Friedrich Otterbach

Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases

Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P<0.05) and was related to reduced tumor apoptosis (P<0.05). No association was found between pAkt or pERK1/2 with cell proliferation assessed by Ki67 and mitotic count (MC). Survival analysis of receptor status, HER2/neu expression, histological grading, MC and pAkt immunoexpression showed a significant correlation with decreased OS, but only pAkt reached statistical significance in the multivariate Cox regression analysis (P=0.015). Activation of Akt in node-negative breast cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor of OS. The determination of pAkt status may be of value in identifying high-risk patients, who would benefit from adjuvant therapy, and gives a rationale to investigate new therapy strategies by specific inhibition of the Akt signaling pathway in breast cancer.

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

IntroductionFocal 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.MethodsWe 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.ResultsElevated 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).ConclusionsImmunohistochemical 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.

Whole-body FDG PET/CT is more accurate than conventional imaging for staging primary breast cancer patients

PurposeThis retrospective study aimed (1) to compare the diagnostic accuracy of whole-body FDG PET/CT for initial breast cancer staging with the accuracy of a conventional, multimodal imaging algorithm, and (2) to assess potential alteration in patient management based on the FDG PET/CT findings.MethodsPatients with primary breast cancer (106 women, mean age 57u2009±u200913xa0years) underwent whole-body FDG PET/CT and conventional imaging (X-ray mammography, MR mammography, chest plain radiography, bone scintigraphy and breast, axillary and liver ultrasonography). The diagnostic accuracies of FDG PET/CT and a conventional algorithm were compared. Diagnostic accuracy was assessed in terms of primary tumour detection rate, correct assessment of primary lesion focality, T stage and the detection rates for lymph node and distant metastases. Histopathology, imaging or clinical follow-up served as the standards of reference.ResultsFDG PET/CT was significantly more accurate for detecting axillary lymph node and distant metastases (pu2009=u20090.0125 and pu2009<u20090.005, respectively). No significant differences were detected for other parameters. Synchronous tumours or locoregional extraaxillary lymph node or distant metastases were detected in 14 patients (13%) solely by FDG PET/CT. Management of 15 patients (14%) was altered based on the FDG PET/CT findings, including 3 patients with axillary lymph node metastases, 5 patients with extraaxillary lymph node metastases, 4 patients with distant metastases and 3 patients with synchronous malignancies.ConclusionFull-dose, intravenous contrast-enhanced FDG PET/CT was more accurate than conventional imaging for initial breast cancer staging due to the higher detection rate of metastases and synchronous tumours, although the study had several limitations including a retrospective design, a possible selection bias and a relevant false-positive rate for the detection of axillary lymph node metastases. FDG PET/CT resulted in a change of treatment in a substantial proportion of patients.

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

IntroductionThe 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.MethodsBM 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.ResultsLOH 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 (≥ 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.ConclusionsThe 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 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.

ERCC1-Positive Circulating Tumor Cells in the Blood of Ovarian Cancer Patients as a Predictive Biomarker for Platinum Resistance

BACKGROUNDnPlatinum 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.nnnMETHODSnThe 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.nnnRESULTSnAt 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.nnnCONCLUSIONSnThe presence of ERCC1(+)CTC can serve as a blood-based diagnostic biomarker for predicting platinum resistance at primary diagnosis of ovarian cancer.

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.

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.

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 3.48u2005mm (1.1 to 9.8u2005mm). In all cases (n=20) biopsies (0.3–2.1u2005mm 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.

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.