ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance.

BACKGROUND: Platinum resistance constitutes one of the most recognized clinical challenges for ovarian cancer. Notably, the detection of the primary tumor-based excision repair cross-complementation group 1 (ERCC1) protein by immunohistochemistry was recently shown to be inaccurate for the prediction of platinum resistance. On the basis of the previous finding that circulating tumor cells (CTC) in the blood of ovarian cancer patients are prognostically significant, and given our hypothesis that the negative prognostic impact of CTC may arise from a cellular phenotype associated with platinum resistance, we asked whether expression of the excision repair cross-complementation group 1 (ERCC1) gene in the form of the ERCC1 transcript in CTC may be a suitable blood-based biomarker for platinum resistance. METHODS: The presence of CTC was analyzed by immunomagnetic CTC enrichment (n = 143 patients) targeting the epithelial epitopes epithelial cell adhesion molecule (EPCAM) (also known as GA733-2) and mucin 1, cell surface associated (MUC1), followed by multiplex reverse-transcription PCR to detect the transcripts EPCAM, MUC1, and mucin 16, cell surface associated (MUC16) (also known as CA125), including ERCC1 transcripts in a separate approach. ERCC1 expression in primary tumors was comparatively assessed by immunohistochemistry, using the antibody 8F1. RESULTS: At primary diagnosis, the presence of CTC was observed in 14% of patients and constituted an independent predictor of overall survival (OS) (P = 0.041). ERCC1-positive CTC (ERCC1(+)CTC) were observed in 8% of patients and constituted an independent predictor, not only for OS but also for progression-free survival (PFS) (P = 0.026 and P = 0.009, respectively). More interestingly, we discovered the presence of ERCC1(+)CTC at primary diagnosis to be likewise an independent predictor of platinum resistance (P = 0.010), whereas ERCC1 expression in corresponding primary tumor tissue predicted neither platinum resistance nor prognosis. CONCLUSIONS: The presence of ERCC1(+)CTC can serve as a blood-based diagnostic biomarker for predicting platinum resistance at primary diagnosis of ovarian cancer.

Comparison of the diagnostic value of FDG-PET/CT and axillary ultrasound for the detection of lymph node metastases in breast cancer patients.

BACKGROUND: FDG-PET/CT is increasingly being used for breast cancer staging. Its diagnostic accuracy in comparison to ultrasound as the standard non-invasive imaging modality for the evaluation of axillary lymph nodes has yet not been evaluated. PURPOSE: To retrospectively compare the diagnostic value of full-dose, intravenously contrast-enhanced FDG-PET/CT and ultrasound for the detection of lymph node metastases in breast cancer patients. MATERIAL AND METHODS: Ninety patients (one patient with a bilateral carcinoma) (89 women, one man; mean age, 55.5 +/- 16.6 years) suffering from primary breast cancer underwent whole-body FDG-PET/CT and axillary ultrasound. The ipsilateral axillary fossa (n = 91) was evaluated for metastatic spread. The sensitivity, specificity, the positive predictive value (PPV), negative predictive value (NPV), and accuracy of both methods were calculated. The sensitivity and accuracy were statistically compared using the McNemar Test (P <0.05). Analyses were made on a patient basis. The number of patients with extra-axillary locoregional lymph node metastases exclusively detected by FDG-PET/CT was evaluated. For axillary lymph node metastases histopathology served as the reference standard. RESULTS: The sensitivity, specificity, PPV, NPV, and accuracy of FDG-PET/CT for the detection of axillary lymph node metastases were 54%, 89%, 77%, 74%, and 75%, respectively. For ultrasound it was 38%, 78%, 54%, 65%, and 62%, respectively. FDG-PET/CT was significantly more accurate than ultrasound for the detection of axillary lymph node metastases (P = 0.019). There was no statistically significant difference between the sensitivity of both modalities (P = 0.0578). FDG-PET/CT detected extra-axillary locoregional lymph node metastases in seven patients (8%) that had not been detected by another imaging modality. CONCLUSION: Though more accurate compared to ultrasound for evaluating the axillary lymph node status FDG-PET/CT is only as sensitive as ultrasound when it comes to the detection of axillary lymph node metastases. Due to the low sensitivity FDG-PET/CT cannot act as a substitute for Sentinel lymph node biopsy. FDG-PET/CT is able to detect previously unknown locoregional extra-axillary lymph node metastases.

