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Supplementary Figure S1 and Supplementary Tables S1-S3.

Purpose: A preliminary study performed on a small cohort of multifocal prostate cancer (PCa) detected BRCA1 allelic imbalances among circulating tumor cells (CTC). The present analysis was aimed to elucidate the biological and clinical roles of BRCA1 losses in metastatic spread and tumor progression in PCa patients.Experimental Design: To map molecular progression in PCa outgrowth, we used fluorescence in situ hybridization analysis of primary tumors and lymph node sections, and CTCs from peripheral blood.Results: We found that 14% of 133 tested patients carried monoallelic BRCA1 loss in at least one tumor focus. Extended molecular analysis of chr17q revealed that this aberration was often a part of larger cytogenetic rearrangement involving chr17q21 accompanied by allelic imbalance of the tumor suppressor gene PTEN and lack of BRCA1 promoter methylation. The BRCA1 losses correlated with advanced T stage (P < 0.05), invasion to pelvic lymph nodes (P < 0.05), as well as biochemical recurrence (P < 0.01). Their prevalence was twice as high within 62 lymph node metastases (LNM) as in primary tumors (27%, P < 0.01). The analysis of 11 matched primary PCa-LNM pairs confirmed the suspected transmission of genetic abnormalities between these two sites. In four of seven patients with metastatic disease, BRCA1 losses appeared in a minute fraction of cytokeratin- and vimentin-positive CTCs.Conclusions: Small subpopulations of PCa cells bearing BRCA1 losses might be one confounding factor initiating tumor dissemination and might provide an early indicator of shortened disease-free survival. Clin Cancer Res; 16(13); 3340–8. ©2010 AACR.

IntroductionLiquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics.MethodsIn total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters.ResultsDetection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection.ConclusionOur study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine.

Aldehyde dehydrogenase 1 (ALDH1) characterizes tumor-initiating cells in solid tumors; however, little is known about its expression in intratumoral stromal cells. Herein, we aimed to dissect its potential dual relevance in prostate cancer (PCa). ALDH1 expression was evaluated immunohistochemically in tumor and stromal cells in primary PCa and metastases. It was correlated to clinico-pathologic parameters, patients' outcome, and selected proteins (CK5/6, CK14, CK8/18, CK19, EpCAM, Ki-67, E-cadherin, N-cadherin, and vimentin). ALDH1 protein was detected in tumor and stromal cells in 16% and 67% of 348 primary PCa, respectively. Tumor cell ALDH1 expression was associated with advanced T stage (P = 0.009), higher Gleason score (P = 0.016), shorter time to biochemical recurrence (TBR P = 0.010) and CK14 expression (P = 0.023). Stromal cell ALDH1 expression correlated to lower T stage (P = 0.008) and Gleason score (P = 0.016), N0 stage (P = 0.017), and longer TBR (P = 0.017). It occurred to be an independent predictor of good prognosis in the subgroup of d'Amico high-risk patients (multivariate analysis, P = 0.050). ALDH1-positive stromal cells were found in tumors characterized frequently by CK8/18 (P = 0.033) or EpCAM expression (P < 0.001) and rarely by epithelial-mesenchymal transition defined as CK8/18(-)vimentin(+) phenotype (P = 0.003). ALDH1-positive tumor and stromal cells were detected in 33% and 41% of hormone naive lymph node metastases (n = 63), 52% and 24% of castration resistant bone metastases, as well as 89% and 28% of castration resistant visceral metastases (n = 21), respectively. We have determined that contrary to tumor cell ALDH1, the presence of stromal ALDH1 is associated with epithelial phenotype of primary PCa, improved clinical outcome, and is less frequent in PCa metastases.

The pandemic spread of the potentially life-threatening disease COVID-19 requires a thorough understanding of the longitudinal dynamics of host responses. Temporal resolution of cellular features associated with a severe disease trajectory will be a pre-requisite for finding disease outcome predictors. Here, we performed a longitudinal multi-omics study using a two-centre German cohort of 13 patients (from Cologne and Kiel, cohort 1). We analysed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. The results from single-cell and bulk transcriptome analyses were validated in two independent cohorts of COVID-19 patients from Bonn (18 patients, cohort 2) and Nijmegen (40 patients, cohort 3), respectively. We observed an increase of proliferating, activated plasmablasts in severe COVID-19, and show a distinct expression pattern related to a hyperactive cellular metabolism of these cells. We further identified a notable expansion of type I IFN-activated circulating megakaryocytes and their progenitors, indicative of emergency megakaryopoiesis, which was confirmed in cohort 2. These changes were accompanied by increased erythropoiesis in the critical phase of the disease with features of hypoxic signalling. Finally, projecting megakaryocyte- and erythroid cell-derived co-expression modules to longitudinal blood transcriptome samples from cohort 3 confirmed an association of early temporal changes of these features with fatal COVID-19 disease outcome. In sum, our longitudinal multi-omics study demonstrates distinct cellular and gene expression dynamics upon SARS-CoV-2 infection, which point to metabolic shifts of circulating immune cells, and reveals changes in megakaryocytes and increased erythropoiesis as important outcome indicators in severe COVID-19 patients.

Circulating tumour cells (CTC) were proven to be prognostically relevant in cancer treatment, e.g., in colorectal cancer (CRC). This study validates a molecular detection technique through using a novel cell imaging approach for CTC detection and enumeration, in comparison to a size-based cellular and correlated the data to clinico-pathological characteristics. Overall, 57 CRC patients were recruited for this prospective study. Blood samples were analysed for CTCs by three methods: (1) Epithelial marker immunofluorescence staining combined with automated microscopy using the NYONE&reg, cell imager, (2) isolation by size using membrane filtration with the ScreenCell&reg, Cyto IS device and immunofluorescence staining, (3) detection by semi-quantitative Cytokeratin-20 RT-qPCR. Enumeration data were compared and correlated with clinic-pathological parameters. CTC were detected by either approach, however, with varying positivity rates: NYONE&reg, 36.4%, ScreenCell&reg, 100%, and PCR 80.5%. All methods revealed a positive correlation of CTC presence and higher tumour burden, which was most striking using the ScreenCell&reg, device. Generally, no intercorrelation of CTC presence emerged amongst the applied techniques. Overall, enumeration of CTC after isolation by size demonstrated to be the most reliable strategy for the detection of CTC in CRC patients. Ongoing studies will have to unravel the prognostic value of this finding, and validate this approach in a larger cohort.

