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A population-based series of 122 patients with pregnancy-associated breast carcinomas was histologically revised and the relationship between hormone receptors, cerbB-2, BRCAl, p27, cyclin E, and cyclin D1 was studied. The [...]

The aim of this study was to investigate protein expression of adenomatous polyposis coil (APC) in primary and metastatic serous ovarian carcinoma. The expression of β-catenin and E-cadherin was additionally [...]

Expression of EGF, HB-EGF, TGF-α, HRG-α, HRG-β1, and HRG-β3 in 100 frozen breast carcinoma materials was immunohistochemically studied. Among these tumors, 67% were positive for EGF, 53% for HB-EGF, 57% [...]

Background. Small cell carcinoma of the cervix is a rare and aggressive tumor. Most gynecologic oncology centers have little experience with this tumor, and only small series have been published. Methods. Twenty-six patients with small cell carcinoma of the uterine cervix were treated at the Norwegian Radium Hospital. Clinical data, immunohistochemical characteristics, and infection with human papilloma-virus were studied. Results. Twelve tumors were of oat cell type and 14 of intermediate cell type. Twelve tumors were associated with other forms of carcinoma: squamous cell carcinoma (6 tumors), adenocarcinoma (5 tumors), and adenocarcinoma in situ (1 tumor). Neuroendocrine differentiation was expressed in 79% of the tumors. Human papilloma-virus (HPV)-18 was detected in 40% of the tumors and HPV-16 in 28%. Fifteen patients had Stage I disease, 7 had Stage II, 2 had Stage III, and 3 had Stage IV. Fourteen patients with Stage I and II disease underwent radical hysterectomy with pelvic lymph node dissection. In four, the operation was preceded by intracavitary radiation treatment. The patients with Stage II, III, and IV disease were treated with a combination of intracavitary radium, external beam radiation therapy, and chemotherapy. The 5-year survival rate was 14%. Four patients are alive, one with recurrent disease 50 months after diagnosis. Three patients free of disease have been followed up 26, 54, and 101 months, respectively. Conclusions. Small cell carcinoma of the cervix is an aggressive tumor with a propensity for rapid recurrence; it is associated with high mortality. Cancer 1994; 73: 672-7.

Background. This study attempted to determine if there is a pattern of matrix/matrix-degrading-enzyme immunoreactivity related to the morphologic types of gastric carcinoma or to their invasiveness. Methods. The authors performed an immunohistochemical study of the basement membrane antigens laminin and collagen IV, fibronectin, and Type IV collagenase in a series of 87 gastric carcinomas and their respective nodal metastases (n = 329). Results. Laminin expression was observed almost exclusively in carcinomas of the intestinal type. The expression of collagen IV was significantly higher in intestinal (52%) and atypical (44%) carcinomas than in diffuse (10%) carcinomas; collagen IV expression also was significantly correlated with lymphatic invasion and aneuploidy. Ninety percent of the carcinomas expressed fibronectin, mostly in the connective tissue at the invading edge of the tumors; fibronectin expression was significantly related to the expanding growth pattern of the neoplasms. Eighty-six percent of the tumors expressed Type IV collagenase, regardless of the histologic type or invasive properties. No relationship was observed between the expression of any of the antigens and the S-phase fraction of the tumors. No significant differences were found between the immunohistochemical profile of the primary tumors and their metastases. Conclusions. The authors conclude that the expression of basement membrane antigens is related to the type of gastric carcinomas, rather than the cell differentiation or proliferative activity of the tumors. The putative prognostic meaning of the relationship between collagen IV immunoreactivity and aneuploidy and lymphatic invasiveness of the carcinomas remains to be clarified. Cancer 1994; 73:518-27.

Background: In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors. Purpose: To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression. Methods: Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M [NaPO.sub.4]) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis. Results: An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the [beta]-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7). Conclusions: Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other. [J Natl Cancer Inst 84:1100-1108, 1992]

As is the case for many types of cancer, the term endometrial cancer describes not a single type of cancer, but rather a set of cancers all affecting the endometrium (lining) of the uterus. Undifferentiated carcinoma of the endometrium is a relatively uncommon form of endometrial cancer, and accounts for only about 1 percent of all endometrial cancers. There have been few studies, however, which describe the characteristics of this type of endometrial cancer. In a review of 1,985 cases of endometrial cancer, 31 (1.6 percent) were found to be of the undifferentiated type. These could be further divided into two types: the large cell type, which accounted for 48 percent of the cancers, and the intermediate/small cell type, accounting for the other 52 percent. While these cancers appeared different under the microscope, there were no statistically significant differences in survival between the two types. The average age at diagnosis for the undifferentiated carcinoma was 64 years, and the five-year and 10-year survival rates were 58 and 48 percent, respectively. As is often the case, the patients with the earlier stage cancer had a significant survival advantage. Cancer of the endometrium often invades the myometrium, which is the smooth uterine muscle surrounding it. For the patients whose cancer invaded only half the depth of the myometrium, the survival at five years was 100 percent. For the patients with greater invasion of the muscle, the five-year survival was only 46 percent. A similar effect on survival was found for the invasion of blood vessels by the cancer. The five-year survival of patients whose cancer had not invaded blood vessels was 89 percent, in contrast with a five-year survival of 54 percent for the patients in whom blood vessel invasion had occurred. (Consumer Summary produced by Reliance Medical Information, Inc.)