FDG-PET/CT for the early prediction of histopathological complete response to neoadjuvant chemotherapy in breast cancer patients: initial results.

BACKGROUND: Up to about one-quarter of patients treated with neoadjuvant chemotherapy do not adequately respond to the given treatment. By a differentiation between responders and non-responders ineffective toxic therapies can be prevented. PURPOSE: To retrospectively test if FDG-PET/CT is able to early differentiate between breast cancer lesions with pathological complete response (pCR) and lesions without pathological complete response (npCR) after two cycles of neoadjuvant chemotherapy (NACT). MATERIAL AND METHODS: In this retrospective study 26 breast cancer patients (mean age, 46.9 years ± 9.9 years) underwent a pre-therapeutic FDG-PET/CT scan and a subsequent FDG-PET/CT after the second cycle of NACT. Histopathology of resected specimen served as the reference standard. Maximum standardized uptake values (SUVmax) of cancer lesions before and after the second cycle of NACT were measured. Two evaluation algorithms were used: (a) pCR: Sinn Score 3 and 4, npCR: Sinn Score 0-2; (b) pCR: Sinn Score 4, npCR: Sinn Score 0-3. The absolute and relative decline of the SUVmax (ΔSUVmax, ΔSUVmax(%))was calculated. Differences of the SUVmax as well as of the SUVmax decline between pCR lesions and npCR lesions were tested for statistical significance P < 0.05. To identify the optimal cut-off value of ΔSUVmax(%) to differentiate between pCR lesions and npCR lesions a receiver-operating curve (ROC) analysis was performed. RESULTS: Using evaluation algorithm A the ΔSUVmax was 13.5 (pCR group) and 3.9 (npCR group) (P = 0.006); the ΔSUVmax(%) was 79% and 47%, respectively (P = 0.001). On ROC analysis an optimal cut-off ΔSUVmax(%) of 66% was found. Using evaluation algorithm B the ΔSUVmax was 17.5 (pCR group) and 4.9 (npCR group) (P = 0.013); the ΔSUVmax(%) was 89% and 51%, respectively (P = 0.003). On ROC analysis an optimal cut-off ΔSUVmax(%) of 88% was found. CONCLUSION: FDG-PET/CT may be able to early differentiate between pCR and npCR of primary breast cancer lesions after two cycles of NACT.

New insight into the SDF-1/CXCR4 axis in a breast carcinoma model: hypoxia-induced endothelial SDF-1 and tumor cell CXCR4 are required for tumor cell intravasation.

The SDF-1/CXCR4 axis has been implicated in breast cancer metastasis. In contrast to its well-established role in organ-specific homing and colonization of tumor cells, the involvement in intravasation, especially in a hypoxic environment, is still poorly understood. Initially, we detected both, the chemokine SDF-1 and its receptor CXCR4 in microvessels in invasive ductal cancer samples. To elucidate the role of the SDF-1/CXCR4 axis in vascular endothelium for tumor intravasation, we evaluated the effects of CXCR4 activation in human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC) and in cultured mammary carcinoma cells (MDA MB231, and MCF7). We observed an upregulation of SDF-1 and CXCR4 in HUVECs in hypoxia, which led to proliferation, migration, and tube formation. Hypoxia induced adhesion of tumor cells to endothelial cells and stimulated transendothelial migration. The effects of hypoxia were dependent on the activity of the transcription factor hypoxia-inducible factor. Adhesion to and migration through a HUVEC monolayer were significantly reduced by lentiviral inhibition of CXCR4 in breast carcinoma cells or treatment of endothelial cells with an anti-SDF-1 neutralizing antibody. These data show that the interaction of SDF-1 secreted by ECs with tumor cell CXCR4 is sufficient to stimulate transendothelial migration of the tumor cells. Our results suggest that the SDF-1/CXCR4 axis is important in angiogenesis and tumor cell intravasation. Because both proteins were readily identifiable in a significant fraction of human breast cancer samples by immunohistochemistry, CXCR4 may constitute a molecular target for therapy when both, SDF-1, and CXCR4 are expressed.