Background: This is the first study investigating the safety and efficacy of the trifunctional antibody catumaxomab administered i.p. at the end of cytoreductive surgery and postoperatively prior to standard chemotherapy in patients with primary epithelial ovarian cancer (EOC). Methods: Patients received i.p. catumaxomab 10 μg intraoperatively and 10, 20, 50 and 150 μg on days 7, 10, 13 and 16, respectively, postoperatively. After the study, patients received standard chemotherapy and were followed for 23 months. The primary endpoint was the rate of postoperative complications. Results: Forty-one patients entered the study and were evaluable for safety and 34 were alive at 24 months. Complete tumour resection rate was 68%. Postoperative complications were observed in 51%, the most common anastomotic leakage (7%) and wound infections (5%). The most common catumaxomab-related adverse events were abdominal pain, nausea, vomiting and pyrexia. Thirty-nine percent discontinued catumaxomab therapy, and 98% received chemotherapy post study. Kaplan–Meier estimates of disease-free and overall survival after 24 months were 56% and 85%, respectively. Conclusions: Intra- and close postoperative catumaxomab seems feasible, but efficacy and safety were limited by postsurgical complications. In the future prospective trials are needed to investigate the best schedule of integration of catumaxomab into current treatment strategies for EOC.

Basal transcription regulation of the epidermal growth factor receptor is dependent upon a CA simple sequence repeat polymorphism in the intron-1 (CA-SSR-1). Here, we evaluate the role of CA-SSR-1 in complete resected esophageal cancer (EC) patients without neoadjuvant or adjuvant treatment. Genomic DNA was extracted from peripheral blood leukocytes of 241 patients. To determine the number of the CA repeats in the CA-SSR-1, DNA was amplified by polymerase chain reaction and sequenced. The results were correlated with clinicopathological parameters and clinical outcome. Three genotypes were defined based on cut-off points for short allele (S) with ≤18 and long allele (L) >18 CA repeats. A steadily increasing risk was evident between LL, SL, and SS genotype for larger tumor size, presence of lymph node metastases, and disseminated tumor cells in bone marrow as well as tumor recurrence (P

Background Molecular breast cancer subgroups show differences with regard to prognosis and response to chemotherapy. In vitro chemotherapy sensitivity and resistance assays (CSRAs) are a means to directly evaluate tumour cell response to a given drug. Methods Using tissue microarray (TMA) analysis, 550 invasive breast cancers were investigated for the expression of oestrogen receptor (ER), progesterone receptor (PR), vimentin, Ck5, Ck14, Ck19, HER2 and Mib-1/Ki-67. All carcinomas were subjected to in vitro CSRA analysis using three separate chemotherapy combinations. In vitro chemotherapy sensitivity was established using an adenosine triphosphate (ATP) bioluminescence assay. Results of immunohistochemical staining and in vitro response were correlated. Results Mib-1 expression was associated with sensitivity against Paclitaxel/Epirubicin (p = 0.014) and Docetaxel/Epirubicin (p = 0.014). Mib-1 expression was positively/negatively correlated with expression of basal/luminal markers, respectively. Hierarchical clustering revealed three subtypes, resembling luminal, basal-like and HER2-like breast cancer subtypes. In vitro response for Paclitaxel/Epirubicin was most frequently observed for cases in the basal category (40.3%) compared to HER2-like (25.8%) and luminal cases (28.6%). In multivariate analysis expression of Mib-1 was not a significant independent predictor of in vitro response to chemotherapy. Conclusion Breast cancer molecular subclasses show differences regarding in vitro chemotherapy sensitivity. These may in part explain differences observed between breast cancer molecular subtypes in vivo.

HER2 signalling by heterodimerisation with EGFR and HER3 in breast cancer is associated with worst outcome of the afflicted patients, which is attributed not only to the aggressiveness of such tumours but also to therapy resistance. Thus, in the present study we investigated the role of EGFR, HER2 and HER3 lateral signalling in cell migration by applying the MDA-MB-468-HER2 (MDA-HER2) breast cancer cell line, representing a valid model system. Knockdown of HER3 expression by siRNA resulted in decreased phosphorylated AKT (pAKT) levels, abrogated epidermal growth factor (EGF)-mediated PLC-γ1 activation and a diminished EGF-induced migratory activity, depicting the interplay of EGF receptor (EGFR)/HER2/PLC-γ1 and HER2/HER3/PI3K signalling in mediating the migration of EGFR/HER2/HER3-expressing breast cancer cells. Since therapy failure usually arises from metastatic cells, we further investigated whether HER3 signalling was active in established breast cancer disseminated tumour cell (DTC) lines as well as in primary DTCs derived from breast cancer patients. EGF treatment of DTC lines resulted solely in increased pAKT S473 levels, whereas in MDA-HER2 cells both pAKT S473 and pAKT T308 levels were increased upon EGF stimulation. Moreover, despite active HER3 molecules, as indicated by pTyr1222 staining, about 90% of analysed breast cancer patient DTCs exhibited very low or even no detectable pAKT S473 levels, suggesting that these cells might have fallen into dormancy. In summary, our data indicate the important role in EGFR, HER2 and HER3 lateral signalling in breast cancer cell migration. Moreover, our data further show that primary tumour cells and DTCs can vary in their HER activation status, which is important to know in the context of cancer therapy.

