Stromal markers AKR1C1 and AKR1C2 are prognostic factors in primary human breast cancer.

BACKGROUND: Stromal fibroblasts influence tumor growth and progression. We evaluated two aldo-keto reductases, AKR1C1 and AKR1C2, in stromal fibroblasts and carcinoma cells as prognostic factors in primary human breast cancer. They are involved in intratumoral progesterone metabolism. METHODS: Immunohistochemistry was performed on tissue microarrays from 504 core biopsies from breast cancer patients. Primary endpoints were disease-free (DFS) and overall (OS) survival. RESULTS: AKR1C1 and AKR1C2 expression in fibroblasts and tumor cells correlated with favorable tumor characteristics, such as small tumor size and negative nodal status. In univariate analysis, AKR1C1 expression in carcinoma cells correlated positively with DFS und OS; AKR1C2 expression in both fibroblasts and tumor cells also showed a positive correlation with DFS and OS. In multivariate analysis, AKR1C1 expression in carcinoma cells was an independent prognostic marker. CONCLUSION: It can be assumed that our observations are due to the independent regulatory function of AKR1C1/2 in progesterone metabolism and therefore provide a basis for new hormone-based therapy options for breast cancer patients, independent of classic hormone receptor status.

Final safety and efficacy analysis of a phase I/II trial with imatinib and vinorelbine for patients with metastatic breast cancer.

BACKGROUND: Imatinib is a tyrosine kinase inhibitor of BCR-ABL, ABL, PDGFR-α and -ß, KIT, and DDR. In solid tumors, it inhibits proliferation and invasiveness and facilitates higher intratumoral cytotoxic drug concentrations. Vinorelbine has good tolerability and efficacy in metastatic breast cancer (MBC). This study evaluates the safety and efficacy of imatinib and vinorelbine in combination. METHODS: In a prospective, open-label, phase I/II trial, 400 mg imatinib p.o. daily (corrected from 600 mg) was combined with an escalating dose of vinorelbine i.v. weekly in four dose levels of 10, 15, 20, and 25 mg/m(2) (each n ≥ 5) to treat patients with MBC (expressing PDGFR-α and/or -ß, and/or KIT). The last patient of each level was treated for >28 days, before enrolment for the next dose level started. Study endpoints were feasibility and tolerability, incidence of hematological and nonhematological toxicity, and clinical efficacy (data cutoff: November 18, 2011). A total of 33 patients have been enrolled, and all dose levels have been fully recruited. One patient is still on study medication. A translational subprotocol is ongoing. RESULTS: All 33 included patients are evaluable for safety (32 within the ITT population). Eleven patients were excluded early from the study (progressive disease, toxicity, and withdrawal of consent). Twenty-two patients participated in the study for >28 days ('ITT >28'). Within the ITT population, the response rate [complete response (CR) and partial response (PR)] was 9.4% (n = 3), the clinical benefit rate (CBR; CR+PR+stable disease) 50% (n = 16), and the median time to progression (TTP) 155 days. A total of 21.3% of the patients were on study medication for >6 months, and 15.2% for >12 months (mean 140 days, range 15-643). Within 'ITT >28', the response rate was 13.6%, CBR 72.7%, and median TTP 176 days. The response was independent of the receptor status (PDGFR-α, -ß, and KIT). Toxicities were as follows (safety population): 21.6% severe leukopenia, 9.1% severe neutropenia (with 1 febrile neutropenia), 1 case of bowel perforation, 36% diarrhea (3% severe), 84.8% nausea (severe 15.2%), 48.5% vomiting (severe 9.1%), 27.3% infections (severe 6.1%), 12.1% peripheral neuropathy (severe 9.1%), and 36.4% dyspnea (3% severe). Four patients on trial died (nondrug-related). CONCLUSION: The combination of imatinib and vinorelbine in MBC appeared to be feasible and tolerable. A CBR of 50% (ITT) in pretreated patients suggests that this combination may be active. Although toxicities were frequent, they appeared to be manageable.