Loss of heterozygosity proximal to the M6P/IGF2R locus is predictive for the presence of disseminated tumor cells in the bone marrow of ovarian cancer patients before and after chemotherapy.

Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of ovarian cancers before surgery and after chemotherapy and are associated with worse prognosis. A molecular biomarker in the primary tumor predicting tumor cell spread would be highly desirable. The purpose of the study was to investigate loss of heterozygosity (LOH) in primary ovarian tumors at four ovarian cancer-relevant chromosomal loci involved in apoptosis, platinum sensitivity, or DNA-repair, to assess the prognostic value of LOH and to correlate LOH with DTC occurrence before surgery and after chemotherapy. Primary tumor DNA of 88 patients was analyzed for LOH at four polymorphic microsatellite markers using PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. LOH at the entire marker set correlated with tumor grading (P = 0.0001) and histology (P = 0.004). LOH at marker D10S1765 correlated with FIGO stage (P = 0.046) and grading (P = 0.05), whereas LOH at D17S855 significantly associated with grading (P = 0.023) and histology (P = 0.012), respectively. DTC were detected in 49% of patients before surgery and in 50% of patients after chemotherapy. Interestingly, LOH proximal to D6S1581 significantly correlated with DTC presence before surgery (P = 0.05) and after chemotherapy (P = 0.022). Conclusively, our data suggest that allelic loss at D6S1581 (proximal to M6P/IGF2R locus) serves as a molecular biomarker for the presence of DTC in the BM before and after chemotherapy.

Intraocular biopsy using special forceps: a new instrument and refined surgical technique.

AIM: The aim was to investigate the Essen biopsy forceps as a new instrument and surgical approach for biopsy of intraocular tumours. Biopsy is indicated for assessment of any uncertain intraocular process or confirmation for presumed diagnosis before treatment. There is increasing interest for further genetic and immunocytological information in order to characterise the neoplasm, especially grading and prognosis of micrometastasis in uveal melanoma. The authors have developed a new surgical technique using special intraocular biopsy forceps. METHODS: Twenty patients with uncertain intraocular subretinal tumour underwent biopsies carried out using the special Essen biopsy forceps. Biopsies were obtained through sutureless 23-gauge three-port vitrectomy. A small retinotomy tumour specimen was taken by the forceps branches. For further processing, the specimens were flushed out into a sterile tube and then sent to pathologists. RESULTS: The prebioptical tumour had a mean thickness of 3.48 mm (1.1 to 9.8 mm). In all cases (n=20) biopsies (0.3-2.1 mm in size) were obtained, in 19 cases (95%) allowing precise histological and immunohistochemical typing of the lesions following cytoblock embedding. Uveal melanoma was diagnosed in 50% (n=10), choroidal metastasis in 15% (n=3) and choroidal naevus in 15% (n=3); other diagnoses (n=3) included choroidal haemangioma, B cell lymphoma and old subretinal haemorrhage. Apart from three patients with temporary punctual bleeding on the surface, there were no intra- and postoperative complications. CONCLUSIONS: Biopsy using special forceps is a promising new approach and precise surgical procedure. Especially for small intraocular tumours, this technique has the advantage in providing enough tissue for improved histological examination and presenting a low risk for complications.

The XbaI G>T polymorphism of the glucose transporter 1 gene modulates 18F-FDG uptake and tumor aggressiveness in breast cancer.

