoma. We analyzed the expression of TFF3 by immunohistochemistry in a cohort of 137 BOT and its association with histopathological features. Overall expression rate of TFF3 was 21.9%. None of the BOT with serous and endometrioid histology displayed strong TFF3 expression. On the other hand, TFF3 was highly expressed in 61.4% of MBOT cases and 33.3% of BOT with mixed histology (P < 0.001) suggesting a potential function of the protein in that subtypes. Associations of TFF3 expression with FIGO stage and micropapillary pattern were significant in the overall cohort but confounded by their correlation with histological subtypes. The highly specific expression of TFF3 in MBOT may help to further clarify potential relationships of tumors with mucinous histology and warrants further studies.

Association Between Genomic Metrics and Immune Infiltration in Triple-Negative Breast Cancer.

Importance: Why some triple-negative breast cancers (TNBCs) have high and others have low immune cell infiltration is unknown. Understanding how immune surveillance shapes the cancer genome could help in the selection of patients and the development of more effective immunotherapy strategies. Objective: To examine the association between genomic metrics and the extent of immune infiltration in TNBCs. Design, Setting, and Participants: This study, performed from June 1, 2015, through January 31, 2017, used DNA and RNA sequencing data and messenger RNA expression results from The Cancer Genome Atlas (TCGA) breast cancer data set (n = 1215) to calculate previously described immune metagene expression values and histologic lymphocyte counts to quantify immune infiltration and assign prognostic categories to TNBCs. It used the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data set as an independent validation cohort. The study compared clonal heterogeneity, somatic total mutational load, neoantigen load, and somatic copy number alteration levels between immune-rich TNBC cohorts with good prognosis and immune-poor TNBC cohorts with poor prognosis. The study also compared the distribution of mutations in 119 canonical cancer genes. Main Outcomes and Measures: Correlation between immune prognostic category and genomic metrics of the cancer. Results: This study of 193 TNBC samples with patient survival information found an inverse association between clonal heterogeneity and immune metagene expression (ρ = −0.395, P = 2 × 10−8). The study also found an inverse association between immune metagene expression and somatic copy number alteration levels (ρ = −0.484, P = 2 × 10−10). Lymphocyte-rich TNBCs with good prognosis had significantly lower mutation and neoantigen counts than did lymphocyte-poor TNBCs with poor prognosis. The robustness of the study results was confirmed by using various immune metagenes in the same TCGA data set and in the independent METABRIC data set. Conclusions and Relevance: This study suggests that immune-rich TNBCs may be under an immune surveillance that continuously eliminates many immunogenic clones, resulting in lower clonal heterogeneity. These cancers may also represent the subset of TNBCs that could derive benefit from immune checkpoint inhibitor therapy to tilt the balance in favor of the immune system.

IMP3 Expression in Borderline Tumors of the Ovary.

BACKGROUND/AIM: Borderline ovarian tumors (BOTs) have a less aggressive behavior than invasive epithelial ovarian tumors. Still some patients relapse or succumb to disease. Molecular markers that reliably predict prognosis are lacking. Insulin-like growth factor II mRNA-binding protein (IMP3) has been suggested as a prognostic marker in colorectal, hepatocellular, and ovarian clear-cell carcinomas. MATERIALS AND METHODS: We analyzed the expression of IMP3 by immunohistochemistry in a cohort of 140 BOT and its association with histopathological features. RESULTS: We found no association of IMP3 expression with patients' age, FIGO stage, microinvasion, and presence of implants. In contrast, IMP3 expression correlated to mucinous subtype of BOTs (42.2% vs. 9.5% among other subtypes) (p<0.001). IMP3 expression was found to be associated with the presence of in situ carcinoma in MBOT, but not in other subtypes (p=0.021). CONCLUSION: Expression of IMP3 in BOT is associated with the mucinous subtype and may serve as an early indicator for the development of malignant features.

Fascin-1 expression as stratification marker in borderline epithelial tumours of the ovary.

AIMS: To evaluate the actin-bundling protein fascin-1 (FSCN1) as marker for borderline ovarian tumours (BOTs). METHODS: We analysed a retrospective cohort of 140 BOTs with validated diagnosis by an independent pathologist. Immunohistochemical detection of FSCN1 was quantified as combined immunoreactive score (CIS) blinded to clinical patient data. Analyses were first performed for FSCN1 positive versus negative, and then verified using three categories derived from the observed distribution (negative, weak, strong; CIS 0, 1-2, 3-9). RESULTS: We detected FSCN1 positivity in 51.4%, and strong expression (CIS 3-9) in 14.3% of the samples. FSCN1 positivity was associated with serous subtype (p<0.001) and micropapillary pattern (p<0.001). Correlation with micropapillary pattern remained significant within the serous BOT (SBOT) subgroup (p=0.022). Strong FSCN1 expression (CIS 3-9) was associated both with the presence of implants (p=0.022), and a higher International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.020). CONCLUSIONS: Our analysis links FSCN1 with SBOT with micropapillary pattern. Strong expression is associated with higher FIGO stage and the presence of implants, both related to elevated risk of recurrence. Hence, FSCN1 is an interesting marker worth further analyses of its prognostic value in BOTs.

Acid ceramidase is associated with an improved prognosis in both DCIS and invasive breast cancer.

Acid ceramidase (ASAH1) a key enzyme of sphingolipid metabolism converting pro-apoptotic ceramide to sphingosine has been shown to be overexpressed in various cancers. We previously demonstrated higher expression of ASAH1 in ER positive compared to ER negative breast cancer. In the current study we performed subtype specific analyses of ASAH1 gene expression in invasive and non invasive breast cancer. We show that expression of ASAH1 is mainly associated with luminal A - like cancers which are known to have the best prognosis of all breast cancer subtypes. Moreover tumors with high ASAH1 expression among the other subtypes are also characterized by an improved prognosis. The good prognosis of tumors with high ASAH1 is independent of the type of adjuvant treatment in breast cancer and is also detected in non small cell lung cancer patients. Moreover, even in pre-invasive DCIS of the breast ASAH1 is associated with a luminal phenotype and a reduced frequency of recurrences. Thus, high ASAH1 expression is generally associated with an improved prognosis in invasive breast cancer independent of adjuvant treatment and could also be valuable as prognostic factor for pre-invasive DCIS.