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Background PR interval prolongation is a preliminary stage of atrial cardiomyopathy which is considered as an intermediate phenotype for atrial fibrillation (AF). AF is a known risk factor for cerebrovascular adverse outcomes including stroke. Cerebral ischemia is one cause of white matter hyperintensities (WMHs), and cognitive dysfunction. Aim To analyze the relationship between PR interval and WMHs. Materials and methods We performed a cross-sectional analysis with individuals from the LIFE-Adult-Study (a population-based cohort study of randomly selected individuals from Leipzig, Germany) with available brain MRI and ECG. The Fazekas stages were used to quantify WMHs (0 = none; 1 = punctate foci; 2 = beginning confluence; 3 = large confluent areas). Stages 2–3 were defined as advanced WMHs. The PR interval was measured from resting 12-lead ECG. PR duration >200ms was defined as PR interval prolongation. We used a binary logistic regression for statistical analysis. We examined the relationship between MRI and ECG measures and adjusted them for clinical risk factors. Results We included 2464 individuals (age 59±15 years, 47% women) into analyses. The median PR interval was 160ms (interquartile range 143–179), and 319 (13%) individuals with advanced WMHs, were significantly older, had more cardiovascular comorbidities and risk factors compared to individuals without WMHs (all p Conclusion PR interval duration is associated with advanced WMHs beside advanced age, hypertension, and history of stroke. Further research is needed to determine whether changes in PR interval indices are clinically relevant for changes in WMHs.

Borderline ovarian tumors (BOT) are uncommon but not rare epithelial ovarian neoplasms, intermediate between benign and malignant categories. Since BOT were first identified > 40 years ago, they have inspired controversies disproportionate to their incidence. This review discusses diagnostic criteria for the histologic subtypes of BOT, highlighting areas of diagnostic challenges, ongoing controversies, and changes in terminology implemented by the recent 2014 WHO Classification of Tumours of the Female Genital Organs. Emerging knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Serous borderline tumors (SBT) share molecular and genetic alterations with low-grade serous carcinomas and can present at higher stages with peritoneal implants and/or lymph node involvement, which validates their borderline malignant potential. All other (non-serous) subtypes of BOT commonly present at stage I confined to the ovary(ies) and are associated with overall survival approaching that of the general population. An important change in the WHO 2014 classification is the new terminology of non-invasive implants associated with SBT, as any invasive foci (previously called "invasive implants") are now in line with their biological behavior considered peritoneal low-grade serous carcinoma (LGSC). The controversy regarding the terminology of non-serous borderline tumors, called by some pathologists "atypical proliferative tumor" in view of their largely benign behavior, has not been resolved. The concepts of intraepithelial carcinoma and microinvasion may evolve in further studies, as their presence appears to have no prognostic impact and is subject to considerable inter-observer variability.

Clinical outcome of patients with stage IV breast cancer is dependent on tumor biology, extent, and localization of metastases. Routine imaging diagnostics for distant metastasis is not recommended by the national guidelines for breast cancer follow-up. In this study, we evaluated different patterns of metastases of cancer subtypes in order to generate hypotheses on individualization of follow-up after breast cancer in the adjuvant setting. Patients of the Regional Breast Cancer Center Dresden diagnosed within the years 2006-2011 were classified into the five intrinsic subtypes luminal A (ER+, Her2-, G1/2), luminal B/Her2 negative (ER+, Her2-, G3), triple positive (ER+, PR+, Her2+), Her2-enriched (ER-, Her2+), and triple negative (ER-, PR-, Her2-) and with a median follow-up of 45 months. Tumor stage at time of first diagnosis of breast cancer as well as time and site of metastasis at first diagnosis of distant metastatic disease was analyzed. Tumor specimen of 2284 female patients with primary breast cancer was classified into five subtypes. Distant recurrence-free survival at 3 years was most unfavorable in Her2-enriched (66.8 %), triple negative (75.9 %), and triple-positive breast cancer (81.7 %). The same subtypes most frequently presented with visceral metastases only at first presentation: Her2-enriched 46.9 %, triple negative 45.5 %, and triple-positive breast cancer 37.5 %. Longest median survival of 2.3 years was seen in luminal A and in Her2-enriched metastatic disease, respectively. Median survival was significantly better in the luminal A, Her2-enriched, and triple-positive subtype compared to triple-negative breast cancer (p < 0.005). Differences in time to metastatic disease, first localization of metastases, and overall survival after diagnosis of metastatic disease were shown. Considering new targeted therapies and the option of surgery of oligometastases, screening for visceral metastases might be reasonable after diagnosis of Her2-positive subtypes.