Bacteriophages (phages) are ubiquitous elements in nature, but their ecology and role in animals remains little understood. Sponges represent the oldest known extant animal-microbe symbiosis and are associated with dense and diverse microbial consortia. Here we investigate the tripartite interaction between phages, bacterial symbionts, and the sponge host. We combined imaging and bioinformatics to tackle important questions on who the phage hosts are and what the replication mode and spatial distribution within the animal is. This approach led to the discovery of distinct phage-microbe infection networks in sponge versus seawater microbiomes. A new correlative in situ imaging approach (‘PhageFISH-CLEM‘) localised phages within bacterial symbiont cells, but also within phagocytotically active sponge cells. We postulate that the phagocytosis of free virions by sponge cells modulates phage-bacteria ratios and ultimately controls infection dynamics. Prediction of phage replication strategies indicated a distinct pattern, where lysogeny dominates the sponge microbiome, likely fostered by sponge host-mediated virion clearance, while lysis dominates in seawater. Collectively, this work provides new insights into phage ecology within sponges, highlighting the importance of tripartite animal-phage-bacterium interplay in holobiont functioning. We anticipate that our imaging approach will be instrumental to further understanding of viral distribution and cellular association in animal hosts.

Thick electrode production is a key enabler for realizing high energy density Lithium-ion batteries. Therefore, the investigation of tortuosity as a crucial limiting parameter was conducted in this work. A thickness threshold (>150 μm) for a drastic increase in tortuosity for aqueous processed LiNi0.8Mn0.1Co0.1O2(NMC811) electrodes was determined. Symmetrical cells, under blocking conditions, were analyzed via electrochemical impedance spectroscopy. To counteract this phenomenon, multi-layer coated electrodes with varying binder concentrations were investigated. This novel coating method, combined with the reduction of binder material, leads to a tortuosity decrease of more than 80%, when compared to high-loading electrodes (>8.5 mAhcm−2) coated with the conventional doctor-blade technique. Additionally, a simplified transmission line model is opposed to a linear fitting method for analyzing the impedance data.

Background. Juvenile papillomatosis is a proliferative epithelial lesion of the female breast, and Rosen et al. introduced it in 1980 as an entity. Results. The authors present a case consistent with juvenile papillomatosis of the male breast, occurring in a 33-year-old man, who had noticed a swelling of his right breast for approximately 17 years. On review of the literature regarding epithelial proliferations of the male breast, the authors found three cases with similar histologic features. Conclusions. The case studied indicates a benign behavior of this lesion in the male breast, although no data concerning long-term prognosis are available. Cancer 1992; 70:126-128.

Zhenhe Suo [*] Huihua Yang [+] Quiang Mei [++] Eva Skovlund [ss] Jianguo Cui [++] Jahn M. Nesland [*] Immunostaining for epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3, c-erbB-4, ER, [...]

Therapeutic drug monitoring is of major importance in the case of medication with a narrow therapeutic range as well as when pharmacokinetic/pharmacodynamic variability is suspected. Methotrexate (MTX), an antifolate antibiotic, proved to be toxic regardless of the chosen treatment schedule. In this contribution, a new analytical method was used for the detection of MTX. A linear response was achieved in the 0.2–2 μM concentration range, with a limit of detection 0.17 μM. The lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) approach combines the fingerprint specificity and high sensitivity of SERS with the high sample throughput of a microfluidic platform. Additionally, it is shown that due to the chemical affinity of the MTX molecules towards Ag nanostructures, the pH value of the solving medium highly affects the obtained SERS signal. More specifically, SERS signals with well resolved bands can be obtained from deprotonated MTX molecules due to their binding to the metallic surface viathe amine groups of the aromatic ring.

AbstractRadioarsenic labelled radiopharmaceuticals could add special features to molecular imaging with positron emission tomography (PET). For example the long physical half-lives of 72As (T1/2=26 h) and 74As (T1/2=17.8 d) in conjunction with their high positron branching rates of 88% and 29%, respectively, allow the investigation of slow physiological or metabolical processes, like the enrichment and biodistribution of monoclonal antibodies in tumour tissue or the characterization of stem cell trafficking. A method for separation and purification of no-carrier-added (nca) arsenic from irradiated metallic germanium targets based on distillation and anion exchange is developed. It finally converts the arsenic into an *As(III) synthon in PBS buffer and pH 7 suitable for labelling of proteins viaAs-S bond formations. The method delivers radioarsenic in high purity with separation factors of 106from germanium and an overall yield from target to labelling synthon of >40%. In a proof-of-principle experiment, the monoclonal antibody Bevacizumab, directed against the human VEGF receptor, was labelled with a radiochemical yield >90% within 1 h at room temperature with nca 72/74/77As.