Transcription factors associated with epithelial-mesenchymal transition and cancer stem cells in the tumor centre and margin of invasive breast cancer.

Although tumor surgery aims for a complete resection respecting tumor-specific safety distance, in many cases the most peripheral part, the invasion front, remains in situ. Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) has been suggested to be of prime importance for tissue and vessel invasion. Recently, features of EMT were shown to be linked to cells with tumor-founding capability, so- called cancer stem cells (CSC). In this study we show that transcription factors associated with EMT markers Snail, Slug, Twist and Zeb1 are differentially expressed between normal breast epithelium, ductal carcinoma in situ and invasive breast cancer. Both invasive and in situ carcinoma expressed less Slug and Twist and more Zeb1 compared to normal epithelium. Using fluorescence multi-staining the number of potential CSC among invasive cancer cells varied dramatically depending on the staining combination used (18.5% for CD44(+)/CD24(-) and 2.4% for CD49f(+)/CD24(+)). Interestingly, neither transcription factors associated with EMT nor potential CSC counts varied between tumor centre and invasion front. No association of these features with clinical outcome was detected. Our results suggest that reliable in situ markers for EMT are missing for invasive breast cancer. Alternatively, the process of EMT might be activated in tumor cells at the margin as well as the centre. Furthermore, our data show that the bio-markers of CSC detect very variable cell populations within breast cancer, challenging the assumption of a hierarchical organization of CSC in these tumors.

Pediatric and adolescent gynecology- a current overview

In pediatric and adolescent gynecology we encounter a number of diseases that occur solely during a specific phase of physical development. The diseases need some experience in the field, as well as an accurate diagnosis and are therefore often diagnosed somewhat late. The separation and traction technique is a painless method of inspecting the child's genitals. It is also effective and easy to perform. In contrast to a routine investigation in adults, very specific diagnostic questions require the insertion of a speculum, vaginoscopy, taking swabs for analysis, ultrasound investigations, or blood sampling in children. A number of diseases that occur frequently in prepubertal girls will be discussed. The etiology, clinical characteristics, treatment and prognosis of the following diseases will be addressed in detail: vulvovaginitis, lichen sclerosus, labial adhesions, ovarian torsion, abnormal uterine bleeding, uterine fibroids, and hypertensive disorders of pregnancy.

Diagnosis and management of an unilateral giant fibroadenoma of the breast in pregnancy.

BACKGROUND: Fibroadenomas represent the vast majority of breast pathologies in young women. 2-4% of the fibroadenomas exceed 5 cm in size or 500 g in weight and are called "giant fibroadenomas". Due to their excessive growth they are usually enucleated to clarify a malignant origin, to differentiate from phyllodes tumor and to prevent persisting deformities of the breast. CASE: We present a case of a 17-year-old female who was pregnant in the 24th week and suffered from a giant fibroadenoma in the right breast. Besides the massive swelling no other illnesses were found. The patient was clinically asymptomatic and had noticed the tumor just 8 weeks ago. On clinical examination we found a tumor of more than 10 cm in size which fulfilled the criteria of a benign process. A prior performed biopsy and an ultrasound investigation could not definitely differentiate the mass from phyllodes tumor. Because of the rapid growth and the progressive deformation of the breast a lumpectomy was indicated and performed without complications in consideration of the gestational stage. CONCLUSION: We present a rare case of giant fibroadenoma in pregnant young women. Because of the progressive structural damage of the breast immediate surgical enucleation was indicated. Safety of the fetus was provided by perioperative monitoring. The pre-operative differentiation from phyllodes tumor is still challenging.

Biomechanics of the female pelvic floor: a prospective trail of the alteration of force-displacement-vectors in parous and nulliparous women.