Hereditary spastic paraplegias (HSPs) represent a clinically and genetically heterogeneous group of diseases. Major symptoms comprise progressive bilateral leg stiffness, spasticity at rest and diffuse muscle weakness. Complex forms are characterized by additional symptoms like dementia, cerebellar dysfunction or seizures. Autosomal dominant, autosomal recessive, X-linked recessive and possibly mitochondrial inheritance have been described in familial HSP. The most frequently mutated gene in familial cases of uncomplicated autosomal dominant HSP is SPAST, however de novo mutations in SPAST are rarely found. Here, we report on the clinical and genetic findings in a family with three children afflicted by complex HSP and their unaffected parents. Although autosomal dominant inheritance seemed unlikely in this family, genetic testing revealed a novel SPAST mutation, c.1837G>C (p.Asp613His), in a heterozygous state in all affected individuals and somatic mosaicism of this mutation in the unaffected mother. Our study thus expands the knowledge on SPAST-associated HSP and emphasizes that de novo mutations and somatic mosaicism should be taken into consideration in HSP families presenting with a family history not suggestive for an autosomal dominant inheritance pattern.

Identification of protein targets that play a role in breast cancer invasion may help to understand the rapid progression of cancer and may lead to the development of new biomarkers for the disease. In this study, we compared two highly invasive and two poorly invasive breast cancer cell lines using comparative label-free LC-MS profiling in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Forty-five proteins were found to be upregulated, and 34 proteins, downregulated. UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell lines. In poorly invasive breast cancer cell lines, siRNA-mediated downregulation of RAD23B subsequently led to an increase in invasion and adhesion in vitro. Immunohistochemistry analysis of 164 specimens of invasive breast cancer showed that having a high percentage (>80%) of RAD23B positive nuclei was significantly associated with histopathological grades 1 and 2 breast cancer and with low mitotic activity. In addition, a high staining intensity for RAD23B in the cytoplasm was significantly associated with histopathological grade 3 breast cancer. This study suggests a potential role of RAD23B in breast cancer progression and may further imply a tumor suppressor role of nuclear RAD23B in breast cancer.

Diagnosis of the origin of metastasis is mandatory for adequate therapy. In the past, classification of tumors was based on histology (morphological expression of a complex protein pattern), while supportive immunohistochemical investigation relied only on few "tumor specific" proteins. At present, histopathological diagnosis is based on clinical information, morphology, immunohistochemistry, and may include molecular methods. This process is complex, expensive, requires an experienced pathologist and may be time consuming. Currently, proteomic methods have been introduced in various clinical disciplines. MALDI imaging MS combines detection of numerous proteins with morphological features, and seems to be the ideal tool for objective and fast histopathological tumor classification. To study a special tumor type and to identify predictive patterns that could discriminate metastatic breast from pancreatic carcinoma MALDI imaging MS was applied to multitissue paraffin blocks. A statistical classification model was created using a training set of primary carcinoma biopsies. This model was validated on two testing sets of different breast and pancreatic carcinoma specimens. We could discern breast from pancreatic primary tumors with an overall accuracy of 83.38%, a sensitivity of 85.95% and a specificity of 76.96%. Furthermore, breast and pancreatic liver metastases were tested and classified correctly.

We performed homozygosity mapping in a consanguineous Pakistani family segregating autosomal-recessive congenital cataracts and identified linkage to a 3.03 Mb locus on chromosome 6p24 containing the GCNT2 gene. GCNT2 encodes glucosaminyl (N-acetyl) transferase 2, an enzyme responsible for the formation of the blood group I antigen. Rare biallelic GCNT2 mutations have been shown to cause the association of congenital cataracts and the adult i blood group, making GCNT2 the prime candidate gene for the observed phenotype. Indeed, we identified a homozygous deletion segregating with cataracts that encompasses exons 1B, 1C, 2 and 3 of GCNT2. Long-range polymerase chain reaction and breakpoint sequencing revealed that affected individuals in this and in a second, apparently unrelated Pakistani family segregating congenital cataracts are homozygous for the same 93 kb deletion. The deletion is flanked by Alu repeats of the AluS family on both sides and microsatellite genotyping suggested that its occurrence in the two families was the product of recurrent Alu-Alu repeat-mediated nonhomologous recombinations or an old founder effect. Subsequently, we showed that cataract-affected individuals in both families have the adult i blood group, whereas unaffected individuals have blood group I as the vast majority of the population. Because the GCNT2 locus is rich in Short INterspersed Elements (SINE repeats) and thus likely prone to genomic rearrangements, microdeletions or microduplications at this locus might cause a larger than currently anticipated fraction of apparently isolated autosomal-recessive cataracts.