UNLABELLED: We investigated the relevance of single-nucleotide polymorphisms (SNPs) in the glucose transporter 1 (GLUT1) gene to the uptake of (18)F-FDG and tumor aggressiveness in breast cancer. METHODS: In 52 individuals with breast cancer, a diagnostic PET/CT scan was obtained, and the standardized uptake value was determined as a measure of (18)F-FDG uptake using a region-of-interest technique. Three GLUT1 SNPs (XbaI G>T, HpyCH4V A>T, and HaeIII T>C) were investigated in genomic DNA that was isolated from the paraffin-embedded specimens of all patients. Tumors were typed and graded according to the World Health Organization classifications. RESULTS: The GG genotype of the XbaI G>T SNP was associated with increased tumor uptake of (18)F-FDG, with a mean standardized uptake value of 11.7 (TT/GT genotypes, 5.9; P = 0.03). Furthermore, the GG genotype was positively related to enhanced tumor proliferation (mitotic count, P = 0.01). In line with this finding, the GG genotype was absent in grade 1 carcinomas and increasingly prevalent in tumors with higher malignancy (grade 2, 28.0%; grade 3, 50%; P = 0.04). CONCLUSION: This study found that the XbaI G>T SNP of the GLUT1 gene is associated with an increased (18)F-FDG uptake and a more advanced tumor grade or growth in breast cancer. Thus, this genetic variant might favor aggressive phenotypes by modulating the efficiency of cancer cells to recruit glucose and escalate growth rate, suggesting the XbaI G>T SNP as a proliferation-related prognostic factor.

The CYP1A1 Ile462Val polymorphism and platinum resistance of epithelial ovarian neoplasms.

The role of estrogens in ovarian carcinogenesis and progression of ovarian cancer is unclear. Cytochrome P450 is involved in estrogen metabolism, and polymorphisms have been associated with functional changes and risk for ovarian cancer. In this study, we investigated the impact of the CYP1A1 Ile462Val polymorphism upon tumor risk and disease progression in ovarian cancer patients. One hundred and eleven ovarian cancer patients who had been treated at the University Hospital of Essen between 1999 and 2007 and 119 age-matched healthy female controls were enrolled in this study. Genotyping was performed using PCR-RFLP. The distribution of genotypes was statistically significant different between ovarian cancer patients and healthy controls. We observed a significant association of the Ile allele with ovarian cancer (OR 2.6, 95% CI 1.5-4.7, p = 0.001). Clinical parameters such as overall survival, FIGO stage, grading, and age at diagnosis did not differ significantly. We observed a statistically significant association between the 462Val allele and platinum resistance, which was defined as a time interval < 6 months to disease progression after administration of a platinum-based primary chemotherapy (OR 5.9, 95% CI 1.5-23.2, p = 0.005). We observed a significant association between the presence of the 462Ile allele with ovarian cancer. While there is uncertainty about the potential involvement of CYP1A1 in the metabolism of platinum-containing agents, our findings suggest an association between the 462Val allele and the development of platinum resistance in ovarian tumors. If confirmed in a larger, independent collective, our findings would have important relevance with respect to the clinical consequences for the primary chemotherapy of ovarian cancer patients.

Aquaporin 1 (AQP1) expression is a novel characteristic feature of a particularly aggressive subgroup of basal-like breast carcinomas.

Aquaporin1 (AQP1) is a water channel protein that facilitates water flux across cell membranes. It is widely expressed in epithelial and endothelial cells in several tissues. AQP1 is also associated with angiogenesis, cell migration and metastasis in some human malignancies. In this study the immunohistochemical expression of AQP1 in 203 invasive breast carcinomas with long-term follow up was investigated. AQP1 expression was demonstrated in 11 tumours (5.4%) and showed highly significant correlation with high tumour grade, medullary-like histology, "triple-negativity", cytokeratin 14 and smooth muscle actin expression. In univariate analysis, AQP1 was significantly associated with poor prognosis. In multivariate analysis, AQP1 expression proved to be an independent prognostic marker if stratified by age, tumour size, lymph node status, histological grade, ER status and CMF therapy. Our results strongly suggest that AQP1 expression is a new characteristic feature of a particularly aggressive subgroup of basal-like breast carcinomas.

Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer.

INTRODUCTION: The origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients. METHODS: BM aspirates of 81 primary breast cancer patients were analyzed for the presence of disseminated tumor cells (DTC) by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. PCR-based fluorescence microsatellite analysis was performed for detection of loss of heterozygosity (LOH) at 6 polymorphic markers using cell-free serum DNA. The data were correlated with established risk factors, and patients were followed-up over 6-10 years. RESULTS: LOH was detected in 33.5% of blood samples. The occurrence of LOH at the entire microsatellite marker set correlated with histopathology (P = 0.05) and grading (P = 0.006) of the primary tumor. The genomic region characterized by marker D9S171 was only affected by LOH in patients with increased tumor stages (pT2-4, P < 0.05) and older age (> or = 55 years, P = 0.05). Kaplan-Meier analysis showed that LOH at D3S1255 (P = 0.009) and D9S171 (P = 0.001) were significantly associated with tumor relapse. In BM, DTC were detected in 39.5% of the patients, and this finding correlated with distant metastases (P < 0.05). Patients with DTC-positive BM had higher DNA yields in their blood than patients with DTC-negative BM (P < 0.05). However, no significant correlations were found between the presence of DTC in BM and the detection of marker-specific LOH on blood DNA. CONCLUSIONS: The detection of LOH on cell-free tumor DNA in blood is unrelated to BM micrometastasis and provides independent information on breast cancer progression.

The AA genotype of a L1C G842A polymorphism is associated with an increased risk for ovarian cancer.

BACKGROUND: Recent studies proposed L1CAM (L1 cell adhesion molecule) expression as a negative prognostic marker in epithelial ovarian cancer (EOC). The gene L1C was screened for single nucleotide polymorphisms (SNPs) which could impact upon EOC risk or disease progression. PATIENTS AND METHODS: Overlapping DNA fragments, including the promoter region, intron 1 and all the exons of 10 healthy volunteers were analyzed to detect SNPs. EOC patients (n=103) and age-matched controls (n=104) were subsequently genotyped by restriction fragment length polymorphism (RFLP). Quantitative real-time PCR was carried out to detect potential associations of SNPs with L1C mRNA expression. RESULTS: One SNP was found in intron 1 (L1C G842A). Genotyping of the EOC patients and age-matched controls revealed an association of EOC with the homozygous AA genotype (OR 7.4, CI 1.6-33.5; p=0.003). The L1C mRNA expression levels and clinical parameters did not differ significantly between the L1C G842A genotypes. CONCLUSION: The L1C 842 AA genotype may be a predisposing factor for EOC.

Diagnostic value of full-dose FDG PET/CT for axillary lymph node staging in breast cancer patients.

PURPOSE: The aims of this study were (1) to evaluate FDG PET/CT and CT for the detection of axillary lymph node metastases in breast cancer (BC) patients and (2) to evaluate FDG PET/CT as a pre-test for the triage to sentinel lymph node biopsy (SLNB) versus axillary lymph node dissection (ALND). METHODS: The sensitivity, specificity, positive and negative predictive value (PPV, NPV), and accuracy of FDG PET/CT and CT for axillary lymph node metastases were determined in 61 patients (gold standard: histopathology). According to the equation "NPV = specificity (1-prevalence) / [specificity (1-prevalence) + (1-sensitivity) prevalence]" FDG PET/CT was evaluated as a triage tool for SLNB versus ALND. RESULTS: The sensitivity, specificity, PPV, NPV and accuracy of FDG PET/CT was 58, 92, 82, 77 and 79% and of CT 46, 89, 72, 71 and 72%, respectively. Patients with an up to approximately 60% risk for axillary lymph node metastases appear to be candidates for SLNB provided that the axilla is unremarkable on FDG PET/CT. CONCLUSION: FDG PET/CT cannot replace invasive approaches for axillary staging but may extend the indication for SLNB.