OBJECTIVE: We hypothesize that the holistic and multiplanar depiction of pelvic floor structures by dynamic MRI is of particular value in rendering information about the extent of functional changes that can lead to pelvic floor dysfunction. METHODS: 134 women were prospectively included for assessment of their pelvic floor function. RESULTS: Study groups differed significantly in the direction of their force-displacement-vectors. A shift from ventral to dorsal is present depending on parity, mode of delivery and age. Maternal age and body height correlated to the force-displacement-vector, whereas maternal weight did not. Pressing direction proved to be dependent on the inclination of the pelvis and the aperture of the levator hiatus while remaining independent from the aperture of the abdominal wall. CONCLUSION: Biomechanical data interpretation uncovered the pathogenetic relevance of progressive retroflection of the force-displacement-vector. This is responsible for the onset of a vicious cycle of trauma-related force deflection perpetuating pelvic floor traumatization.

In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells.

BACKGROUND: Breast cancer treatment is based on a combination of adjuvant chemotherapy followed by radiotherapy effecting intracellular signal transduction. With the tyrosine kinase inhibitors new targeted drugs are available. Imatinib mesylate is a selective inhibitor of bcr-abl, PRGFR alpha, beta and c-kit. The purpose of this study was to determine whether Imatinib has an influence on the effectiveness of radiotherapy in breast cancer cell lines and if a combination of imatinib with standard chemotherapy could lead to increased cytoreduction. METHODS: Colony-forming tests of MCF 7 and MDA MB 231 were used to study differences in cell proliferation under incubation with imatinib and radiation. Changes in expression and phosphorylation of target receptors were detected using western blot. Cell proliferation, migration and apoptosis assays were performed combining imatinib with doxorubicin. RESULTS: The combination of imatinib and radiotherapy showed a significantly stronger inhibition of cell proliferation compared to single radiotherapy. Differences in PDGFR expression could not be detected, but receptor phosphorylation was significantly inhibited when treated with imatinib. Combination of imatinib with standard chemotherapy lead to an additive effect on cell growth inhibition compared to single treatment. CONCLUSIONS: Imatinib treatment combined with radiotherapy leads in breast cancer cell lines to a significant benefit which might be influenced through inhibition of PDGFR phosphorylation. Combining imatinib with chemotherapy enhances cytoreductive effects. Further in vivo studies are needed to evaluate the benefit of Imatinib in combination with radiotherapy and chemotherapy on the treatment of breast cancer.

Living with Male Breast Cancer: A Qualitative Study of Men's Experiences and Care Needs.

BACKGROUND: Male breast cancer (MBC) is rare, and most previous studies limited their focus on clinical aspects of the disease. Psychosocial implications and care needs of MBC patients are poorly understood. OBJECTIVES: The aim of this study is to explore the experiences of men living with breast cancer and to identify supportive care needs. METHODS: Eighteen men were interviewed using qualitative, semi-structured telephone interviews. Qualitative content analysis was used to analyze the data. RESULTS: The majority of men did not have negative feelings about having a "women's disease," although some felt that stigmatization threatened their masculinity. Male sex was perceived as hindering access to adequate care. Patients identified key barriers including (1) a lack of awareness and experience of treating males among health professionals; (2) treatment and available information were based on evidence for females; and (3) lacking support services. CONCLUSION: To improve MBC care, it is important to raise awareness of the disease and to adapt treatment strategies, patient information, and support services to meet the needs of men.

Cytoplasmic levels of high mobility group A2 determine survival prognoses in breast cancer patients.

BACKGROUND: High mobility group A proteins are involved in chromatin remodeling, thereby influencing multiple fundamental biological processes. HMGA2 has been linked to oncogenic traits among a variety of malignancies. OBJECTIVE: To determine the prognostic implications of subcellular distribution patterns of HMGA2 in breast cancer. METHODS: Nuclear and cytoplasmic HMGA2 was evaluated in 342 breast cancer specimens and matched with clinico-pathological parameters. RESULTS: Overall and cytoplasmic, but not nuclear, levels of HMGA2 correlated with better survival prognoses in our collective (hazard ratio (HR) 0.34, P = 0.001 and HR 0.34, P < 0.001, respectively). The protective effect of cytoplasmic HMGA2 persisted in the Luminal A and triple negative breast cancer subgroups. Evaluating Luminal A and B subgroups jointly, only cytoplasmic, but not overall or nuclear HMGA2 levels were associated with better survival (HR 0.42, 95% confidence interval 0.21, 0.86, P = 0.017), irrespective of tumor size and node status. The addition of HMGA2 overall and cytoplasmic scores strengthened the prognostic selectivity in a model of conventional breast cancer risk factors. No predictive significance with regard to endocrine or chemoendocrine therapies was observed. CONCLUSION: Unexpectedly, we found a favorable survival probability upon overall levels of HMGA2 in our breast cancer collective, which was predominantly determined by the presence of HMGA2 in the cytoplasm.