Purpose There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

A whole chromosome arm loss of 16q belongs to the most frequent and earliest chromosomal alterations in invasive and in situ breast cancers of all common subtypes. Besides E-cadherin, several putative tumour suppressor genes residing on 16q in breast cancer have been investigated. However, the significance of these findings has remained unclear. Thus, other mechanisms leading to gene loss of function (eg haploinsufficiency, or distortion of multiple regulative subnetworks) remain to be tested as a hypothesis. To define the effect on gene expression of whole-arm loss of chromosome 16q in invasive breast cancer, we performed global gene expression analysis on a series of 18 genetically extensively characterized invasive ductal breast carcinomas and verified the results by quantitative real-time PCR (qRT-PCR). The distribution of the differential genes across the genome and their expression status was studied. A second approach by qRT-PCR in an independent series of 30 breast carcinomas helped to narrow down the observed effect. Whole-arm chromosome 16q losses, irrespective of other chromosomal changes, are associated with decreased expression of a number of candidate genes located on 16q (eg CDA08, CGI-128, SNTB2, NQO1, SF3B3, KIAA0174, ATBF1, GABARAPL2, KARS, GCSH, MBTPS1 and ZDHHC7) in breast carcinomas with a low degree of genetic instability. qRT-PCR provided evidence to suggest that the expression of these genes was reduced in a gene dosage-dependent manner. The differential expression of the candidate genes according to the chromosomal 16q-status vanished in genetically advanced breast cancer cases and changed ER status. These results corroborate previous reports about the importance of whole-arm loss of chromosome 16q in breast carcinogenesis and give evidence for the first time that haploinsufficiency, in the sense of a gene dosage effect, might be an important contributing factor in the early steps of breast carcinogenesis.

International audience; The factors determining the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients are largely unknown. Here we compared the specificity and clinical performance of two antibodies frequently used for DTC detection. Reactivities of antibodies A45-B/B3 (A45) and AE1/AE3 (AE) for selected cytokeratins (CK) were assessed by 2-DE Western Blot analysis. Using these antibodies bone marrow aspirates from 391 breast cancer patients (M, pT1-3, pN0-3) were screened for the presence of DTC. To obtain prognostic information, patients were followed up over a median of 83 months for time to relapse and 99 months for time to death. Among the analyzed CK, AE detected CK5, CK7, CK8, and CK19, whereas A45 recognized CK7 and CK18. In total, 24 of 391 patients (6.1%) were DTC-positive for A45, and 41 (10.5%) for AE. Although concordance between the two antibodies was 84.4%, overlap among positive cases was only 3.2%. DTC-positivity with AE and A45 was more frequent in patients of higher nodal status ( = 0.019 and = 0.036, respectively). Nearly all patients with A45-positive DTC had hormone receptor-positive tumors (23/24), while detection of AE-positive DTC was more frequent among hormone receptor negative patients ( = 0.006). Survival analyses of all patients revealed shorter distant disease-free survival ( = 0.039) for patients with A45-positive DTC, whereas the prognostic relevance of AE-positive DTC was restricted to node-positive patients. The clinical utility of immunocytochemical (ICC) DTC detection depends on the anti-CK antibody used, which may reflect the complex CK composition of DTC.

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.

Clinical and animal studies have shown that coexpression of the receptor tyrosine kinases HER2 and epidermal growth factor (EGF) receptor (EGFR) indicates a highly metastatic phenotype of breast cancer. In a cellular model of this phenotype using differential gene expression analysis, we identified TOB1 to be up-regulated depending on EGF stimulation and transduction through phosphorylation of HER2 tyrosine 1248. mRNA expression analysis of breast cancers from a cohort of node-negative patients showed significantly shortened distant metastasis-free survival for patients with high TOB1 expression. In subsequent tissue microarray studies of 725 clinical samples, high HER2 and EGF protein levels were significantly correlated with TOB1 expression in breast cancer, whereas EGFR and EGF levels correlated with TOB1 phosphorylation. We did not observe a correlation between TOB1 expression and cyclin D1, which was previously suggested to mediate the antiproliferative effect of unphosphorylated TOB1. A positive correlation of TOB1 phosphorylation status with proliferation marker Ki67 suggests that elevated TOB1 phosphorylation might abrogate the antiproliferative effect of TOB1 in breast cancer. This suggests a new regulatory role for TOB1 in cancer progression with particular significance in HER2- and/or EGFR-positive breast cancers. [Cancer Res 2009;69(12):5049–56]

Intratumor genetic heterogeneity, a well-known characteristic of numerous cancers, often confounds a precise diagnosis and leads to therapy resistance. This study deals with such chromosomal variability, which may be due to an inherent genetic instability affecting heterogeneity and clonal effects. Subpopulations of the breast cancer cell line MDA-MB-468 were isolated according to epidermal growth factor receptor (EGFR) expression by FACS. Whole genome profiling (CGH; mapping arrays) and determination of egfr gene amplification (fluorescence in situ hybridisation, FISH; qPCR) were done directly after sorting or after several passages of cell culture. Subpopulations differed in the amplification of the egfr-locus 7p11-14 showing egfr gene amplification rates of up to 60-fold in high-level expressing populations and less than 2-fold in low-level expressing populations. However, after several passages the original low-level cells showed a new amplification of the egfr gene, which was as heterogeneous as the original amplification detected in MDA-MB-468. Additional, spontaneously expressed fragile sites could be shown in FISH analyses which may affect cell culture heterogeneity. Understanding the precise chromosomal process would clarify mechanisms in vivo and improve both diagnosis and therapy of corresponding cancers.

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT–PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P

Cancer develops and progresses by genetic aberrations expressed as many changes in chromosomes and DNA. Thereby, on the one hand microscopically detectable increases or decreases in the numbers of entire chromosomes as well as gains and losses of chromosomal stretches occur. On the other hand at the molecular level of DNA deletions, amplifications and base exchanges are also observed that have an impact on gene function and expression. It is still under debate which process of the two is the dominant cancer cause and promoter of cancer progression. Nevertheless, clonal selection during cancer development leads to the establishment of a dominant cancer cell subpopulation which harbours distinct genetic aberrations which can be used to classify cancer for prognosis and therapy prediction. The non-ambiguous relationship between the genotype of distinct loci and the phenotype is physiologically a rare event and therefore, highly selective in cancer. Although contemporary genetics have shown that quantitative trait loci exist [1], it is unlikely that the gene dosage sensitivity for a single locus significantly changes the phenotype of normal somatic cells. However, specific gene families, e.g. those involved in essential signal transduction systems, show such dosage sensitivity in cancer [2]. The most prominent example of those genes is the HER receptor family, most prominently HER2, which induces signal transduction for survival, proliferation and migration [3]. The interaction of the gene products is often the reason for such gene dosage sensitivity. Subsequent direct interactions or downstream pathway crossings with other proteins might lead to a modification of the effect of gene dosages and result in a subtle variety of phenotypes (Figure 1). To find the decisive phenotype requires enlightening the structure of the genomic ‘chaos’ in cancer by the determination of striking genomic events on the background of genetic noise.