The haplotype of two FSHR polymorphisms in ovarian cancer--a potential role of ethnology in risk modification.

OBJECTIVES: The precise role of gonadotropins in the carcinogenesis of epithelial ovarian cancer remains uncertain. Recently, the haplotype of two single nucleotide polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), has been described as a risk factor for ovarian cancer in Chinese women. In this study we investigated the impact of this haplotype regarding the risk to develop ovarian cancer as well as possible effects upon the clinical course in a Caucasian patient sample. SUBJECTS AND METHODS: Determination of genotypes in 115 patients with primary epithelial ovarian cancer and 115 age-matched controls was performed by Pyrosequencing for Thr307Ala and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for Asn680Ser. RESULTS: Analysis of the genotypes revealed almost complete linkage disequilibrium of both SNPs. The distribution of genotypes was not statistically significant different between ovarian cancer patients and age-matched controls. Clinical parameters such as overall survival, CA12-5 elevation at primary diagnosis, age at diagnosis, FIGO stage, grading, and platinum resistance were not statistically significantly different regarding genotypes. CONCLUSIONS: We could not confirm the FSHR Ala307-Ser680 haplotype as a risk factor for epithelial ovarian cancer in Caucasian women. Hence, the modification of tumor risk may be affected by the ethnology of the patient collective. We could not find any associations of clinical parameters or course of the disease with the different genotypes.

Association of the AA genotype of the BCL2 (-938C>A) promoter polymorphism with better survival in ovarian cancer.

BACKGROUND: Bcl-2 plays a key role in the regulation of apoptosis. Recently, a novel regulatory single nucleotide polymorphism (-938C>A) in the inhibitory P2 BCL2 promoter was described. In this study we investigated its potential association with survival in epithelial ovarian cancer. EXPERIMENTAL DESIGN: Patients (n=110) with primary epithelial ovarian cancer were retrospectively genotyped by pyrosequencing. RESULTS: Genotype distribution was not significantly different between 110 ovarian cancer patients and 120 healthy controls, suggesting that genotypes of this polymorphism do not increase the susceptibility to ovarian cancer. Kaplan-Meier curves showed a significant association of the AA genotype with increased survival (p=0.002). Multivariate analysis revealed that the BCL2-938AC/CC genotype (hazard ratio 4.5; p=0.003) was an independent prognostic factor compared to other prognostic factors such as age, histological grade or tumor stage. CONCLUSION: The results suggest a role for the BCL2-938C>A polymorphism as a marker for survival in patients with epithelial ovarian cancer.

Prognostic relevance of the AQP5 -1364C>A polymorphism in primary breast cancer.

Besides known prognostic factors in breast cancer, disseminated tumor cells are regarded as a surrogate marker for minimal residual disease in breast cancer. Their prognostic significance is comparable to that of lymph node status. However, the mechanism by which the primary tumor directs cells to disseminate into lymph nodes or into blood vessels is unclear. Aquaporins (AQPs) are small integral membrane proteins that provide a major pathway for water transport throughout several organs, and have been shown to be of potential importance in various types of cancer. Here, we analyzed the single nucleotide polymorphism (SNP) A(-1364)C in AQP5 in 107 patients with early stage breast cancer in order to test the hypothesis that this polymorphism is associated with lymphogenous and hematogenous tumor cell dissemination and overall survival. Paraffin-embedded tumor tissue was dewaxed, and DNA was extracted from tumor tissues using the QIAamp Blood DNA Mini Kit. The quantification and quality of the extracted DNA were determined spectrophotometrically. Genotyping of the -1364A>C polymorphism was performed by Pyrosequencing. Cytokeratin-positive (CK+) bone marrow (BM) cells were isolated by density gradient centrifugation followed by immunocytochemistry, applying the pan CK antibody A45-B/B3. There was no evidence of an association between the AQP5C>A genotypes and an increased risk of developing breast cancer, nor between the genotypes and tumor size, lymph node involvement, distant metastasis, grade, histopathology, expression of estrogen receptor and HER2, or the dissemination of tumor cells to BM. In contrast, when comparing C-allele carriers with patients carrying the AA genotype, expression of the progesterone receptor differed significantly between these genotype groups (mean AA genotype 51%, mean AC and CC genotype 73%; p=0.039). Additionally, a significant correlation was found between progesterone receptor expression and adjuvant chemotherapy (p=0.021) and adjuvant endocrine therapy (p=0.017), respectively. SNPs in AQP5 are not associated with hematogenous or lymphogenous tumor cell spread. However, we observed for the first time an association between SNPs in AQP5 and progesterone receptor positivity, which might have implications for future adjuvant treatment. Further investigations must include more than one AQP as well as factors promoting angiogenesis to elucidate the different modes of tumor cell dissemination.