In einer randomisierten klinischen Studie erhielten 30 schwerverbrannte Patienten, die fakultativ ein Inhalationstrauma hatten, entweder nur eine standardisierte intensivmedizinische Behandlung oder an den Behandlungstagen 3, 5,7,10 und 13 zusätzlich 250 mg/kg Pseudomonas-Immunglobulin. Das Verbrennungstrauma wurde nach dem «Abbreviated Burn Severity Index» klassifiziert. Erstes Ziel der Untersuchung war die Häufigkeit von Bakteriämien in den Untergruppen mit einem Inhalationstrauma, der Verbrennungsindex dieser Untergruppen und der Vergleich verstorbener und überlebender Patienten. Als Ergebnis war der Verbrennungs-Score der verstorbenen Patienten der mit Pseudomonas-Immunglobulin behandelten Patienten mit einem Inhalationstrauma und/oder einer Bakteriämie mehr als 1 Punkt höher als in der Kontrollgruppe. Zweites Ziel der Untersuchung waren die IgG-Serumkon-zentrationen und die spezifischen Antikörpertiter gegen die Fisher-Immuntypen 1 und 4 in diesen Untergruppen. Ungeachtet der Substitution mit IgG waren die IgG-Serumkonzentrationen der Patienten mit einer Bakteriämie und/oder mit einem Inhalationstrauma, die an den Folgen der Verbrennungsverletzungen verstarben, am 10. und 13. Behandlungstag niedriger als bei überlebenden Patienten. Dies gilt auch für den Fisher-Immuntyp 1, nicht aber für den Immuntyp 4. Zusammenfassend wurde unter Berücksichtigung der Verletzungsschwere das Letalitätsrisiko bei den mit Pseudomonas-Immunglobulin behandelten Patienten gesenkt. Der erneute Abfall der Immuntyp-1-Antikörper und der IgG-Serumkonzentration ist bei sterbenden Patienten eventuell ein Hinweis auf die irreversible Schädigung der körpereigenen Abwehr von schwerverbrannten Patienten.

The aim of the present study was to identify seed treatment methods for eradicating Phoma valerianellaefrom lamb`s lettuce seeds in organic vegetable production. Using seeds naturally infested with the pathogen, the effect of three physical methods (hot water, aerated steam, electron treatment) and different agents of natural origin (micro-organisms, plant derived products, resistance inducers) was tested on moist filter paper, in seed trays under controlled conditions and in the field. In an initial screening, none of the tested putative resistance inducers prevented infection by P. valerianellae, while two out of seven formulated micro-organism preparations and six out of 16 experimental microbial strains were effective. When selected agents and the three physical seed treatment methods were compared in blotter and seed tray tests, the physical methods were generally the most effective treatments, while the micro-organism treatments were clearly less efficacious. However, in field experiments with the same seed lots and the same treatments, a statistically significant increase in plant stand was not obtained with any of the treatments. Combinations of the three physical treatment methods with selected non-chemical agents did not perform better than the physical treatments alone. The most effective alternative seed treatments identified in the present study, aerated steam, hot water, electron treatment and thyme oil (0.1%), can be recommended for eradication of P. valerianellaefrom lamb`s lettuce seeds in organic farming. Because their efficacy was generally as high as that of the chemical fungicide Aatiram (active ingredient thiram), they are also potentially suited for use in conventional vegetable production. Ziel der vorliegenden Untersuchung war es, für den ökologi-schen Landbau geeignete Saatgutbehandlungsverfahren zur Bekämpfung von Phoma valerianellaean Feldsalat zu finden. Dazu wurde die Wirksamkeit von drei physikalischen Verfah-ren (Heißwasserbehandlung, Heißluftbehandlung, Elektronenbehandlung) und verschiedenen Agenzien natürlicher Herkunft (Mikroorganismen, pflanzliche Präparate, Resi-stenzinduktoren) mit natürlich infiziertem Saatgut auf Filter-papier, unter kontrollierten Bedingungen in Saatschalen-testen sowie in Feldversuchen untersucht. In der ersten Über-prüfung konnte keines der Mittel, bei denen eine Resistenz-induktion als Wirkmechanismus angenommen wurde, die Infektion mit P. valerianellaeverhindern, während zwei von sieben formulierten Mikroorganismenpräparaten und sechs der 16 überprüften experimentellen Mikroorganismen Wirk-samkeit zeigten. Bei einem Vergleich ausgewählter nicht-che-mischer Mittel und der drei physikalischen Verfahren in Filter-papier- und Saatschalentesten waren die physikalischen Ver-fahren am effektivsten und die Mikroorganismen am wenigsten wirksam. Allerdings konnte in Feldversuchen mit dem glei-chen Saatgut und den gleichen Behandlungen mit keinem der Verfahren ein signifikanter Anstieg der Pflanzenzahl erreicht werden. Kombinierte Behandlungen aus physikalischen Ver-fahren und ausgewählten nicht-chemischen Mitteln erbrach-ten keine besseren Ergebnisse als die alleinige Anwendung der physikalischen Verfahren. Die wirksamsten in der vorlie-genden Untersuchung gefundenen Varianten (Heißluftbe-handlung, Heißwasserbehandlung, Elektronenbehandlung, Thymianöl [0.1%]) können für die Sanierung von Feld-salatsaatgut im Ökoanbau bei Befall mit P. valerianellaeempfohlen werden. Da ihre Wirksamkeit in der Regel ähnlich hoch war wie die des chemischen Saatbeizmittels Aatiram (Aktivsubstanz: Thiram) erscheinen sie auch für den konven-tionellen Gemüseanbau potentiell geeignet.