Expression of EGFR in high grade osteosarcomas has been observed to be correlated with an improved prognosis. Yet, the underlying mechanism remained unclear since amplifications of EGFR have rarely been described. Recently, the length of a polymorphic CA repeat located at a 5′-regulatory sequence in the intron 1 of the EGFR gene (SSR I) has been shown to be associated with its basal transcriptional activity. We therefore determined the allelic length of CA SSR-I in 219 cases of high grade osteosarcoma and correlated the results with EGFR expression in 34 cases, the presence of amplifications within the CA SSR-I repeat in 59 cases, and clinical follow-up. Our results confirm that in osteosarcoma patients short alleles are more frequent than longer ones, 16 CA repeats being the most frequent. The allele composition differed significantly from the one recently described in a healthy control population (P < 0.01). Short alleles tended to be associated with increased expression of EGFR. Amplifications of the EGFR gene were seen in 13.5% of cases. Significant correlations between allele length composition and neoadjuvant chemotherapy response or long term clinical outcome could not be established. While we were able to show that high frequency of EGFR expression in osteosarcomas is associated with predominantly short alleles of EGFR-CA SSR I, persisting shortcomings in the correspondence with clinical data point toward the existence of additional, putatively more important transcription control mechanisms for EGFR in osteosarcomas which might account for the good prognostic value of EGFR expression. © 2008 Wiley-Liss, Inc.

Purpose: The expression of the epidermal growth factor receptor (EGFR) in osteosarcomas has repeatedly been described. With the introduction of anti-EGFR–targeted therapies in clinical practice, these findings regain increased attention. Experience with anti-EGFR–targeted therapies in other cancers has made clear that besides the expression status of EGFR, a detailed knowledge about gene mutations is of major predictive power. We therefore aimed to explore the EGFR expression and gene mutation status in high-grade osteosarcomas. Experimental Design: We investigated tumor samples of osteosarcoma patients of all age groups by means of immunohistochemistry (n = 111) and egfr fluorescence in situ hybridization (n = 39). Sixty-three patients were treated according to the Cooperative Osteosarcoma Study Group protocols and complete clinical follow-up was available in these cases. Results: Ninety-one of 111 (81%) of osteosarcomas revealed an expression of EGFR. EGFR expression showed a dose-response relation with improved event-free and overall survival. This was independent of the degree of tumor regression due to neoadjuvant chemotherapy. Nine of 39 (23%) osteosarcomas showed egfr amplifications by means of fluorescence in situ hybridization. All these cases expressed EGFR. When comparing EGFR expression between primary biopsy and resection specimen (n = 19), viable residual tumor cells in resection specimens revealed a lower EGFR expression and a tendency toward membranous staining compared with the initial biopsy. Conclusions: In conclusion, expression and amplification of EGFR are frequently observed in high-grade osteosarcomas and are associated with improved prognosis in a dose-responsive way. This implies that low EGFR expression possibly predicts lack of response to conventional treatment in high-grade osteosarcomas and may warrant a more intensive therapeutic approach, although not based on EGFR targeting.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. For predictive cancer diagnostics and therapeutic targeting of EGFR, it is important to know how the expression level of EGFR is controlled and related to receptor signaling. A novel transcriptional regulation mechanism has been described that depends on the length of a CA repeat in intron 1 [CA simple sequence repeat 1 (CA SSR I)] of the EGFR gene. Thereby, the number of CA repeats is inversely correlated to pre-mRNA synthesis. Indirect evidence for the importance of this mechanism includes the preferential occurrence of amplifications in cancer tissue harboring short CA repeats in this sequence and the discovery of distinct alleles in young breast cancer patients with a family history of the disease and in Japanese breast cancer patients. It can be postulated that the length of the CA repeat influences DNA bendability and, in consequence, the binding of repressor proteins. In summary, it seems that the CA SSR I represents an inherited variable for response to anti-EGFR therapies that could be determined before therapy. Moreover, the potential for synergistic effects with other polymorphism [e.g., EGFR R497K (HER-1 497K) and CCND1 A870G] leading to a simultaneous increase of EGFR signaling activity and expression should be investigated. From a practical perspective, assessment of the CA SSR I number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it is a constant feature that does not change over time and can be easily measured in normal and cancer tissues (blood cells, skin, and tumor biopsies) in an assay that is technically simple, objective, and even quantitative.

The aim of this study was to determine polymorphism of repeated sequences (CA)(n) in the ERBB-1 gene. The study group included 197 breast cancer patients and 180 healthy women. DNA was isolated from fresh-frozen tumour tissue and from peripheral blood. ERBB-1 (CA)(n) microsatellite polymorphism was examined by polymerase chain reaction (PCR). A polymorphic simple sequence repeat region of 9-23 CA repeats was detected in both groups. Homozygotes comprised 22% and 34% of breast cancer patients and controls, respectively (P=0.009). An allelic imbalance (AI), mostly in the shorter allele, was found in 27% of breast cancer patients. AI occurrence was associated with the lack of oestrogen receptors in tumour cells (P=0.05); otherwise, there were no correlations between histoclinical features and (CA)(n) microsatellite polymorphism of ERBB-1. It was concluded that an allelic imbalance is a common feature in breast cancer patients and may coincide with the lack of oestrogen receptors in tumour cells. The clinical relevance of ERBB-1 microsatellite polymorphism in breast cancer remains to be established.

Background: New targeted cancer treatments acting against growth factor receptors such as the epidermal growth factor receptor (EGFR) necessitate selecting patients for treatment with these drugs. Besides carcinomas, soft tissue sarcomas (STS) express EGFR and might thereby be a promising target for this new therapeutic strategy. Objective: To test and compare different EGFR antibodies to determine the frequency of EGFR expression in STS. Methods: 302 consecutive specimens of STS were examined using the tissue microarray technique. EGFR expression levels were assessed by immunohistochemistry using five different commercially available antibodies. Gene amplification status was measured by fluorescence in situ hybridisation (FISH). Immunoreactivity and amplification status were correlated with clinicopathological features and follow up data available in 163 cases. Results: EGFR expression frequency ranged between 0.3% and 52.9%, depending on the antibody and scoring method used. In all, 3.5% of the tumours showed egfr gene amplification by FISH, which correlated with EGFR expression for three antibodies. Only one antibody had independent prognostic value in multivariate analysis and correlated with an unfavourable outcome; egfr gene amplification status showed no correlation with clinical features. Conclusions: Frequency of EGFR immunopositivity in STS strongly depends on the antibody used, and only one of five antibodies tested predicted an unfavourable clinical outcome. This indicates that choice of primary antibody and scoring system have a substantial impact on the determination of EGFR immunoreactivity.