The AA genotype of the regulatory BCL2 promoter polymorphism ( 938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients.

PURPOSE: Expression of the antiapoptotic and antiproliferative protein Bcl-2 has been repeatedly shown to be associated with better clinical outcome in breast cancer. We recently showed a novel regulatory (-938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter generating significantly different BCL2 promoter activities. EXPERIMENTAL DESIGN: Paraffin-embedded neoplastic and nonneoplastic tissues from 274 patients (161 still alive after a follow-up period of at least 80 months) with primary unilateral invasive breast carcinoma were investigated. Bcl-2 expression of tumor cells was shown by immunohistochemistry; nonneoplastic tissues were used for genotyping. Both the Bcl-2 expression and the (-938C>A) genotypes were correlated with the patients' survival. RESULTS: Kaplan-Meier curves revealed a significant association of the AA genotype with increased survival (P = 0.030) in lymph node-negative breast cancer patients, whereas no genotype effect could be observed in lymph node-positive cases. Ten-year survival rates were 88.6% for the AA genotype, 78.4% for the AC genotype, and 65.8% for the CC genotype. Multivariable Cox regression identified the BCL2 (-938CC) genotype as an independent prognostic factor for cancer-related death in lymph node-negative breast carcinoma patients (hazard ratio, 3.59; P = 0.032). Immunohistochemical Bcl-2 expression was significantly associated with the clinical outcome of lymph node-positive but not of lymph node-negative breast cancer patients. In lymph node-negative cases, the (-938C>A) SNP was both significantly related with the immunohistochemically determined level of Bcl-2 expression (P = 0.044) and the survival of patients with Bcl-2-expressing carcinomas (P = 0.006). CONCLUSIONS: These results suggest the (-938C>A) polymorphism as a survival prognosticator as well as indicator of a high-risk group within patients with lymph node-negative breast cancer.

Influence of platinum-based chemotherapy on disseminated tumor cells in blood and bone marrow of patients with ovarian cancer.

OBJECTIVE: We evaluated (1) the prevalence of disseminated tumor cells (DTC) before and after first-line chemotherapy with carboplatin and paclitaxel in the bone marrow (BM) and peripheral blood (PB) of 57 patients with primary ovarian cancer and (2) the coexpression of the epithelial antigen EpCAM on DTC including the determination of apoptotic cells. METHODS: DTC were detected by immunocytochemistry applying the anti-cytokeratin (CK) antibody A45-B/B3. For double-labeling of DTCs, the antibodies M30 (apoptosis), HEA-125-FITC/Ber-EP4-FITC (EpCAM) were used. RESULTS: Before chemotherapy, we identified DTC in 12/57 PB samples (21%) with a median number of 2 cells/20 ml (range 1-8) and in 25/46 BM samples (54%) with a median number of 5 cells/9x10E6 BM cells (range 1-28). Analysis of DTC in PB and BM before and after therapy was performed in 30 patients. In this subgroup, we identified DTC in 5/30 PB samples (16%) and in 15/30 BM samples (50%) before chemotherapy. After chemotherapy, DTC in PB were only detected in one patient but in the BM of 15/30 patients (50%). After chemotherapy, BM analysis revealed evidence that no DTC were detectable any longer in 9 patients, no significant change in DTC was documented in 14 patients and a significant enhancement of DTC was shown in 10 patients, including 8 patients who had no DTC before chemotherapy. DTC, still present after chemotherapy, co-expressed EpCAM and were non-apoptotic. In a univariable analysis, patients with a marked increase of DTC showed a significantly reduced PFS (p=0.041). A corresponding multivariable Cox regression analysis was not feasible due to the limited number of events. No correlation of DTC in BM and PB was found with patient's and tumor characteristics. CONCLUSION: DTC were present in 50% of patients after first-line chemotherapy in ovarian cancer. It has to be considered whether patients with persisting EpCAM/CK-positive BM cells probably might benefit from an additive immunotherapy e.g. targeting EpCAM.

