M. Orlowska-Volk

Specific induction of pp125 focal adhesion kinase in human breast cancer.

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT–PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51±0.84 (mean±standard deviation), which was strongly induced to 2.91 (±1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0±0.63 vs 2.27±0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.

Expression of the transcriptional coregulator FHL2 in human breast cancer: a clinicopathologic study.

Objective: Although the Four and a Half LIM domain protein 2 (FHL2) has been suggested to play an important role in tumor development, this has not been investigated in breast cancer. Methods: Paraffin-embedded tissues from patients (n = 85) with primary breast cancer were submitted to immunohistochemical investigation of FHL2 expression and subsequent correlation with clinicopathologic parameters and patient survival. Results: The expression of FHL2 was confined to the cytoplasm of the tumor cells. Forty (47%) of 85 samples showed weake expression of FHL2, whereas high expression was found in 45 tumors (53%). A statistically significant positive correlation was observed between FHL2 and androgen receptor expression (P = .029). Patients with tumors expressing low amounts of FHL2 were characterized by a significantly better survival compared to those with high intratumoral FHL2 expression (P = .0215, log-rank test). The additional stratification according to adjuvant tamoxifen treatment revealed a significantly improved survival rate for patients receiving tamoxifen and being diagnosed with a tumor expressing high amounts of FHL2. This might indicate that tamoxifen is at least partially capable of reversing the negative prognostic impact of high FHL2 expression. Multivariate Cox regression analysis revealed FHL2 expression as a significant indepenent predictor of survival. Conclusion: The specific in tumor tissue points to an important functional role of FHL2 in human breast cancer. Our survival data indicate that the expression of FHL2 in primary breast cancer is a potentially relevant prognostic factor. Further studies are warranted to elucidate whether analysis of FHL2 expression is suitable to predict response to antihormonal treatment with tamoxifen.

Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection

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

Expression levels of hnRNP G and hTra2-beta1 correlate with opposite outcomes in endometrial cancer biology

HnRNP G is a member of heterogeneous nuclear ribonucleoprotein (hnRNP) family with potent tumor suppressive activities. Human transformer‐2‐beta1 (hTra2‐beta1) belongs to the arginine‐serine rich like proteins and is found over‐expressed in various human cancers. It was recently shown that hnRNP G and hTra2‐beta1 exert antagonistic effects on alternative splicing. In our study we explored the impact of these two factors in tumor biology of endometrial cancer (EC). EC tissues (n = 139) were tested for hnRNP G and hTra2‐beta1 expression on mRNA level by real time PCR and on protein level by immunohistochemistry. HTra2‐beta1 mRNA level was found being induced in advanced International Federation of Gynecology and Obstetrics (FIGO) stages (p = 0.016). HnRNP G protein nuclear expression was found more prominent in patients without distant organ metastases (p = 0.033) and in FIGO Stages I/II group (p < 0.001). HTra2‐beta1 protein nuclear levels were elevated in poorly differentiated (p = 0.044) and lymph node metastases (p = 0.003) cancers. Kaplan‐Meier survival curves revealed that elevated hnRNP G mRNA (p = 0.029) and protein (p = 0.022) levels were associated with a favorable patient outcome. Multivariate Cox‐regression analyses identified nuclear hnRNP G level [hazard ratio (HR) 0.468, p = 0.026) as well as hTra2‐beta1 level (hazard ratio 5.760, p = 0.004) as independent prognostic factors for EC progression‐free survival. Our results indicate that the antagonistic functional effects of hnRNP G and hTra2‐beta1 on alternative splicing correlate directly to their opposite clinical effects on EC patient outcome.

HNRNP G and HTRA2-BETA1 regulate estrogen receptor alpha expression with potential impact on endometrial cancer

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

Abstract PD07-06: Basal-Like Molecular Subtype Predicts Response to Neoadjuvant Chemotherapy with Epirubicine, Cyclophosphamide, and Docetaxel in Patients with Primary Breast Cancer

Background: Gene expression profiling provides an opportunity to predict response to specific regimens of neo-adjuvant chemotherapy (NAC) in breast cancer patients. Materials and Methods: In a prospective cohort study of 32 women with primary invasive breast cancer with a measurable lesion, we obtained a tumor specimen by high speed core biopsy before and after 4 cycles of NAC with epirubicine 90mg/m2 and cyclophosphamide 600mg/m2 every 3 weeks, followed by 4 cycles of docetaxel 100mg/m2. Total RNA was extracted from tumor specimens and the whole transcriptome was quantified with Agilent9s 44K single color microarray interrogating 44000 unique human genes. Tumor lesions were ultrasonographically measured to assess response using Sinn criteria. Data analysis was performed by GeneSpring v11 and IBM SPSS v18. Results: Using three single sample predictors, 10 tumors were classified as basal-like and 22 tumors were classified as non basal-like. We found that gene expression-based molecular subtype (basal-like vs. non basal-like) (p=0.003), but not tumor grade (p=0.07), estrogen receptor (p=0.1), progesterone receptor (p=0.6), and HER2 status (p=0.4) predicted response to NAC. Specifically, 7/10 basal-like tumors responded to NAC, whereas 19/22 non basal-like tumors did not respond. Comparing gene expression signatures before and after 4 cycles of NAC, we found that all patients with an initial non basal-like tumor retained this tumor type, whereas 5/7 basal-like tumors, including all responders, lost this molecular subtype. Using regression models based on centroid predicition gene sets, complete prediction of response to NAC based on the initial tumor biopsy as well as on the change of gene expression between two tumor biopsies was achieved with a 21 gene list (p=0.000008) and a 23 gene list (p=0.000007), respectively. Of note, both the expression and upregulation of a single gene, ie HER4, predicted response to NAC in 26/32 (81%; p=0.002) and in 23/25 (92%; P Conclusions: Basal-like molecular subtype and therapy-induced HER4 gene upregulation predict response to NAC with epirubicine, cyclophosphamide, and docetaxel. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-06.