Metastasis development requires the migratory activity of tumor cells. It is therefore important to understand the molecular mechanisms of this migration in order to prevent metastasis development, which is the pernicious step in most solid tumor diseases. A lot of methods have been invented to investigate tumor cell migration, but not all are equally suited and no method alone is able to deliver a complete picture of tumor cell migration. We herein suggest a combination of three-dimensional in vitro and in vivo methods for the investigation of tumor cell migration and summarize the knowledge, which has been reached so far.

Extravasation has been described as a rate-limiting step in the process of hematogeneous metastasis formation. Thereby, transendothelial migration of tumor cells consists of a complex series of events involving multiple cell-cell and cell-matrix interactions. 3D-extravasation assays are valuable tools for the identification of genes, which are the key players at switchboards of the intracellular signaling pathways. In consequence, the combination of 3D-modeling and whole genome expression analysis lead to unravel molecular parameters which descripe distinct clinical phenotypes of cancer and therefore, work as prognosticators, predictors of therapy and new therapy targets.

In the field of local application of antimicrobials, a number of novel drugs and/or new drug delivery systems have been developed in recent years. The present study aimed to investigate hydroxyapatite cement (HAC) as a carrier for vancomycin in the treatment of chronic osteomyelitis due to Staphylococcus aureus strains with various mechanisms of resistance. The release of vancomycin from standard test cylinders was determined in vitro and the efficacy of the delivery system was measured in vivo using a rabbit model of chronic osteomyelitis. First, powdered HAC was mixed with vancomycin at 80, 160 and 240 mg/g. After hardening, formed cylinders were eluted in phosphate buffer and antibiotic release was measured by agar diffusion. High levels of release (1512+/-318 to 1937+/-336 microg/ml) were obtained for 12 to 20 days depending on the dosage of vancomycin. Additionally, bone infection was induced in the tibia of 30 New Zealand white rabbits by injecting either a methicillin-resistant S. aureus strain (MRSA) or a S. aureus strain with a small colony variant (SCV) phenotype. After 3 weeks (chronic infection), all animals were treated by debridement. Moreover, group 1 (challenged with SCVs) and group 2 (challenged with MRSA) were treated by filling the marrow with HAC alone, whereas in groups 3 (SCVs) and 4 (MRSA) the marrow was filled with HAC/vancomycin (160 mg/g). After 6 weeks all animals were sacrificed. At 3 weeks, pathogens were detected in 24 of 30 animals. All swabs of the control groups, positive for S. aureus on day 21, were also positive on day 42 and S. aureus strains recovered were shown to be clonal to the strains used for induction of osteomyelitis. By contrast, no growth was found in the treatment group following 7 days of incubation in BHI bouillon. HAC/vancomycin-treated animals showed no histological evidence of infection on day 42. In the other groups, different stages of chronic osteomyelitis were found histologically. No local or systemic side effects due to HAC or vancomycin were seen. HAC is an effective carrier material for antibiotic compounds even in refractory infections due to MRSA or S. aureus SCVs.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.

Vimentin expression is a rather rare finding in invasive breast cancer, and is associated with high tumour invasiveness and chemoresistance. It is currently explained by two different biological theories: direct histogenetic derivation from myoepithelial cells, and epithelial-mesenchymal transition (EMT) reflecting the end-stage of breast cancer dedifferentiation. In this study we aimed to obtain further insights into the biological hallmarks of these vimentin-expressing breast cancers. We applied immunohistochemistry for vimentin and 15 other differentiation markers to a series of 364 invasive breast cancer cases, using tissue microarray technology. 7.7% of all tumours expressed vimentin. Almost all of these cases (19/21) were Grade 3 invasive ductal carcinomas, and the majority (13/21) of these were associated with a ductal in situ component. Vimentin expression was also seen in the respective in situ components and correlated positively with the expression of SMA, CD10, CK 5, p53, Mib-1 and EGFR. A negative correlation was seen for the expression of CK 8/18 and the oestrogen receptor. Vimentin-expressing carcinomas revealed a significantly higher average absolute number of cytogenetic alterations per case, but a significantly lower frequency of chromosome 16q losses compared to vimentin-negative cases. Our present results demonstrate that, despite analogies between vimentin-positive breast cancers and myoepithelial cells in their expression of differentiation-related proteins, neither myoepithelial histogenesis nor EMT can exclusively explain the biology of these distinct tumours. This is mainly supported by the significantly higher incidence of vimentin-expressing breast cancers compared to any other myoepithelial breast tumours and the fact that vimentin is already observed in ductal in situ components. We therefore propose the alternative hypothesis that vimentin-expressing breast carcinomas may derive from breast progenitor cells with bilinear (glandular and myoepithelial) differentiation potential.

A study was performed to investigate the effectiveness of hydroxyapatite cement (HAC) as a new carrier system in the treatment of chronic, posttraumatic osteomyelitis. In the in vitro study, release of gentamicin from standard cylinders of HAC were measured by agar diffusion test. As a representative for mechanical properties, compression strength was measured in order to detect changes when mixing HAC with gentamicin. In the in vivo study, bone infection was induced according to the model of Norden by injection of 1 ml Na-morrhuat and 3×106 CFU Staphylococcus aureus. After 3 weeks, when chronic stage of infection was obtained, 17 animals were treated by debridement and filling the marrow either with HAC alone or HAC mixed with gentamicin (32 mg/g). Animals of the control groups were left untreated. After 6 weeks, all animals were sacrificed. Hematological, radiological, microbiological and histological examinations were carried out by covered investigation. Best evidence of the efficiency of treatment was observed in histopathological and microbiological findings. In all swabs of the control groups, taken 6 weeks following infection S. aureus were detected which were clonal to the strain used for induction of osteomyelitis. In HAC/gentamicin-treated animals, no growth was detectable after 7 days of culturing in BHI bouillon. In the HAC/gentamicin-treated group, there was no histopathological evidence of infection. In all other groups different stages of chronic osteomyelitis were found. No side effect was observed, neither locally nor systemically by HAC or gentamicin. Therefore, HAC is considered to be a very effective carrier for antibiotics in treatment of chronic, posttraumatic osteomyelitis.