Actual management of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) represents a premalignant, non-invasive intraductal carcinoma of the breast. About 30% of all mammographically detected breast cancers contain DCIS. Due to the increased use of mammography during the last 20 years the incidence of DCIS has dramatically risen. Histologically it represents a heterogenous group of potentially malignant lesions. The prognosis of DCIS is excellent, but the optimal management of the disease still remains controversial. This review summarizes the results of the latest randomized trials and retrospective analyses investigating the optimal therapeutic strategies in the treatment of DCIS. In addition, it presents a range of treatment options on the basis of the guidelines of the German gynecological oncology group (AGO) 2008.

Heterogeneous intracellular TRAIL-receptor distribution predicts poor outcome in breast cancer patients.

Upon ligand binding, plasma membrane-located TNF-related apoptosis-inducing ligand (TRAIL)-receptors 1 and 2 induce apoptosis as well as cancer-promoting signaling in cancer cells. TRAIL-R3 and TRAIL-R4 are believed to negatively regulate TRAIL-mediated apoptosis. Intracellular localization of TRAIL-receptors, as observed in many tumor cells, has been associated with oncogenic features, which are distinct from membrane-associated TRAIL-R signaling. Here, analyzing a panel of 354 breast cancer specimens, we found that an unfavorable outcome correlating with cancer-promoting properties of TRAIL-R1, TRAIL-R2, and TRAIL-R4 was most significantly defined by their intracellular distribution and mutual co-expression. A nuclear or cytoplasmic heterogeneous expression pattern correlated with markedly decreased overall survival and discriminated high-risk breast cancer patients from low-risk patients with a homogeneous distribution of expression, i.e., nuclear and cytoplasmic expression. The homogeneous TRAIL-R expression was associated with favorable breast cancer surrogate markers corresponding with excellent survival prognoses at 5 years after diagnosis (hazard ratio, 0.043) and over the complete course of follow-up (hazard ratio, 0.098; both p < 0.001). No associations with specific intrinsic breast cancer subtypes were found. Our data suggest that the determination of intracellular co-expression patterns of TRAIL-R1, TRAIL-R2, and TRAIL-R4 provides an innovative and robust method for risk stratification in breast cancer patients beyond conventional prognostic markers. KEY MESSAGES: A total of 70% of breast cancer specimens show comparably high levels of intracellular TRAIL-Rs. Nuclear or cytoplasmic TRAIL-R co-expression occurs in the majority of tumors. A total of 25% of tumors show a heterogeneous expression of cytoplasmic or nuclear TRAIL-Rs. Patients with a heterogeneous TRAIL-R expression present with poor prognoses. Additive TRAIL-R-based risk stratification comprises different breast cancer subtypes.

HSulf-1 inhibits angiogenesis and tumorigenesis in vivo.

We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.

Activation of the orphan receptor GPR55 by lysophosphatidylinositol promotes metastasis in triple-negative breast cancer.

The orphan G protein-coupled receptor GPR55 has been directly or indirectly related to basic alterations that drive malignant growth: uncontrolled cancer cell proliferation, sustained angiogenesis, and cancer cell adhesion and migration. However, little is known about the involvement of this receptor in metastasis. Here, we show that elevated GPR55 expression in human tumors is associated with the aggressive basal/triple-negative breast cancer population, higher probability to develop metastases, and therefore poor patient prognosis. Activation of GPR55 by its proposed endogenous ligand lysophosphatidylinositol confers pro-invasive features on breast cancer cells both in vitro and in vivo. Specifically, this effect is elicited by coupling to Gq/11 heterotrimeric proteins and the subsequent activation, through ERK, of the transcription factor ETV4/PEA3. Together, these data show that GPR55 promotes breast cancer metastasis, and supports the notion that this orphan receptor may constitute a new therapeutic target and potential biomarker in the highly aggressive triple-negative subtype.

