A clinically relevant gene signature in triple negative and basal-like breast cancer.

INTRODUCTION: Current prognostic gene expression profiles for breast cancer mainly reflect proliferation status and are most useful in ER-positive cancers. Triple negative breast cancers (TNBC) are clinically heterogeneous and prognostic markers and biology-based therapies are needed to better treat this disease. METHODS: We assembled Affymetrix gene expression data for 579 TNBC and performed unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC. We used n = 394 cases for discovery and n = 185 cases for validation. Sixteen metagenes emerged that identified basal-like, apocrine and claudin-low molecular subtypes, or reflected various non-neoplastic cell populations, including immune cells, blood, adipocytes, stroma, angiogenesis and inflammation within the cancer. The expressions of these metagenes were correlated with survival and multivariate analysis was performed, including routine clinical and pathological variables. RESULTS: Seventy-three percent of TNBC displayed basal-like molecular subtype that correlated with high histological grade and younger age. Survival of basal-like TNBC was not different from non basal-like TNBC. High expression of immune cell metagenes was associated with good and high expression of inflammation and angiogenesis-related metagenes were associated with poor prognosis. A ratio of high B-cell and low IL-8 metagenes identified 32% of TNBC with good prognosis (hazard ratio (HR) 0.37, 95% CI 0.22 to 0.61; P < 0.001) and was the only significant predictor in multivariate analysis including routine clinicopathological variables. CONCLUSIONS: We describe a ratio of high B-cell presence and low IL-8 activity as a powerful new prognostic marker for TNBC. Inhibition of the IL-8 pathway also represents an attractive novel therapeutic target for this disease.

SATB1 gene expression and breast cancer prognosis.

Recently it has been shown that the genome organizer SATB1 plays an important role in breast cancer progression and predicts a poor prognosis. However its prognostic value compared to markers as the estrogen receptor is currently unclear. The expression levels of SATB1 mRNA from Affymetrix microarray in a cohort of 2058 breast cancer samples and its prognostic impact were analyzed. There was no significant difference in disease-free survival among ER negative cancers but instead a benefit for high SATB1 expression among ER positive tumors (p = 0.042). However, even in ER positive cancer no independent prognostic value in multivariate analysis with standard parameters was observed. Thus the use of SATB1 as target or prognostic marker for breast cancer should be viewed with caution and a possible confounding effect of the estrogen receptor status of the tumor should be taken into account when analysing new markers as SATB1.

Homogeneous datasets of triple negative breast cancers enable the identification of novel prognostic and predictive signatures.

BACKGROUND: Current prognostic gene signatures for breast cancer mainly reflect proliferation status and have limited value in triple-negative (TNBC) cancers. The identification of prognostic signatures from TNBC cohorts was limited in the past due to small sample sizes. METHODOLOGY/PRINCIPAL FINDINGS: We assembled all currently publically available TNBC gene expression datasets generated on Affymetrix gene chips. Inter-laboratory variation was minimized by filtering methods for both samples and genes. Supervised analysis was performed to identify prognostic signatures from 394 cases which were subsequently tested on an independent validation cohort (n = 261 cases). CONCLUSIONS/SIGNIFICANCE: Using two distinct false discovery rate thresholds, 25% and <3.5%, a larger (n = 264 probesets) and a smaller (n = 26 probesets) prognostic gene sets were identified and used as prognostic predictors. Most of these genes were positively associated with poor prognosis and correlated to metagenes for inflammation and angiogenesis. No correlation to other previously published prognostic signatures (recurrence score, genomic grade index, 70-gene signature, wound response signature, 7-gene immune response module, stroma derived prognostic predictor, and a medullary like signature) was observed. In multivariate analyses in the validation cohort the two signatures showed hazard ratios of 4.03 (95% confidence interval [CI] 1.71-9.48; P = 0.001) and 4.08 (95% CI 1.79-9.28; P = 0.001), respectively. The 10-year event-free survival was 70% for the good risk and 20% for the high risk group. The 26-gene signatures had modest predictive value (AUC = 0.588) to predict response to neoadjuvant chemotherapy, however, the combination of a B-cell metagene with the prognostic signatures increased its response predictive value. We identified a 264-gene prognostic signature for TNBC which is unrelated to previously known prognostic signatures.