Distinct parallel cytogenetic pathways in breast carcinogenesis could be identified in recent years. Nevertheless, it remained unclear as to which tumours may have progressed in grade or which patterns of cytogenetic alteration may define the switch from an in situ towards an invasive lesion. In order to gain more detailed insights into cytogenetic mechanisms of the pathogenesis of breast cancer, the chromosomal imbalances of 206 invasive breast cancer cases were characterised by means of comparative genomic hybridisation (CGH). CGH data were subjected to hierarchical cluster analysis and the results were further compared with immunohistochemical findings on tissue arrays from the same breast cancer cases. The combined analysis of immunohistochemical and cytogenetic data provided evidence that carcinomas with gains of 7p, and to a lesser extent losses of 9q and gains of 5p, are a distinct subgroup within the spectrum of ductal invasive grade 3 breast carcinomas. These aberrations were associated with a high degree of cytogenetic instability (16.6 alterations per case on average), 16q-losses in over 70% of these cases, strong oestrogen receptor expression and absence of strong expression of p53, c-erbB2 and Ck 5. These characteristics provide strong support for the hypothesis that these tumours may develop through stages of well- and perhaps intermediately differentiated breast cancers. Our results therefore underline the existence of several parallel and also stepwise progression pathways towards breast cancer.

The regulation of the epidermal growth factor receptor (egfr) gene in human cancer is not yet fully understood. Recent data on a polymorphic CA repeat located at the 5′-regulatory sequence in intron 1 of the egfr gene [egfr CA simple sequence repeat (SSR) I] point to a possible inheritance of cancer risk associated with the egfr gene. Furthermore, we have detected frequent allelic imbalances restricted to the egfr CA SSR I in breast cancer tissue and nontumorous breast tissue adjacent to invasive and in situ breast cancer representing amplifications. Therefore, we conducted a population-based case-control study to assess the relationship between the egfr polymorphism and breast cancer risk. Cases with a first primary breast cancer by age 50 years and age-matched population controls provided information on known and suspected risk factors. The allelic length of the egfr CA SSR was determined in 616 cases and 1072 population-sampled controls. Genotypes were categorized for analysis by allele length. Multivariate logistic regression was used to compare genotype distributions, accounting for other risk factors, and to investigate gene-environment interactions. We found a modifying effect, albeit no main effect, of the allelic length of the egfr polymorphism on breast cancer risk. The presence of two long alleles (≥19 CA) was associated with a significantly elevated odds ratio (OR) of 10.4 [95% confidence interval (CI), 1.85–58.70] among women with a first-degree family history of breast cancer (P = 0.015 for interaction). The risk increase associated with high red meat consumption (OR, 10.68; 95% CI, 1.57–72.58) and the protective effect of high vegetable intake (OR, 0.07; 95% CI, 0.004–1.07) was also most pronounced among carriers of two long alleles (≥19 CA). The length of the egfr CA SSR may increase the risk for familial breast cancers, and its effect could be modulated by dietary factors.

Early-stage non-small cell lung cancer (NSCLC) can be cured by surgical resection, but a substantial fraction of patients ultimately dies due to distant metastasis. In this study, we used subtractive hybridization to identify gene expression differences in stage I NSCLC tumors that either did or did not metastasize in the course of disease. Individual clones (n=225) were sequenced and quantitative RT-PCR verified overexpression in metastasizing samples. Several of the identified genes (eIF4A1, thymosin beta4 and a novel transcript named MALAT-1) were demonstrated to be significantly associated with metastasis in NSCLC patients (n=70). The genes' association with metastasis was stage- and histology specific. The Kaplan-Meier analyses identified MALAT-1 and thymosin beta4 as prognostic parameters for patient survival in stage I NSCLC. The novel MALAT-1 transcript is a noncoding RNA of more than 8000 nt expressed from chromosome 11q13. It is highly expressed in lung, pancreas and other healthy organs as well as in NSCLC. MALAT-1 expressed sequences are conserved across several species indicating its potentially important function. Taken together, these data contribute to the identification of early-stage NSCLC patients that are at high risk to develop metastasis. The identification of MALAT-1 emphasizes the potential role of noncoding RNAs in human cancer.

Background. Patients with extensive, long-standing ulcerative colitis have increased risk of colorectal cancer. Aims. To improve the detection of high-risk patients, using a combination of colonic cytology, histology, and DNA image cytometry after segmental colonic lavage. Patients. A series of 16 patients (8 high-risk patients) with ulcerative colitis were investigated. Methods. After segmental lavage step, biopsies were obtained. Gradient centrifugation of the colonic fluid was performed for isolation and purification of epithelial cells. The smears and biopsy specimens obtained were stained for routine interpretation and for DNA image cytometry. Results. Segmental lavage could be performed in all patients. Specimens from two high-risk patients showed low grade dysplasia and atypia by means of histology and cytology, respectively. In one patient, without increased colorectal cancer risk, atypia was detected. Three patients in the high-risk group, two of those diagnosed as positive for dysplasia and atypia, showed aneuploidy histologically and cytologically. DNA aneuploidy, in cytological material, was found exclusively in three low-risk patients, one of those had atypia cytologically. Conclusions. Isolation and purification of epithelial cells after segmental colonic lavage using density gradient centrifugation can be performed as part of routine endoscopy. It provides information about atypical cells and DNA aneuploidy as additional markers of malignant transformation. The combination of cytologic examination and DNA image cytometry might improve the detection of high-risk ulcerative colitis patients.

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)-induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC)-gamma1 due to c-erbB-2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long-term PLC-gamma1 tyrosine phosphorylation and sustained levels of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c-erbB-2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short-term PLC-gamma1 tyrosine phosphorylation and short-term IP3 and DAG turnover. A third cell line expressing a point-mutated c-erbB-2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC-gamma1 activation was attributed to this structure. Neither PLC-gamma1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC-g1 activation time course by c-erbB-2 signaling. Induction of cell migration was solely observable in the c-erbB-2-positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D-collagen lattice. In summary, c-erbB-2 up-regulation switches on the cell migration program by modulating the time course of PLC-gamma1 activation.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.