Simultaneous congener-specific determination of selected organochlorine compounds and nitro musks in human whole blood samples by solid-phase extraction and capillary gas chromatography with electron capture detection.

Chlororganic compounds like pesticides or polychlorinated biphenyls (PCB) and nitro musks are environmental contaminants, which remain public health concerns because of their persistence in humans and their toxicological properties. Many of these substances are associated with endocrine dysfunction or with carcinogenicity. Therefore, a simple method using solid-phase extraction followed by capillary gas chromatography with electron capture detection for the simultaneous determination of both organochlorines and nitro musks in human whole blood samples has been developed. Recovery rates of 13 PCB congeners and of 7 pesticides ranged from 67.5% to 100.4% and from 81.1% to 110.5%, respectively. Recoveries of the 5 nitro musks were consistent and ranged from 90.2% to 98.8%. The accuracy for organochlorines and nitro musks varied from 6.3% to 8.6%. Method detection limits ranged from 0.02 microg/L to 0.11 microg/L for the organochlorines and from 0.04 microg/L to 0.08 microg/L for the nitro musks. The method has a high sensitivity with a low detection limit even in slightly contaminated human blood samples. The time and technical effort is small, so the method is feasible for epidemiological studies with regard to the impact of organochlorines and nitro musks on certain diseases.

Role of cannabinoid receptor CB2 in HER2 pro-oncogenic signaling in breast cancer.

BACKGROUND: Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different cancer models. However, the biological role of these receptors in tumor physio-pathology is still unknown. METHODS: We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen, and Freiburg between 1997 and 2010 and CB2 mRNA expression in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2) rat ortholog (neu) and lacks CB2 and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by colocalization, coimmunoprecipitation, and proximity ligation assays. Statistical tests were two-sided. RESULTS: We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis (decreased overall survival, hazard ratio [HR] = 0.29, 95% confidence interval [CI] = 0.09 to 0.71, P = .009) and higher probability to suffer local recurrence (HR = 0.09, 95% CI = 0.049 to 0.54, P = .003) and to develop distant metastases (HR = 0.33, 95% CI = 0.13 to 0.75, P = .009). We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade and that an increased CB2 expression activates the HER2 pro-oncogenic signaling at the level of the tyrosine kinase c-SRC. Finally, we show HER2 and CB2 form heteromers in cancer cells. CONCLUSIONS: Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and they suggest that CB2 may be a biomarker with prognostic value in these tumors.

Significance of syndecan-1 expression in ductal carcinoma in situ of the breast.

BACKGROUND: Fibroblast growth factor-2 (FGF-2) supports tumor progression in breast cancer. FGF-2 signaling is modulated by heparan sulfate proteoglycans, such as syndecan-1 (CD138). The exact role of CD138 in ductal carcinoma in situ of the breast (DCIS) is still uncertain. Differential expression depending on grading could suggest a role for syndecan-1 during growth and tumor progression. MATERIALS AND METHODS: Samples of 127 cases of breast DCIS associated with follow-up data were included. CD138 staining intensity, number of positive cells, intracellular and tissue localization were examined. RESULTS: Median follow-up was 45.4 months and median recurrence-free survival (RFS) 86 months. Age, menopausal status and previous hormone replacement therapy had no significant influence on RFS. Smoking significantly influenced RFS (p=0.008). Endocrine therapy or radiotherapy did not improve RFS. Grading was not correlated with CD138 staining intensity, but was significantly associated with the percentage of CD138-positive cells (low-vs. high-grade, p=0.043). Estrogen receptor (ER) expression did not influence staining intensity of CD138 (p=0.247), but negatively correlated with the proportion of CD138-positive cells (p=0.032). Progesterone receptor (PR) expression significantly influenced the intensity of staining (p=0.010) and the percentage of CD138-positive cells (p=0.004); both were increased in PR-negative cases. CD138 staining intensity and percentage of positive cells did not correlate with RFS. Nuclear grade and syndecan-1 staining localization were significantly associated (p=0.001). ER-positive, and PR-positive DCIS more often exhibited membrane-bound syndecan-1 than ER- or PR-negative cases (p=0.001). Nuclear grade and tissue localization of CD138 correlated significantly (p=0.005). PR influenced CD138 tissue distribution, while ER did not. Syndecan-1 localization did not statistically impact RFS. CONCLUSION: In DCIS of different nuclear grades, tissue localization of syndecan-1 is significantly divergent, suggesting a specific effect on biology and progression of DCIS.