Der intensive Pflanzenbau erfordert wirksame Pflanzenschutzmaßnahmen. Eine der wirksamsten Maßnahmen ist die Behandlung des Saatgutes gegen Pathogene. Sie erfolgt bisher fast ausschließlich durch chemische Beizmittel (Pestizide), die Anwender und Umwelt gefährden können. Neue Gesetze und Richtlinien in Deutschland und der Europäischen Union fordern die nachhaltige Einschränkung der Anwendung chemischer Pflanzenschutzmittel und den Ersatz durch neue, alternative Verfahren und Methoden.

The objectives of the present study were the determination of treatment threshold values for control of common bunt of wheat (Tilletia tritici) and evaluation of alternative seed treatments. Wheat seed inoculated with the fungus was sown in fields at three sites in Germany from 2002–2004. Seeds of three cultivars — ‘Naturastar’ (highly susceptible), ‘Aron’ (moderately susceptible), and ‘Batis’ (highly susceptible) — were first inoculated with 20, 100 and 1000 spores/seed and treated with Tillecur®, a natural plant strengthener, or hot water before planting. Untreated inoculated seeds were used as control. Bunt infection rates in the resulting wheat plants varied according to inoculum size, cultivar and treatment method, with additional variation between the respective years and sites. Regarding inoculum size, 20 spores/seed sufficed for bunt development in the highly susceptible cultivars. In the moderately susceptible cultivar, 1000 spores/seed were required in the first year compared to only 20 spores/seed in the following years. Tillecur® provided the most effective bunt control: the number of infested ears was low to nil for all sites and inoculum sizes. Hot water was less effective and its effect was not significant except at the high inoculum level. Field data and calculations of the theoretically possible spore load suggest that low-level bunt infection represents a general danger. We conclude that treatment thresholds for control of common bunt must vary depending on the susceptibility of cultivar and the purpose of the harvested seed. When used for seed production, measures for prevention of disease accumulation are imperative. In those cases, we recommend a treatment threshold of one spore/seed for susceptible wheat cultivars and 20 spores/seed for less susceptible cultivars. Zur Bestimmung von Bekämpfungsschwellen für Weizensteinbrand (Tilletia tritici) und zur Untersuchung der Wirkung von alternativen Saatgutbehandlungen wurden in den Jahren 2002–2004 an drei Standorten Feldversuche mit jeweils drei Inokulumstufen (20, 100, 1000 Sporen/Korn), drei Sorten (‘Naturastar’, ‘Aron’, ‘Batis’) und den Behandlungen Tillecur® und Heißwasser angelegt. In allen Jahren und an allen Standorten waren deutliche Unterschiede zwischen den Inokulationsstufen und den Sorten vorhanden. Hochanfällige Sorten hatten in den unbehandelten Kontrollen nach Inokulation mit 20 Sporen/Korn Brandähren, die mittelanfällige Sorte zeigte im ersten Jahr bei 1000 Sporen/Korn, in den anderen Jahren bereits bei 20 Sporen/Korn Befall. Das Pflanzenstärkungsmittel Tillecur® erwies sich als wirkungsvollstes Behandlungsmittel gegen T. tritici. Nach Tillecur®-Behandlung wurden an allen Standorten und in allen Inokulationsstufen keine bis sehr wenige befallene Ähren gefunden. Die Wirkung der Heißwasserbehandlung war insgesamt geringer und eine signifikante Wirkung wurde nur in der höchsten Inokulationsstufe erreicht. Die Feldversuche und eine Berechnung der theoretisch möglichen Sporenbelastung des Ernteguts machten das Gefährdungspotenzial einer geringen Infektion deutlich. Befallstoleranzschwellen sind abhängig von der Sorte und dem Verwendungszweck des Ernteguts. Für die Saatgutproduktion, bei der eine Vermehrung des Erregers vermieden werden muss, müssen anfällige Sorten schon bei einer geringen Kontamination ab einer Spore/Korn behandelt werden. Weniger anfällige Sorten sind ab einem Befall von 20 Sporen/Korn zu behandeln.

Aberrant expression of EPH receptors and their ligands, ephrins, has been reported in a large variety of human cancers, including epithelial cancers from the colon and ovary. Due to the recently reported decrease or loss of EPHBs expression in colorectal carcinomas and the abundance of CpG sites in their promoters, we analyzed the promoter methylation status of three members of the EPHB family, EPHB2, EPHB3 and EPHB4, in a series of 22 colon cancer cell lines, as well as in four ovarian cancer cell lines and 56 ovarian tumor samples. The promoters of the three receptor genes were unmethylated in the vast majority of samples as assessed by methylation-specific polymerase chain reaction (MSP). These results were confirmed by direct bisulphite sequencing. Furthermore, from RT-PCR analyzes and Northern blotting, EPHB2 showed only small variation in RNA expression across ovarian cancer cell lines and clinical samples. We conclude that promoter hypermethylation of EPHB2, EPHB3 and EPHB4 is not a common event in colon and ovarian cancers, and therefore plays no major role in these tumors.