The T393C polymorphism in the gene GNAS1 of G protein is associated with survival of patients with invasive breast carcinoma.

The GNAS1 locus encodes the G(alpha)s protein which stimulates the formation of cyclic AMP (cAMP). Subsequently the cAMP pathway mediates various pleiotropic effects including regulation of apoptosis and proliferation. We have recently shown that genotypes of the single nucleotide polymorphism (SNP) T393C in the gene GNAS1 are associated with survival of patients suffering from bladder, renal cell and colorectal carcinoma. In the present study, the genotypes of the T393C SNP were determined in 279 patients with invasive breast carcinoma. Comparing the genotypes with the overall survival as well as important clinico-pathological parameters showed that carriers of the T allele had a significantly less favourable course of the disease when compared to carriers of the homozygous CC genotype. In multivariate analysis the homozygous TT genotype was independently associated with a decreased overall survival. Our results suggest that the GNAS1 T393C SNP is a novel genetic host factor for disease progression in patients with invasive breast carcinoma.

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome.

INTRODUCTION: Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy. METHODS: We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis. RESULTS: Elevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01). CONCLUSIONS: Immunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

Heat shock protein 72 and apoptosis indicate cardiac decompensation during early multiple organ failure in sheep.

OBJECTIVE: Inducible heat shock protein 72 (HSP 72) preserves myocardial function and prevents apoptosis. We investigated the expression and localization of HSP 72 and apoptosis in our previously described new model of multiple organ failure. DESIGN: Eighteen adult-instrumented sheep and three healthy controls were randomly assigned to one of three groups: (a) norfenefrine-masked hypovolemia plus endotoxemia (NMH+ENDO); (b) norfenefrine-masked hypovolemia without endotoxemia (NMH); (c) recurrent endotoxemia during normovolemia (ENDO); and (d) normovolemia without endotoxemia (CONTROLS). MEASUREMENTS AND RESULTS: Hearts were analyzed by light microscopy, Western blots, immunohistochemistry, and TUNEL staining. HSP 72 expression was approximately threefold increased in NMH+ENDO compared with the other groups ( p<0.05) and was localized mainly in left ventricular cardiomyocytes. HSP 72 was elevated in animals with norfenefrine-refractory shock compared to survivors ( p=0.015). TUNEL-positive cells in the left ventricle were significantly elevated in the NMH+ENDO group ( p=0.05) and correlated with HSP 72 expression (r=0.51, p=0.018). HSP 72 correlated positively with heart rate (r=0.76, p<0.0001), the prefinal hourly dose of norfenefrine (r=0.88, p<0.0001), and negatively with left ventricular stroke work index (r=-0.52, p=0.028). Double staining revealed TUNEL-positive cells with and without HSP 72 expression. Micronecroses were only detectable in NMH and NMH+ENDO without intergroup difference or correlations with hemodynamics. CONCLUSION: HSP 72 overexpression and apoptosis, but not necrosis, indicate cardiovascular decompensation and poor outcome during early multiple organ failure.