Tumorzellen sind strukturiert und hochspezialisiert, was auch beinhaltet, dass sie von den normalen Organzellen und der organspezifischen extrazellularen Matrix Signale empfangen und fur ihr Wachstum nutzen.

Die morphologische Definition eines malignen Tumors als Ansammlung von Zellen, die keine Kontaktinhibition zeigen, invasiv uber Basalmembranen hinweg wachsen und in entfernten Organen Tochtergeschwulste (Metastasen) bilden konnen, ist seit mehr als 100 Jahren diagnostisch relevant.

This meeting report summarizes the presentations of three different groups that are active in the field of flow cytometry (FCM) in relation to diagnosing and classification of proliferative disorders. The report starts with the contribution from Regensburg about the developments in DNA FCM, the progression to dual parameter determinations, and combination of immunophenotyping in combination with DNA. In the second part, the use of FCM for the detection of isolated tumor cells in the peripheral blood from patients with prostate or breast cancer is discussed in a contribution from Munster. In the third part, from Heerlen, the use of multi-parameter FCM on formalin-fixed paraffin-embedded tissues from solid tumors is discussed as a new development and application in routine surgical pathology.

The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation and motility of normal as well as tumor cells. The transduction of extracellular signals to the cytoplasm via the receptor not only depends on ligand binding, but is also determined by the receptor density on the cell surface. Therefore, with regard to cancer diagnosis and therapeutic approaches targeting EGFR it is important to know how the expression level of EGFR is controlled. We found that transcription activity declines with increasing numbers of CA dinucleotides of a highly polymorphic CA repeat in the first intron of the epidermal growth factor receptor gene. In vivo data from cultured cell lines support these findings, although other regulation mechanisms can compensate this effect. In addition, we showed that RNA elongation terminates at a site closely downstream of the simple sequence repeat (SSR) and that there are two separate major transcription start sites. Model calculations for the helical DNA conformation revealed a high bendability in the EGFR polymorphic region, especially if the CA stretch is extended. These data suggest that the CA-SSR can act like a joint, bringing the promoter in proximity to a putative repressor protein bound downstream of the CA-SSR. The data indicate that this polymorphism may be a marker for cancer, linking genetic and epigenetic risk factors. Furthermore, in breast cancer, heterozygous tumors with short CA-SSR showed an elevated EGFR-expression in contrast to tumours with longer CA-SSR. Tumours with loss of heterozygosity in intron 1 of egfr revealed an increased EGFR expression if the longer allele was lost. Moreover, decreased EGFR gene levels were significantly correlated with poor prognosis in breast cancer.

To gain a better understanding of molecular changes in oral squamous cell carcinomas, we tested fresh tumour specimens from 110 patients for erbB-1 and -2 oncogene aberrations using the competitive differential polymerase chain reaction. The significance of established tumour characteristics such as TNM stage, differentiation and oncogene aberrations for tumour progression were analyzed. ErbB-2 amplification with a gene copy number >1.6 in tumour tissue and erbB-1 deletion with a gene copy number

The amplification and overexpression of the erbB-2 oncogene and its involvement in tumorigenesis makes this receptor an appropriate target for specific agents directed towards tumor cells. The purpose of this study was to evaluate the in vitro effect of the bacterially produced recombinant immunotoxin scFv(FRP5)-ETA on the protein synthesis and adenosine triphosphate (ATP) reduction in oral squamous cell carcinoma (OSCC) cells. This agent recognizes the erbB-2 receptor and inhibits protein synthesis in receptor-overexpressing cells. OSCC cells were selected for this study, and amplification and expression levels of the erbB-2 receptor were determined. Cell suspensions were cultured for 6 d with various concentrations of scFv(FRP5)-ETA (1-1000 ng/ml). A431 and MDA-MB468 cell lines were used as controls. Chemosensibility of tumor cells was measured by [3H]leucine incorporation assay and by an ATP luminescence assay. In OSCC cells with amplification and overexpression of erbB-2 inhibition, up to 92% of protein synthesis and 90% of ATP reduction was observed when cells were exposed to 1,000 ng/ml immunotoxin. In OSCC cells showing a deletion of erbB-2 and in erbB-2-negative MDA-MB468 cells, protein synthesis was inhibited by 22% and 8%, respectively. These results indicate that the effectiveness of a recombinant immunotoxin targeting erbB-2 receptors in OSCC cells depends on the level of erbB-2 amplification and expression, that it is highly specific for tumor cells expressing these receptors, and that a dose-dependency can be observed.

Patients with extensive, long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer. In this study, we wanted to establish a method for retrieving cytological material after segmental colonic lavage for further cytopathological investigations and for performing DNA image cytometry.Ten patients with long-standing and extensive ulcerative colitis and 10 patients without macroscopic abnormalities were investigated. After segmental colonic lavage during routine colonoscopy a three-layer (1.146, 1.075, and 1.046 g/ml, respectively) density gradient centrifugation of the retrieved colonic fluid was performed for isolation and purification of the epithelial cells. For identification of the epithelial cells flow cytometry with monoclonal antibody against cytokeratin and counterstaining with propidium iodine was performed. The smears obtained were stained for routine cytopathological interpretation and for DNA image cytometry.In eight of 10 UC patients and in nine of 10 control group patients adequate cytological material could be obtained. The band on top of the density gradient at 1.046 g/ml could be identified as the epithelial cells. Atypical cells were found in smears of three UC patients. In these patients and in one additional patient aneuploid stemlines could be detected. In smears of control group patients neither atypical cells nor aneuploidy were present.Isolation and purification of epithelial cells after segmental colonic lavage by using density gradient centrifugation was performed. This cytological material is adequate for cytopathological interpretation and for DNA image cytometry. Information about atypical cells and DNA aneuploidy as an additional marker of malignant transformation in UC patients was obtained. The combination of cytological examination and DNA image cytometry might improve the detection of UC patients with high risk for colorectal cancer.