T-cell metagene predicts a favorable prognosis in estrogen receptor-negative and HER2-positive breast cancers.

INTRODUCTION: Lymphocyte infiltration (LI) is often seen in breast cancer but its importance remains controversial. A positive correlation of human epidermal growth factor receptor 2 (HER2) amplification and LI has been described, which was associated with a more favorable outcome. However, specific lymphocytes might also promote tumor progression by shifting the cytokine milieu in the tumor. METHODS: Affymetrix HG-U133A microarray data of 1,781 primary breast cancer samples from 12 datasets were included. The correlation of immune system-related metagenes with different immune cells, clinical parameters, and survival was analyzed. RESULTS: A large cluster of nearly 600 genes with functions in immune cells was consistently obtained in all datasets. Seven robust metagenes from this cluster can act as surrogate markers for the amount of different immune cell types in the breast cancer sample. An IgG metagene as a marker for B cells had no significant prognostic value. In contrast, a strong positive prognostic value for the T-cell surrogate marker (lymphocyte-specific kinase (LCK) metagene) was observed among all estrogen receptor (ER)-negative tumors and those ER-positive tumors with a HER2 overexpression. Moreover ER-negative tumors with high expression of both IgG and LCK metagenes seem to respond better to neoadjuvant chemotherapy. CONCLUSIONS: Precise definitions of the specific subtypes of immune cells in the tumor can be accomplished from microarray data. These surrogate markers define subgroups of tumors with different prognosis. Importantly, all known prognostic gene signatures uniformly assign poor prognosis to all ER-negative tumors. In contrast, the LCK metagene actually separates the ER-negative group into better or worse prognosis.

Differential expression of claudins in human endometrium and endometriosis.

Membrane proteins of the claudin superfamily are important components of cellular tight and adherens junctions. Although their exact function remains unclear, these proteins may play a role in tissue remodeling, a process which is associated with several diseases including endometriosis. In the present work we analyzed the expression of 13 members of the claudin family in the endometrium and peritoneum by microarray analysis. Real-time polymerase chain reaction and immunohistochemistry in human endometrium and peritoneal endometriotic lesions were performed for validation of the expression of claudin-1, -3, -4, -5 and -7. Diminished expression of claudin-3, -4 and -7 in ectopic endometrium was frequently observed as indicated by all three methods. In contrast to a higher expression of claudin-5 mRNA detected in bulk biopsies of ectopic endometrium, immunohistochemistry revealed no alteration of claudin-5 protein expression in glandular cells of endometriosis samples. The downregulation of various members of the claudin family may contribute to endometrial cell detachment and increase the number of cells invading pelvic organs.

Microarray analysis of altered sphingolipid metabolism reveals prognostic significance of sphingosine kinase 1 in breast cancer.

Beside their structural role for the cell membrane the family of sphingolipids act as effector molecules in signal transduction with links to various aspects of cancer initiation, progression and treatment response. The "sphingolipid rheostat" balances between apoptosis inducing ceramid and growth promoting sphingosine-1-phosphate. We analyzed gene expression of 43 proteins from this pathway in different subtypes of breast cancer using microarray data of 1,269 tumor samples (test set n=171; validation sets n=1098) and observed significant differences for several genes. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidsynthase (GCS), dihydroceramidsynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a worse outcome of patients with high SPHK1 expression. To avoid a confounding effect of the ER status we also restricted the analysis to 750 patients with ER positive tumors. Again a worse outcome was observed for tumors displaying high SPHK1 expression. While 75.8+/-1.9% of the patients with tumors low in SPHK1 expression were free of metastasis at 5 years, this was the case for only 64.9+/-3.6% of patients with tumors displaying high SPHK1 expression (P=0.008). Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor. The correlation of SPHK1 with a poor prognosis as well as its high expression among ER negative tumors are in line with the antiapoptotic and proliferative properties of its product sphingosine-1-phosphate. Targeting of the sphingolipid rheostat may thus open new treatment options.

Expression of membrane-type 5 matrix metalloproteinase in human endometrium and endometriosis.