The present study addressed the impact of p14, p16, p57, and Ki-67 in a large cohort of uniformly treated patients with stage III ovarian cancer in relation to other clinicopathologic variables and prognosis. We immunohistochemically studied 171 primary tumors from previously untreated patients with advanced ovarian carcinomas for expression of Ki-67, p16, p14, and p57. High protein levels of Ki-67 (>10% positive nuclei) were found in 144 cases (84%), p16 (>50% positive nuclei) in 53 cases (31%), p57 (>10% positive nuclei) in 41 cases (24%), and p14 (any positive nuclei) in 19 cases (11%). A correlation between high Ki-67 expression and presence of residual disease after primary surgery (P= 0.019), ascites (P= 0.006), higher International Federation of Gynecology and Obstetrics substage (P< 0.001), poor differentiation (P< 0.001), and higher Silverberg histopathologic grade (P< 0.0001) was seen. High expression of p16 correlated to poor differentiation (P= 0.033) and higher Silverberg histopathologic grade (P= 0.018). In univariate analysis, high expression of Ki-67 (P= 0.0001) and p16 (P= 0.005) was associated with poor survival. However, in multivariate analysis, only high expression of Ki-67 was significantly associated with shorter survival (P= 0.025). No correlations were seen between expression of p14 and p57 and clinicopathologic parameters. None of the factors studied was able to predict response to chemotherapy. Our results showed that Ki-67 represents an independent prognostic predictor in stage III ovarian cancer. We did not find p16, p14, and p57 to be useful as prognostic markers.

Breast cancer is the most common malignancy in women. Although an increasing number of patients with breast cancer are being cured by surgery, a considerable number of patients suffer relapse in the form of metastases after surgery. E-cadherin and catenins have documented roles in breast cancer progression. Mammography is supposed to decrease breast cancer mortality by detecting tumours while they are small and before they have reached a clinically detectable stage.

Abstract: High carotenoid potato may have particular value for human health due to the antioxidant properties and the therapeutic value for eye health in patients at risk for macular degeneration. Carotenoid concentrations were determined among the progeny of a cross between two high carotenoid lines derived from diploidPapa Amarilla germplasm from South America. The total carotenoid content ranged from 82 to 2686 µg / 100 g fresh weight (FW). The higher values greatly exceeded the mid-parent value of the cross. An index for yellow tuber flesh color was determined for a subset of the progeny. A cleaved amplified polymorphic sequence (CAPS) assay was developed to distinguish the alleles of beta-carotene hydroxylase (bch) in the two high-carotenoid parents. Abch allele (denoted B) common to the high carotenoid parents co-segregated with yellow flesh in the progeny of a white-flesh x yellow-flesh cross, makingbch an excellent candidate for the classicalY locus, required for yellow tuber flesh. The same allele was also present in all other yellow-fleshed potato clones tested. Genotype atbch explained a portion of the variation of total carotenoid (R2=0.42). Clones homozygous for the B allele (BB) contained, on average, slightly more carotenoid than heterozygous Bb clones, which in turn had much more carotenoid than homozygous bb clones, suggesting a partially dominant gene model. Similarly, bb flesh was significantly less yellow than Bb and BB, the latter two being quite close. Total carotenoid varied considerably between progeny in the Bb and BB genotype categories, suggesting that variation at one or more additional loci have a significant effect on total carotenoid levels. Since the total carotenoid levels in manyPapa Amarilla cultivars and progeny are much higher than those in white- and yellow-fleshed tetraploid cultivars, it may be possible to breed for high carotenoid values in tetraploid germplasm by introducing one or more genes derived fromPapa Amarilla germplasm.

The present study addressed the impact of human papillomavirus (HPV), p14, and the product of the retinoblastoma gene (pRb) in vulvar carcinoma in relation to other clinicopathologic variables and prognosis. We immunohistochemically studied 217 primary tumors from patients with vulvar carcinoma for the expression of pRb and p14. By the use of in situ hybridization, the primary tumors and 7 lymph node metastases were studied for the presence of HPV-16, HPV-18, HPV-31, and HPV-33 DNA. HPV-infected cases significantly correlated with high expression of p14 (P < .01) and p16 (P < .01). In HPV- cases with high expression of p53, no p14 expression predicted the poorest disease-specific survival (P < .01). For the first time, we have shown that p14 expression indicates longer disease-specific survival in patients with vulvar carcinoma. In patients with HPV- tumors expressing high levels of p53, low p14 indicated the poorest 5-year disease-specific survival.

Comparison between BRCA1-associated and sporadic ovarian carcinomas is a potential method to identify candidate modifier gene/s involved in the carcinogenic pathway of either or both groups. A previous study identified a significant difference in the frequency of copy number gain at 2q24–q32 by comparing BRCAI-associated and sporadic ovarian tumour specimens using comparative genomic hybridisation (CGH). The present study aimed to investigate the reported allelic imbalance at 2q24–32 by amplification of several microsatellite markers at the region by quantitative microsatellite analysis (QuMA) using Taqman at the same region identified as a site of allelic imbalance.

Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2–9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.

By using tissue microarrays and immunohisto-chemical analysis, we studied protein expression of genes in the erb-b signaling pathway (erb-b1; erb-b2; phosphoinositide-3-kinase, catalytic, a polypeptide [PIK3CA]; phosphatase and tensin homologue [PTEN]; phosphorylated AKT [p-AKT]; and phosphorylated extracellular signal-regulated kinase [p-ERK]) in 118 advanced ovarian carcinomas and related expression to clinicopathologic features and survival. High protein expression was seen in 15.3% of cases for erb-b2, 44.1% for erb-b1, 43.2% for PIK3CA, 51.6% for p-AKT, and 28.0% for p-ERK. Low protein levels of PTEN were seen in 41.5% of the cases and tended to be more common in well-differentiated tumors. In multivariate analysis, only high expression of both erb-b1 and erb-b2 was an independent factor in progression-free and disease-specific survival (P=.009, hazard ratio=2.46; P=.002, hazard ratio=3.023, respectively). The PI3K/AKT and RAS/MEK/ERK pathways seem to be activated in some cases of advanced ovarian carcinomas, although PIK3CA, p-AKT, p-ERK, and PTEN do not seem to be independent prognostic markers in this group of patients.

The protein and mRNA expression of dysadherin was studied in a series of squamous cell cervical carcinomas, and their clinicopathological associations and prognostic value were explored. Immunohisto-chemistry was used to assess protein expression of dysadherin in 206 patients with squamous cell cervical carcinoma, FIGO stage Ia-IVb. Frozen tissues from 20 cases in which the tumors showed variable dysadherin protein expression were used for laser capture microdissection (LCM) and processed for RT-PCR detection of dysadherin mRNA. Immunohisto-chemically, all the dysadherin-positive staining was membranous. Positive cell membranous dysadherin-positive staining was often observed at the edge of tumor nests, although strong immunoreactivity throughout whole tumor nests was also seen in some tumors. Basal cells of the normal cervical epithelia were positive for dysadherin while its expression in the squamous cell cervical carcinomas was variable. Among the 206 tumors, 23 (11.2%) were negative, 53 (25.7%) were scored 1+, 54 (26.2%) were scored 2+ and 76 (36.9%) were scored 3+. In the 20 tumors analyzed, mRNA expression of dysadherin basically corresponded to its protein expression. No significant cor-relation between expression of dysadherin and age, FIGO stage or lymph node status was observed. Higher level of dysadherin expression, however, was significantly associated with shorter overall survival (p=0.04). We conclude that there is dysadherin protein expression in basal and parabasal cells of normal cervical epithelia, and higher level of dysadherin protein expression in squamous cell cervical carcinoma is predictive of a shorter overall survival, indicating that dysadherin may be a valuable prognostic marker in cervical carcinoma.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two major histological types of breast cancer worldwide. Whereas IDC incidence has remained stable, ILC is the most rapidly increasing breast cancer phenotype in the United States and Western Europe. It is not clear whether IDC and ILC represent molecularly distinct entities and what genes might be involved in the development of these two phenotypes. We conducted comprehensive gene expression profiling studies to address these questions. Total RNA from 21 ILCs, 38 IDCs, two lymph node metastases, and three normal tissues were amplified and hybridized to ∼42,000 clone cDNA microarrays. Data were analyzed using hierarchical clustering algorithms and statistical analyses that identify differentially expressed genes (significance analysis of microarrays) and minimal subsets of genes (prediction analysis for microarrays) that succinctly distinguish ILCs and IDCs. Eleven of 21 (52%) of the ILCs (“typical” ILCs) clustered together and displayed different gene expression profiles from IDCs, whereas the other ILCs (“ductal-like” ILCs) were distributed between different IDC subtypes. Many of the differentially expressed genes between ILCs and IDCs code for proteins involved in cell adhesion/motility, lipid/fatty acid transport and metabolism, immune/defense response, and electron transport. Many genes that distinguish typical and ductal-like ILCs are involved in regulation of cell growth and immune response. Our data strongly suggest that over half the ILCs differ from IDCs not only in histological and clinical features but also in global transcription programs. The remaining ILCs closely resemble IDCs in their transcription patterns. Further studies are needed to explore the differences between ILC molecular subtypes and to determine whether they require different therapeutic strategies.

Eph receptor tyrosine kinases and their cell-surfacebound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RT-PCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

Follicular lymphomas (FLs) are a heterogeneous group of tumors, but prognostic factors are evaluated insufficiently in this common hematologic neoplasm. While bcl-6 and CD10 are expressed characteristically in FLs, their significance for biologic behavior of FL has not been studied previously. Samples from 73 patients with FL and clinical follow-up from 7 to 231 months were evaluated by immunohistochemical analysis. Patients with high levels of bcl-6 expression had favorable overall survival (OS) (P = .003), disease-specific survival (DSS) (P = .033), and time to treatment failure (P = .003) compared with patients with low levels of bcl-6 expression. Multivariate analysis showed that the results for OS, DSS, and time to treatment failure were independent of the international prognostic index. Patients with CD10+ FLs also had longer OS (P = .001), DSS (P = .007), and time to treatment failure (P = .004), and grade 1 FL was associated with better OS (P = .01) and a statistical trend for longer DSS (P = .05) and time to treatment failure (P = .05), but these results were not independent of bcl-6 expression or the international prognostic index in multivariate analysis.