Item does not contain fulltext Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere. 01 juli 2010

Background: Nodding syndrome (NS) is a seemingly progressive epilepsy disorder of unknown underlying cause. We investigated association of pyridoxal-phosphate serum levels and occurrence of anti-neuronal antibodies against N-methyl-D-aspartate (NMDA) receptor and voltage gated potassium channel (VGKC) complex in NS patients.Methods: Sera of a Tanzanian cohort of epilepsy and NS patients and community controls were tested for the presence of anti-NMDA-receptor and anti-VGKC complex antibodies by indirect immunofluorescence assay. Furthermore pyridoxal-phosphate levels were measured.Results: Auto-antibodies against NMDA receptor or VGKC (LG1 or Caspr2) complex were not detected in sera of patients suffering from NS (n=6), NS plus other seizure types (n=16), primary generalized epilepsy (n=1) and community controls without epilepsy (n=7). Median Pyridoxal-phosphate levels in patients with NS compared to patients with primary generalized seizures and community controls were not significantly different. However, these median pyridoxal-phosphate levels are significantly lower compared to the range considered normal in Europeans.Conclusions: In this pilot study NS was not associated with serum anti-NMDA receptor or anti-VGKC complex antibodies and no association to pyridoxal-phosphate serum levels was found.Key words: nodding syndrome, epilepsy, anti-neuronal antibodies, pyridoxal-phosphate

Holoprosencephaly is a clinically and genetically heterogeneous midline brain malformation associated with neurologic manifestations including developmental delay, intellectual disability and seizures. Although mutations in the sonic hedgehog gene SHH and more than 10 other genes are known to cause holoprosencephaly, many patients remain without a molecular diagnosis. Here we show that a homozygous truncating mutation of STIL not only causes severe autosomal recessive microcephaly, but also lobar holoprosencephaly in an extended consanguineous Pakistani family. STIL mutations have previously been linked to centrosomal defects in primary microcephaly at the MCPH7 locus. Our results thus expand the clinical phenotypes associated with biallellic STIL mutations to include holoprosencephaly.

Summary We have previously described a seizure disorder characterized by head nodding (HN). In a prospective study in southern Tanzania, we evaluated 62 patients with HN. Here, we report the patients’ clinical characteristics and those of their seizures, which indicate high seizure frequency, unsatisfactory seizure control, a high burden of cognitive impairment and disease-associated barriers to education.

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor.

The study was aimed to detect differences in nuclear morphology between nuclear populations as well as between tumours with different p53 expression in breast cancers with different clinicopathological features, which also reflect the stage of tumour progression. The p53 immunohistochemistry was performed on paraffin sections from 88 tumour samples. After the cells had been localised by means of an image cytometry workstation and their immunostaining had been categorised visually, the sections were destained and stained by the Feulgen protocol. The nuclei were relocated and measured cytometrically by the workstation.There were significant differences in the nuclear features between tumours as well as between nuclear populations with different p53 expression in the most subgroups. The variability of nuclear shape in tumour groups, classified by the tumour size or the lymph node status, increase with the p53 immunoreactive score, whereas in tumours grouped by the Bloom–Richardson grade features of the chromatin distribution were different between the p53 staining categories.The nuclear subpopulations showed differences in the amount and distribution of chromatin in most subgroups.The results demonstrate the relationship between the nuclear morphology and the p53 expression in different stages of breast cancers. The p53 status is an important factor of the biological behaviour but not the only one.