BACKGROUND: The metalloproteinases (MMPs) are a family of proteolytic enzymes involved in tissue remodeling and cell migration. Endometrial tissue remodeling proceeds during the menstrual cycle and requires a temporary and spatially balanced expression of several different MMPs. Various members of the MMPs also seem to play an important role in the invasion process of endometriosis; however, so far only a limited number of studies have focused on membrane-associated MMPs. METHODS: The present study investigated the expression of membrane-type 5 metalloproteinase (MT5-MMP) in the human endometrium and endometriotic lesions by microarray hybridization, real-time polymerase chain reaction (PCR) and immunofluorescence. RESULTS: Both the gene chip expression analyses as well as PCR indicated expression of MT5-MMP in normal human endometrium and strongly elevated transcript levels in most peritoneal endometriosis lesions analyzed. Moreover we detected enhanced MT5-MMP expression in the eutopic endometrium from patients suffering from endometriosis, further supporting a role of MT5-MMP in the formation of endometriosis. Immunohistochemical analysis was used to determine the intracellular localization and tissue distribution of MT5-MMP. While the MT5-MMP antigen expression could be clearly attributed to the membrane of epithelial cells, a highly complex differential immunohistochemical staining of MT5-MMP in the various compartments of endometrial tissue was observed. The strongest staining was seen in luminal epithelial cells, whereas endometrial glands frequently showed partial expression of MT5-MMP. CONCLUSION: Our microarray analysis and real-time PCR of MT5-MMP transcripts may point to an elevated tissue remodeling and cell migration in endometrium from endometriosis patients as implied by the function of related MMPs.

Characterization of WNT7A expression in human endometrium and endometriotic lesions.

OBJECTIVE: To characterize the expression of WNT7A in human eutopic and ectopic endometrium. DESIGN: Experimental study using real-time polymerase chain reaction, laser microdissection, in situ hybridization, and immunofluorescence. SETTING: University-based laboratory. PATIENT(S): Patients with and without endometriosis undergoing surgery for benign indications. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Relative expression values compared with housekeeping genes using real-time polymerase chain reaction. Detection of positive cells by immunofluorescence and in situ hybridization. RESULT(S): In endometriosis, statistically significant higher WNT7A mRNA expression was observed compared with eutopic endometrium. Expression of WNT7A was found in the luminal and glandular epithelial cells as well as stroma cells in endometrium and endometriosis by immunofluorescence, in situ hybridization, and polymerase chain reaction of laser microdissected tissue. CONCLUSION(S): The results of the present study suggest that WNT7A plays a role in the pathophysiology of endometriosis.

Utero-tubal sperm transport and its impairment in endometriosis and adenomyosis.

The uterus is composed of different smooth muscle layers that serve various functions. First, menstrual debris is expulsed at the time of the menses. Second, sperm is transported in the preovulatory phase to maximize fertility, and third, the human embryo is placed in an adequate setting during implantation. Endometriosis is a gynecologic disorder leading to severe pain symptoms such as severe pain during menstruation (dysmenorrhea), chronic pelvic pain, pain during sexual intercourse (dyspareunia), and abnormal uterine bleeding. Besides, endometriosis is often associated with female infertility and exhibits a massive impairment in the physiology of uterine contractility that can be documented by the in vivo examination method of hysterosalpingoscintigraphy (HSSG). In addition, endometriosis is associated in 80-90% of subjects with adenomyosis and our data clearly indicate that sperm transport is disturbed by hyperperistalsis when at least one focus of adenomyosis can be detected via magnetic resonance imaging (MRI) and turns into dysperistalsis (a complete failure in sperm transport capacity) when diffuse adenomyosis affecting all myometrial uterine muscle layers is detected. Hence, dysperistalsis is significantly associated with reduced spontaneous pregnancy rates. We therefore recommend MRI and HSSG in every sterility workup.

Poor outcome in estrogen receptor-positive breast cancers predicted by loss of plexin B1.