Normal mammary fibroblasts induce reversion of the malignant phenotype in human primary breast cancer.

BACKGROUND/AIM: The tumor microenvironment plays a major role in tumor growth and progression. Its manipulation can lead to a reversion of the malignant phenotype. Here we explored the ability of normal mammary fibroblasts (HMFs) to induce reversion of the malignant phenotype of primary breast carcinoma cells (PBCs) in a three-dimensional (3D) context. MATERIALS AND METHODS: PBCs were isolated from 13 primary breast carcinomas and cultured in 3D collagen-I gels as mono- or co-culture with HMFs. RESULTS: In five co-cultures, PBCs exhibited reversion of their malignant phenotype, whereas PBCs in matched monocultures exhibited disorganized growth. Reversion, defined as the restoration of the complete baso-apical polarity axis, was confirmed with established polarity markers. Secretion of the tissue-specific glycoprotein MAM-6 into the acinar lumens and deposition of basement membrane indicated functional differentiation. Gene expression analysis revealed a set of differentially regulated genes which possibly affect the reversion process. These included MAL, ELF5, MAP6, ZMYND11 and SQLE. CONCLUSION: These findings highlight the significant role of fibroblasts in regulating the carcinoma phenotype.

Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracrine signals.

Stromal factors play a critical role in the development of the mammary gland. Using a three dimensional-coculture model we demonstrate a significant role for stromal fibroblasts in the regulation of normal mammary epithelial morphogenesis and the control of tumor growth. Both soluble factors secreted by fibroblasts and fibroblast-derived modifications of the matrix compliance contribute to the regulation of epithelial cell morphogenesis. Readjustment of matrix tension by fibroblasts can even induce a phenotypic reversion of breast carcinoma cells. These data offer a basis to develop new strategies for the normalization of the tumor stroma as an innovative target in cancer therapy.

Neutrophil gelatinase-associated lipocalin (NGAL) predicts response to neoadjuvant chemotherapy and clinical outcome in primary human breast cancer.

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.

3D microchannel co-culture: method and biological validation.

Conventional 3D culture is typically performed in multi-well plates (e.g. 12 wells). The volumes and dimensions necessitate relatively large numbers of cells and fluid exchange steps are not easily automated limiting throughput. 3D microchannel culture can overcome these challenges simplifying 3D culture processes. However, the adaptation of immunocytochemical endpoint measurements and the validation of microchannel 3D culture with conventional 3D culture are needed before widespread adoption can occur. Here we use a breast carcinoma growth model governed by complex and reciprocal interactions between epithelial carcinoma cells and mesenchymal fibroblasts to validate the 3D microculture system. Specifically, we report the use of a 3D microchannel co-culture assay platform to interrogate paracrine signalling pathways in breast cancer. Using a previously validated 3D co-culture of human mammary fibroblasts and T47D breast carcinoma cells, we demonstrate the use of arrayed microchannels to analyze paracrine signalling pathways and screen for inhibitors. Results in both conventional format (multiwell plate) and microchannels were comparable. This technology represents a significant advancement for high-throughput screening in individual patients and for drug discovery by enabling the use of 3D co-culture models via smaller sample requirements and compatibility with existing HTS infrastructure (e.g. automated liquid handlers, scanners).