Summary Objectives Nodding syndrome (NS), a new epilepsy disorder of sub-Saharan Africa, has only recently been classified. In a study conducted in southern Tanzania in 2005, 62 patients with NS were analyzed in great detail. The present study, a follow-up investigation, was conducted to evaluate the progression of NS over time and to obtain serial electroencephalography (EEG) data. Methods Of the 62 NS patients, 53 (85.5%), the majority of whom were currently on some form of antiepileptic treatment, could be reevaluated in 2009 with a standardized questionnaire. A subset of these patients (25/53) underwent EEG investigation. Results In patients with “head nodding (HN) only” in 2005, 10 (43.5%) of 23 remained with the same diagnosis, whereas 5 (21.7%) of 23 had developed “HN plus” (i.e., HN and generalized tonic–clonic seizures). Six patients (26.1%) had seizures other than HN only, and two patients (8.7%) had fully recovered. In the “HN plus” group of 2005, 9 (30.0%) of 30 patients remained “HN plus,” and 15 patients (50.0%) had seizures other than HN only. Four patients (13.3%) reverted to “HN only,” and two patients (6.7%) stopped all seizures. In 11 (44.0%) of 25 patients, electroencephalography (EEG) showed generalized slowing. Six (54.6%) of these 11 abnormal EEG studies further showed generalized epileptiform discharges: (1) ictal electroencephalographic pattern with generalized 2.5 Hz spike and waves in two patients and (2) interictal bursts of 1.5–2 Hz spike and waves in four patients. Significance This follow-up study confirms that HN represents an epilepsy disorder, possibly of the atypical absence type with dynamic development over time.

Introduction: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-β signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer. Methods: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis. Results: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of β-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced. Conclusions: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of β-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth., Deutsche Forschungsgemeinschaft (DFG-Br 1336/3-1), Deutsche Forschungsgemeinschaft (DFG-Br 1336/3-2), Germany. Bundesministerium für Bildung und Forschung

Background Li-Fraumeni-Syndrome (LFS) is an autosomal-dominant, inherited tumour predisposition syndrome associated with heterozygous germline mutations in the TP53 gene. Patients with LFS are at a high risk to develop early-onset breast cancer and multiple malignancies, among which sarcomas are the most common. A high incidence of childhood tumours and close to 100% penetrance has been described. Knowledge of the genetic status of the TP53 gene in these patients is critical not only due to the increased risk of malignancies, but also because of the therapeutic implications, since a higher rate of radiation-induced secondary tumours in these patients has been observed. Case report We report a patient with LFS harbouring heterozygous, pathogenic TP53 germline mutation, who was affected by four synchronous malignancies at the age of 40: a myxofibrosarcoma of the right upper arm, bilateral breast cancer and a periadrenal liposarcoma. Radiological treatments and a surveillance program were adjusted according to recommendations for LFS patients. Conclusion Management of tumour treatment of patients with LFS is different to the general population because of their risk for secondary cancers in the radiation field. Screening procedures should take a possibly elevated risk for radiation induced cancer into account.

Classical Hutchinson-Gilford progeria syndrome (HGPS) is caused by LMNA mutations that generate an alternatively spliced form of lamin A, termed progerin. HGPS patients present in early childhood with atherosclerosis and striking features of accelerated aging. We report on two pedigrees of adult-onset coronary artery disease with progeroid features, who were referred to our International Registry of Werner Syndrome (WS) because of clinical features consistent with the diagnosis. No mutations were identified in the WRN gene that is responsible for WS, among these patients. Instead, we found two novel heterozygous mutations at the junction of exon 10 and intron 11 of the LMNA gene. These mutations resulted in the production of progerin at a level substantially lower than that of HGPS. Our findings indicate that LMNA mutations may result in coronary artery disease presenting in the fourth to sixth decades along with short stature and a progeroid appearance resembling WS. The absence of early-onset cataracts in this setting should suggest the diagnosis of progeroid laminopathy. This study illustrates the evolving genotype-phenotype relationship between the amount of progerin produced and the age of onset among the spectrum of restrictive dermopathy, HGPS, and atypical forms of WS.

The urokinase-type plasminogen activator receptor (uPAR) is a key molecule for pericellular proteolysis in tumour cell invasion and metastasis. The aim was to evaluate the prognostic impact of uPAR in invasive breast cancer dependent on which cell types within the tumour express uPAR.uPAR expression was analysed by immunohistochemistry in 270 tumour tissue specimens of invasive ductal breast carcinomas using tissue microarrays. For evaluation of uPAR immunoexpression we used the epitope-mapped, uPAR domain II-specific monoclonal antibody IID7. High uPAR score values in both tumour cells (uPAR-Tc) and stromal cells were significantly related to high tumour grade (G3), and inversely correlated with oestrogen receptor status. On multivariate analysis, high uPAR-Tc values contributed independent prognostic information for disease-free survival (hazard ratio 1.93, P = 0.007) when adjusted for prognostically relevant clinicopathological parameters, whereas uPAR expression in stromal cells was not related to prognosis. In addition, elevated uPAR-Tc values were found to be prognostic indicators in clinically relevant subgroups of patients with invasive breast cancer.In invasive breast cancer uPAR expression in invasive carcinoma cells, but not in stromal cells, has a significant impact on patients' prognosis, and contributes to a more aggressive tumour phenotype.