We established an RT-PCR method to measure the amount of a 2.3-kb alternatively spliced mRNA of the human c-erbB-2/HER-2 proto-oncogene relative to the 4.6-kb full-length transcript. For the first time, we demonstrated production of c-erbB-2 extracellular domains via alternative splicing in breast cancer tissues, lymph node and bone marrow micrometastases. In 15 c-erbB-2-positive primary breast tumor samples, we found two significantly distinct subgroups: 6/15 had a low level of the extracellular fragment, and 9/15 showed an average 4.4-fold higher amount of the alternatively spliced mRNA. Additionally, six lymph nodes and six bone marrow aspirates from metastatic breast cancer patients were analyzed: 5/6 lymph nodes and 6/6 bone marrow aspirates were found to produce elevated relative amounts of the truncated fragment. The results demonstrate that our method is suitable for sensitive detection of c-erbB-2-positive micrometastasis and strongly suggest that the alternatively spliced c-erbB-2 variant is involved in the development of micrometastasis in breast cancer.

ErbB2(+) breast cancer is an aggressive breast cancer subtype generally associated with lower estrogen receptor alpha (ERα) expression and more aggressive tumor behavior compared to ERα(+)/ErbB2(-) breast cancer. The ErbB2(+) phenotype is associated with resistance to endocrine therapy, e.g. the selective estrogen receptor modulator Tamoxifen. However, the mechanisms underlying endocrine resistance are not fully understood. Here, we investigated the impact of AKT signaling and distinct functional roles of AKT isoforms in ErbB2(+) breast cancer from Balb-neuT mice. AKT isoform specific in vitro kinase assays revealed that AKT3 is activated in Balb-neuT breast tumors in comparison to normal murine breast tissue. Knock-down of AKT3, but not of AKT1 or AKT2, led to reduced expression and tyrosine-phosphorylation of ErbB2 and ErbB3 in Balb-neuT-derived mammary tumor cells. In contrast, expression of ERα was strongly up-regulated and phosphorylation of the AKT substrate Foxo3a which regulates ERα transcription was decreased in AKT3 knockdown cells. These data suggest that ERα expression is down regulated via AKT3/Foxo3a signaling in ErbB2(+) breast cancer cells. Furthermore, up-regulation of ERα after depletion of AKT3 resulted in a significant increase in Tamoxifen responsiveness of Balb-neuT-derived mammary tumor cells. In addition, Tamoxifen resistant human breast cancer cell lines showed increased AKT3 expression and activity in comparison to Tamoxifen responsive MCF-7 cells. Finally, by AKT isoform specific in vitro kinase assays of human breast cancer samples, AKT3 activity was detected in ErbB2(+) and triple negative tumors but not in ERα(+) breast cancer. Our data indicate that AKT3 regulates the expression of ErbB2, ErbB3 and ERα and demonstrate that down-regulation of activated AKT3 can sensitize ErbB2(+) breast cancer cells for treatment with Tamoxifen. Therefore, AKT3 targeting might be a new promising strategy for therapy of ErbB2(+)/ERα(-) breast cancer and might further increase the responsiveness to an endocrine therapy approach.

Characterizing lymphocyte subsets in bronchoalveolar lavage fluid (BALF) by flow cytometry (FC) proper gating of the lymphocyte subpopulation being analyzed is crucial. In order to test lymphocyte gate quality for the first time we used a DNA-dye to evaluate plasmamembrane integrity and thus to mark off fluorescent but not DNA-containing particles (eg, debris). A comparative prospective study between this newly developed FC technique and a standard peroxidase anti-peroxidase (PAP) method was performed. Samples of BALF from 50 patients with various pulmonary diseases were examined. After determination of the total cell yield, a differential cell count was performed. Subsequently, the immunophenotype of pan T lymphocyte CD3-, T-helper lymphocyte CD4-, and T-suppressor lymphocyte CD8-positive lymphocyte subsets was assessed with FC as well as with the PAP method. Both methods showed excellent correlation (CD3: r=0.81; CD4: r=0.97; CD8: r=0.96; p + cells (90.6±1.0% vs 85.8±1.3%). For CD4 (45.0±3.4% vs 44.4± 3.4%) and CD8 (48.1 ±3.5% vs 46.7±3.5%), there was good agreement. In a clinical setting, the reliability of both methods was equivalent, and FC using a DNA-dye to test lymphocyte gate quality offered a rapid and reliable determination of lymphocyte subsets in BAL. (CHEST 1995; 108:464-69)

The ability of primary tumours to metastasize accounts for the majority of cancer deaths. The emergence of circulating carcinoma cells in the peripheral blood is supposed to be an indicator for cancer cell spread. We have focused on this phenomenon in order to develop a sensitive technique for enriching epithelial derived cells on the basis of a two-layer density gradient and subsequent immune magnetic cell sorting. Epithelial cells are possess a cytoskeleton containing an assembly of intermediate filaments. During carcinogenesis these filaments do not undergo modifications of antibody binding epitopes such as occur in the protein domains of surface markers. We have developed a two-layer density gradient in which the epithelial cells form a single density band. This was demonstrated by recovery experiments using [3H]thymidine-labelled epithelial cells which showed epithelial cells were enriched within this first step by a factor of 20. In a second step the MACS system was applied. Cells were stained with a performed FITC-conjugated mouse anti-human cytokeratin antibody bound to a rat anti-mouse antibody coupled to superparamagnetic particles (immune paramagnetic separation complex; IPSC) and subjected to high gradient magnetic fields. The two-step procedure was confirmed by dispersing 50 epithelial cells in 5 x 10(5), 5 x 10(6), 5 x 10(7), 5 x 10(8), 5 x 10(9) peripheral blood leucocytes. Specific binding of the preformed IPSC was demonstrated by flow cytometry, confocal laser, fluorescent and electron microscopy. The specificity of the method was further proved by dual staining with IPSC and anti-human PSA antibody of epithelial prostatic cells separated from peripheral blood in vitro. By means of this double-step separation method it was possible to isolate up to 15-20 cells out of 50 epithelial cells originally suspended into 5 x 10(7) to 5 x 10(9) human peripheral blood leucocytes. This represented an enrichment factor between 20,000 and 200,000, depending on the initial cell number. The immunologically captured epithelial cells can be used for further cytogenetic investigations such as in situ hybridization (ISH) and/or polymerase chain reaction (PCR) to detect cancer cell specific gene aberrations. This sensitive combined buoyant density immune magnetic cell separation technique is capable of detecting free carcinoma cells in the peripheral blood.