NKX3.1is a homeobox gene which exhibits prostate and testis specific expression. Loss of NKX3.1 expression has been implicated in prostate development and tumorigenesis, but the role of NKX3.1 in testis biology is not known. Here we show that NKX3.1 expression is dramatically down-regulated in testicular cancer of germ cell origin. Immunohistochemical analysis on a tissue microarray containing 510 testicular tissue samples indicate that NKX3.1 is expressed at high levels in normal germ cells and in carcinoma in situ, but is sharply decreased or absent in most seminomas and all embryonal carcinomas. However, NKX3.1 is expressed in a subset of the more differentiated nonseminomas. We provide evidence that these changes in NKX3.1 protein levels are mainly due to transcriptional effects. These results suggest that NKX3.1 is essential for normal testis function and that its loss of expression is highly associated with the invasive phenotype of testicular germ cell tumors.

We have developed improved procedures for recovery of haploid and doubled haploid (DH) melon plants, using hybrids derived from crosses of lines with multiple virus resistance. Seeds formed after pollination with irradiated pollen were cultured in liquid medium for 10 days before excision of the embryos for further culture. This made it easier to identify the seeds containing parthenogenetic embryos, thereby reducing the effort required and increasing the percentage of plants recovered. The plants obtained (~175) were transferred to a greenhouse for evaluation. Three fertile lines were identified, and selfed seeds were obtained for evaluating virus resistance. Flow cytometry of leaf tissues showed that two of these lines were spontaneous DH and the third was a mixoploid containing haploid and diploid cells. The other plants remained sterile through the flowering stage. Flow cytometry of 20 sterile plants showed that all were haploid. Attempts to induce chromosome doubling by applying colchicine to greenhouse-grown plants were unsuccessful. Shoot tips from the haploid plants were used to establish new in vitro cultures. In vitro treatment of 167 micropropagated haploid shoots with colchicine produced 10 diploid plants as well as 100 mixoploid plants. Pollen from male flowers that formed in vitro on the colchicine-treated plants was examined. High percentages of viable pollen that stained with acetocarmine were found not only in the diploids but also in >60% of the plants scored as mixoploid or haploid by flow cytometry. Efficient recovery of DH from hybrid melon lines carrying combinations of important horticultural traits will be a valuable tool for melon breeders.

Our study evaluates the prognostic significance of the cytokeratin-positive mononuclear cells (CK+ cells) in the bone marrow (BM) and peripheral blood (PB) as detected by immunocytochemistry in patients with locoregionally confined prostate cancer. BM and PB samples were obtained from 66 newly diagnosed patients with T1-4pN0M0 prostate cancer. All samples were analyzed by standardized immunocytochemical methods (anticytokeratin mononuclear antibody; AE1/AE3) applying a negative immunomagnetic cell enrichment technique. A second sampling was obtained in 60 of the 66 patients ≥2 years after definitive radiotherapy. The median follow-up after high-dose radiotherapy of the patients was 65 months. For the analysis of the postradiotherapy clinical progression-free survival (PFS) treatment, failure was defined as pelvic tumor growth or development of distant metastases. At diagnosis CK+ cells were found in BM in 14 of 66 (21%) prostate cancer patients. This was not associated with an increased risk of progression. On the other hand, the presence of CK+ cells in 12 of 60 (20%) patients at the second BM aspiration was significantly related to a shorterPFS (p = 0.02). In the multivariate analysis, the presence of CK+ cells in the posttreatment BM did not remain as an independent variable of PFS assessment if posttreatment PSA was entered into the analysis. CK+ cells in PB were found in 12% of the patients. After therapy, none of the patients had detectable CK+ cells in PB. The presence of CK+ cells in the posttreatment but not in the pretreatment BM was associated with decreased PFS in patients irradiated for pelvis-confined nonmetastatic prostate cancer. Although this association was not retained in multivariate analysis, our observations indicate that the presence of CK+ cells after local therapy define a group of patients that have a high risk of developing distant metastases. © 2002 Wiley-Liss, Inc.