Microsatellite instability (MSI) in head and neck squamous cell carcinoma (HNSCC) has been reported with a wide range of frequencies. The aim of our study was to disclose the frequency and basis of MSI in HNSCC and to correlate MSI and findings on loss of heterozygosity (LOH) with the clinical data.We analyzed MSI and LOH in 91 tumors. All tumors presenting instability were analyzed for the expression of mismatch repair genes (MMR) proteins.Low-level microsatellite instability (MSI-L) was seen in 7.7% of the HNSCC. None of the MSI-L tumors had aberrant MMR protein expression. LOH rates up to 57% were identified for different regions on chromosome 3p. For the marker D10S197, we found a significant correlation between LOH and tumor stage IV.Our results indicate that MMR gene inactivation is rare among primary HNSCC. In contrast, the MSI-L phenotype plays a role in a small subset of tumors. LOH on chromosome arm 3p and 10p12 seems to be involved in tumorigenesis and progression HNSCC, respectively.

Summary Purpose: In the 1960s in Tanzania, L. Jilek-Aall observed a seizure disorder characterized by head nodding (HN). Decades later, “nodding disease,” reminiscent of what was seen in Tanzania, was reported from Sudan. To date this seizure disorder has not been classified and possible causes still remain obscure. Methods: In a prospective study in southern Tanzania, we evaluated 62 patients with HN. Selected patients underwent blood (n = 51) and cerebrospinal fluid (CSF) (n = 48) analyses. Others were chosen for MRI (n = 12) and EEG (n = 10). Results: Seizure type was classified as “head nodding only” and “head nodding plus,” the latter being combined with other types of seizure (n =34). During HN, consciousness was impaired in 11 patients (17.7%) and supportive signs of epileptic seizures were described by 15 (24.2%) patients. Precipitating factors were confirmed by 11 (17.7%) patients. Fifty-six (90.3%) patients had at least one relative with epilepsy. EEG confirmed interictal epileptic activity in two patients and unspecific changes in four patients. MRI showed hippocampus pathologies (n = 5) and gliotic changes (n = 5). Skin polymerase chain reaction (PCR) positivity for Onchocerca volvulus was significantly associated with lesions on MRI. However, PCR of the CSF was negative in all cases. Conclusions: We present a comprehensive clinical description of the “HN syndrome,” possibly a new epilepsy disorder in sub-Saharan Africa. MRI lesions and their association with positive skin PCR for O. volvulus despite negative PCR of the CSF is intriguing and deserves attention. Furthermore, the high prevalence of hippocampus sclerosis and familial clustering of epilepsy may point toward other potential pathogenetic mechanisms.

Recurrent respiratory infections in a 9-year-old girl prompted a chest radiograph and a CT scan, which showed a right middle lobe consolidation. Bronchoscopy revealed a tumor that totally obstructed the middle lobe. Open lung biopsy revealed a low-grade mucoepidermoid carcinoma. Middle and lower right lung lobectomy was performed, followed by an uneventful recovery. Cytogenetic investigation of tumor cells exhibited the translocation t(11;19). This case shows that further diagnostic modalities such as CT scanning should be performed early in children with recurrent lower respiratory tract infections who have suspicious radiographic findings such as persistent atelectasis or recurrent unifocal infiltration. Bronchial mucoepidermoid carcinoma is infrequent, and molecular investigations might shed additional light on the prognosis.