PURPOSE: A common characteristic of mammary carcinomas is an inverse relationship between the estrogen receptor (ER) status and the proliferative activity of the tumor. Yet, a subset of ER-positive breast cancers is characterized by a high proliferation, suggesting malfunctions in ER responsiveness that influence the biological and therapeutic behavior of tumor cells. The expression of several ER-dependent genes seems to be dysregulated among those "uncoupled" tumors. One of those genes is plexin B1, a cell-surface receptor for the semaphorin Sema4D (CD 100). However, the biological role of plexin B1 in breast cancer is largely unknown. EXPERIMENTAL DESIGN: Expression data of plexin B1 were obtained from Affymetrix microarray analysis of n = 119 breast cancer specimens. Validation was done by quantitative real-time PCR and protein expression was evaluated by immunohistochemistry. Expression data were compared with clinical characteristics as well as follow-up data of the disease. RESULTS: Low plexin B1 expression levels characterize a more aggressive tumor phenotype. The expression of plexin B1 is strongly correlated with the ER status. However, even among ER-positive tumors, loss of plexin B1 is associated with an impaired prognosis of breast cancer patients in both univariate (all patients, P = 0.0062; ER positive, P = 0.0107) and multivariate analyses (all patients, P = 0.032; ER positive, P = 0.022). Immunohistochemistry reveals that the tumor cells themselves and not the endothelial cells are the major source of plexin B1 expression in the tumor. CONCLUSION: Plexin B1 acts not only as a new important prognostic but should also represent a predictive marker indicating an endocrine resistance. These data give a new insight in markers that could be involved in endocrine dysregulation of breast cancer.

Endometriosis may be generated by mimicking the ontogenetic development of the female genital tract.

OBJECTIVE: To compare the expression of genes playing a decisive role during the embryonic development of the female genital tract (WNT4, WNT5A, WNT7A, PAX8) in the peritoneum of patients with endometriosis and control patients. DESIGN: Experimental study using real-time polymerase chain reaction and in situ hybridization. SETTING: University-based laboratory. PATIENT(S): Patients with and without endometriosis undergoing surgery for benign indications. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Percentage of samples positive for gene expression by using real-time polymerase chain reaction, as well as relative expression values compared with housekeeping genes. Confirmation of results by in situ hybridization. RESULT(S): Expression of WNT7A and PAX8 was found in the normal peritoneum in approximately half of the patients with endometriosis in contrast to the controls. In patients with endometriosis WNT7A and PAX8 in histologically normal peritoneum (with no evidence of endometriosis, endosalpingiosis, or other changes) were confirmed by in situ hybridization. CONCLUSION(S): The expression of these genes in the normal peritoneum suggests that endometriosis can arise through metaplasia and can in the process make use of the developmental steps involved in the embryonic development of the female genital tract.

Uterine contractility and directed sperm transport assessed by hysterosalpingoscintigraphy (HSSG) and intrauterine pressure (IUP) measurement.

BACKGROUND: Uterine peristalsis sustains sperm transport and can be detected by hysterosalpingoscintigraphy (HSSG). This study is the first to be designed to investigate utero-tubal transport function by HSSG and uterine contractility by intrauterine pressure measurement (IUP) consecutively on the same day in the periovulatory phase. METHODS: Twenty-one female subjects (mean age 28.4 years) without a gynecologic history were examined sequentially by HSSG and IUP on the same day to evaluate uterine contractility in relation to the utero-tubal transport function. In HSSG, intact transport function was visualized by the rapid uptake of 99m-technetium-marked albumin aggregates through the female genital tract. In IUP, the frequency of uterine contractions (UC/min), amplitude of uterine contractions and basal pressure tone were detected via a intrauterine catheter. HSSG and IUP were embedded in cycle monitoring with measurement of LH and estradiol. RESULTS: In HSSG, a positive transport of inert particles was assessed in 20 of 21 subjects, in 76% to the side of the dominant follicle or on both sides of the oviduct, and in 19% a strict contralateral transport could be observed. In only one subject (5%), no transport was assessed. The mean value of uterine contractions was 3.4 UC/min (SD +/- 0.7), the mean amplitude was 12.0 mmHg (SD +/- 4.25 mmHg). Basal pressure tone was 70.7 mmHg. There was a statistically significant correlation with estradiol levels: none of the subjects with less than 3 UC/min showed an estradiol level higher than 100 pg/mL; nearly every patient (one exception) with more than 3 UC/min had an estradiol level higher than 100 pg/mL (p < 0.0001, Fisher's exact test). CONCLUSIONS: Intact periovulatory utero-tubal transport function can be documented by HSSG and is caused by directed uterine contractility, measured consecutively by IUP. Uterine contractility is influenced by rising estradiol levels. Directed uterine contractility and intact utero-tubal transport function are considered necessary for intact sperm transport, mainly to the side bearing the dominant follicle to maximize fertility.