Imbalance between pro-apoptotic and anti-apoptotic proteins, causing altered apoptosis, may lead to tumour development and tumour progression, and reduced response to adjuvant therapy. In this study, we evaluated the expression patterns of Bcl-2, Bcl-xL, and Bax protein in 126 primary invasive breast carcinomas, and the association with other clinicopathological parameters. We used immunohistochemical methods to evaluate protein expression. Reduced expression of both Bax and Bcl-2 was associated with lymphnode metastases in univariate analyses (one-way ANOVA) as well as in multivariate analysis (binary logistic regression) (Bcl-2 p=0.003 univariate, p=0.01 multivariate, Bax p=0.05 univariate, p=0.03 multivariate). Bcl-2 overexpression showed an inverse association with cyclin A (p=0.05), while expression of Bcl-xL showed an association only with cyclin D3 (p=0.04). Bcl-xL expression also showed a highly significant association with oestrogen receptor status (p=0.009). Bcl-2 and Bcl-xL showed an association with different D-type cyclins, indicating different pathways of pathogenesis. Expression of Bcl-2 was associated with better patient survival in univariate analysis (Kaplan meyer p=0.04), but lost its prognostic value in multivariate analysis (Cox regression p=0.2).

We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent.

The present study analysed by immunohistochemistry the protein level of cyclin A and Ki‐67 in a panel of paraffin‐embedded tissue obtained from 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, and ten benign naevi. Since cyclin A exists in the same quaternary complex in the S‐phase of the cell cycle as the cdk inhibitor p21WAF1/CIP1, the levels of the two proteins were compared. Cyclin A and Ki‐67 were heterogeneously expressed in the malignant tumours, whereas in benign naevi, only rare positive cells were detected. In superficial spreading melanomas, the cyclin A level was related to tumour thickness, with less expression in thinner lesions (p<0.00001), and to Ki‐67 (p<0.00001) and p21WAF1/CIP1(p=0.01) scores. Multivariate analysis showed that in addition to the depth of the primary tumour, the protein level of cyclin A was an independent indicator of relapse‐free period (thickness, p<0.00001; cyclin A, p=0.0003). In contrast, in nodular melanoma, the cyclin A level was associated with Ki‐67 expression, but neither cyclin A nor Ki‐67 was related to tumour thickness (cyclin A, p=0.06; Ki‐67, p=0.61) and neither had any impact on relapse‐free (cyclin A, p=0.64; Ki‐67, p=0.32) or overall (cyclinA, p=0.94; Ki‐67, p=0.45) survival. In conclusion, the results indicate that cyclin A is a strong prognostic factor for patients with superficial spreading melanomas. In nodular melanomas, the proliferation rate seems to have little impact on disease progression. Copyright © 2001 John Wiley & Sons, Ltd.

Objective.The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors.

The use of automated microscopy has reached the maturity necessary for its routine use in the clinical pathology laboratory. In the following study we compared the performance of an automated microscope system (MDS™) with manual method for the detection and analysis of disseminated tumor cells present in bone marrow preparations from breast carcinoma patients. The MDS System detected rare disseminated tumor cells among bone marrow mononuclear cells with higher sensitivity than standard manual microscopy. Automated microscopy also proved to be a method of high reproducibility and precision, the advantage of which was clearly illustrated by problems of variability in manual screening. Accumulated results from two pathologists who had screened 120 clinical slides from breast cancer patients both by manual microscopy and by use of the MDS System revealed only two (3.8%) missed by the automatic procedure, whereas as many as 20 out of 52 positive samples (38%) were missed by manual screening. Cytometry (Comm. Clin. Cytometry) 46:215–221, 2001. © 2001 Wiley-Liss, Inc.

Production of protoporphyrin IX (PpIX) in human B-cell leukemia cell line (Reh) and T-cell lymphoma cell line (HPB-ALL) was studied by flow cytometry after incubation with 5-aminolevulinic acid (ALA) or its hexylester in vitro. Cell survival and cell death pathway were also investigated in these two cell lines by cell growth curves, flow cytometry, and electron microscopy after ALA hexylester-mediated photodynamic therapy. Both ALA and its hexylester could induce PpIX production in the two cell lines, but ALA hexylester was about 100 times more efficient than ALA. Reh cells appear to be more sensitive than HPB-ALL cells to ALA hexylester-mediated phototoxicity. Apoptosis was the major cell death pathway of Reh cells, while necrosis played a major role in the case of HPB-ALL cells.

Progression through the mammalian cell cycle is facilitated by cyclin–cyclin-dependent kinase (cdk) complexes, which are activated at specific points during the cell cycle. Alteration in cyclin–cdk complexess may lead to altered cell cycle and tumorigenesis. In this study, we analyzed expression of cyclins A, D1, D3 and E in tumor tissue from 170 patients with primary invasive breast carcinomas. Immunohistochemical methods were used to detect protein expression of these cyclins. We detected positive immunoreactivity in 55 (32%), 22 (13%), 38 (22%) and 37 (21.8%) of the samples for cyclins A, D1, D3 and E, respectively. A highly statistically significant association was observed between expression of cyclin A and early relapse (p = 0.001 univariate analysis, p = 0.006 multivariate analysis) as well as cancer-specific death (p &lt; 0.0001) during the follow-up time. No association was observed between cyclin D1 or cyclin E, respectively, and relapse of disease or survival, while cyclin D3 over-expression was associated with development of metastases during follow-up (p = 0.005 univariate analysis, p = 0.01 multivariate analysis). However, cyclin D3 did not show any statistically significant association when cancer-specific death was examined in a multivariate analysis (Cox regression for survival function). © 2001 Wiley-Liss, Inc.