Background. ZAC/Lot1 is a previously identified candidate tumor suppressor gene. The gene maps to the human chromosome 6q24-q25, a region frequently deleted in squamous cell carcinomas of the head and neck and other solid tumors. Methods. We have used a model of head and neck squamous cell carcinoma (HNSCC) and cell lines to analyze the role of the candidate tumor suppressor gene ZAC/Lot1 in oral carcinogenesis. We analyzed the expression in 11 cell lines, and we performed loss of heterozygosity (LOH)- and sequence analyses in 51 primary tumors. Results. Three (27.3%) of 11 cell lines showed a distinctly reduced expression of ZAC/Lot1 compared with expression levels of the gene in the normal oral mucosa. In addition, we analyzed 51 primary squamous cell carcinomas of the head and neck for LOH with seven microsatellite markers flanking ZAC/Lot1. We detected an average LOH rate of 31.4% in the region of interest. Sequence analysis revealed no mutations for the ZAC/Lot1 coding exons, including the exon/intron boundaries. Conclusions. These data could suggest a minimal role for ZAC/Lot1 in a subgroup of HNSCC tumors. © 2004 Wiley Periodicals, Inc. Head Neck26: 338–344, 2004

The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.

In this study, we asked whether overexpression of caspase-3, a central downstream executioner of apoptotic pathways, might sensitize breast cancer cells with acquired drug resistance (MT1/ADR) to drug-induced apoptosis. As control, we employed caspase-3 negative and caspase-3-transfected MCF-7 cells. Whereas mock-transfected MCF-7 cells were resistent to epirubicin, etoposide and paclitaxel (taxol), the same drugs led to breakdown of nuclear DNA in caspase-3-transfected MCF-7 cells. MT1/ADR cells express low levels of wild type caspase-3 but show defective caspase activation and apoptosis upon drug exposure. These cells also display a less efficient activation of the mitochondrial permeability transition. Caspase-3-transfected MT1/ADR clones showed a 2.8-fold increase in the protein level and a 3.7-fold higher specific enzyme activity. Procaspase-3 overexpression was not toxic and did not affect background apoptosis. Interestingly, procaspase-3-transfected MT1/ADR cells were more sensitive to cytotoxic drugs as compared with vector-transfected controls and DNA fragmentation nearly reached the levels of the original drug sensitive MT1 cells. Thus, overexpression of caspase-3 enhances chemosensitivity especially in situations where activation of the mitochondrial apoptosome is disturbed.

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.Figures onhttp://www.esacp.org/acp/2000/20-2_3/friedrich.htm.

Hitherto, the relationship between malignancy-associated morphological features in single tumour cells and the expression of markers indicating functional properties of these cells remained widely unknown. This study was aimed at describing differences in the size, shape and chromatin structure between tumour cells with different marker expression for progesterone receptors (PgR) and p53. Two series of breast cancers, consisting of 50 PgR-positive, and 39 p53-negative and 49 p53-positive mammary carcinomas, were investigated. The immunohistochemical staining was performed on paraffin sections using 3-amino-9-ethylcarbazole as the chromogenic substrate. By means of a cytometry workstation equipped with a computer-controlled motorised scanning stage, about 500 positive and negative tumour cells in each case were localised in the microscope and categorised by a scoring system for their staining intensity. After destaining, the tissue sections were Feulgen-stained. Then, all the tumour cells were relocated automatically and analysed by high resolution image cytometry. Among the numerous size, shape, and texture features used in the system, several variables of the nuclear contour and chromatin structure were found to be significantly different between the positive and negative tumour cell populations. Nuclei without PgR had more malignancy-associated morphological features than PgR-positive cells. Whereas p53-negative nuclei had a higher degree of regularity, their positive counterparts exhibited higher DNA ploidy values.

The expression of p53 and bcl-2 was immunohistochemically investigated in 61 formalin-fixed, paraffin-embedded invasive breast carcinomas. The study was aimed to elucidate the relationship between both markers and the correlation of p53 and bcl-2, respectively, to clinicopathological variables, to hormone receptor status and to DNA-ploidy. Twenty tumors showed a positive reaction with the monoclonal antibody DO-1 against p53 protein. Its immunohistochemical demonstration was significantly correlated with a tumor size larger than 2 cm, a low estrogen receptor status and DNA-aneuploidy. Bcl-2 was demonstrated in 51 breast cancers. Bcl-2 was preferably seen in low grade and hormone receptor positive tumors. We found a negative correlation between the immunoreactive scores of p53 and bcl-2, but in 17 carcinomas a coexpression of both proteins was seen. Cases with this coexpression did not differ significantly from the other tumors in clinicopathological parameters. In eight of these cases more than 10% of the cells were found to be positive for both markers. In four cases we could show many cells to exhibit both markers as it was assessed by an immunofluorescence double labeling technique.