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1.  O6.05ERBB2/HER2-SPECIFIC NATURAL KILLER CELLS FOR ADOPTIVE IMMUNOTHERAPY OF GLIOBLASTOMA 
Neuro-Oncology  2014;16(Suppl 2):ii13.
BACKGROUND: While EGFRvIII appears a logical target for immunotherapy, only a subpopulation of tumor cells express EGFRvIII and immune escape has been demonstrated. ErbB2 is overexpressed in a substantial proportion of glioblastomas and has been successfully utilized in immunotherapies. Natural killer (NK) cells are the first line of defense against viral infections and malignant cells. The continuously growing cytotoxic cell line NK-92 holds promise for cancer immunotherapy. Safety of infusion of high doses of NK-92 was established in previous phase I clinical trials utilizing irradiated cells to prevent permanent engraftment. METHODS: To provide NK-92 cells with pre-determined tumor-cell specificity, we generated a lentiviral second generation chimeric antigen receptor (CAR) construct (5.28.z) employing the ErbB2 (HER2)-specific scFv(FRP5) antibody fragment for target cell recognition, and human CD28-CD3 ζ as a composite signaling moiety. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards ErbB2-expressing tumor cells of various origins in vitro. We evaluated the cytotoxicity of NK-92/5.28.z cells against a panel of glioblastoma cell lines and primary glioblastoma cultures with different levels of Erb2 expression in vitro and in vivo. RESULTS: 13 out of a total of 15 established glioblastoma cell lines and 5 out of 5 primary glioblastoma cell lines were specifically lysed by NK-92/5.28.z cells, although to varying degrees. ErbB2 expression was necessary for cytotoxicity of NK-92/5.28.z cells towards glioblastoma cells, and ectopic overexpression of ErbB2 enhanced their cytotoxicity. In subcutaneous glioblastoma xenograft models, injections of NK-92/5.28.z induced prolonged regression in NOD-scid/gamma (NSG) mice with grafts of LN-319 cells and retarded the growth of LNT-229 grafts. In an orthotopic intracranial model utilizing LN-319 cells, tumor size in mice intratumorally injected with NK-92/5.28.z cells weekly was substantially smaller compared to controls as assessed by MR imaging. Injections of NK-92/5.28.z cells also conferred an impressive advantage in symptom free survival in two independent experiments (median 73 days in controls versus 140 days in NK-92/5.28.z treated animals in experiment A, p < 0,05, and median 77 days in controls versus 163 days in NK-92/5.28.z treated animals in experiment B, p < 0,01). In preparation of a phase I trial, GMP-compliant protocols for expansion of the NK-92/5.28.z line were established. CONCLUSIONS: Adoptive immunotherapy with application of ErbB2-specific NK-92/5.28.z cells may be a promising new immunotherapy approach for ErB2 positive glioblastoma. A phase I trial for glioblastoma patients with local injections of NK-92/5.28.z cells is in preparation.
doi:10.1093/neuonc/nou174.46
PMCID: PMC4185813
2.  Classification of Eating Disturbance in Children and Adolescents: Proposed Changes for the DSM-V 
Childhood and adolescence are critical periods of neural development and physical growth. The malnutrition and related medical complications resulting from eating disorders such as anorexia nervosa (AN), bulimia nervosa (BN) and eating disorder not otherwise specified may have more severe and potentially more protracted consequences during youth than during other age periods. The consensus opinion of an international workgroup of experts on the diagnosis and treatment of child and adolescent eating disorders is that (a) lower and more developmentally sensitive thresholds of symptom severity (e.g. lower frequency of purging behaviours, significant deviations from growth curves as indicators of clinical severity) be used as diagnostic boundaries for children and adolescents, (b) behavioural indicators of psychological features of eating disorders be considered even in the absence of direct self-report of such symptoms and (c) multiple informants (e.g. parents) be used to ascertain symptom profiles. Collectively, these recommendations will permit earlier identification and intervention to prevent the exacerbation of eating disorder symptoms.
doi:10.1002/erv.994
PMCID: PMC4470383  PMID: 20151366
eating disorders; classification; children; adolescents; malnutrition
4.  Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling 
British Journal of Cancer  2010;102(5):815-826.
Background:
The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers.
Methods:
The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERα, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs.
Results:
The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r2=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab.
Interpretation:
Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.
doi:10.1038/sj.bjc.6605553
PMCID: PMC2833247  PMID: 20145614
SP; BC; luminal; HER2; T-ICs
5.  Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer 
British Journal of Cancer  2009;100(12):1949-1956.
Small cell lung cancer (SCLC) is an aggressive, rapidly metastasising tumour. Previously, we demonstrated the influence of CXCL12–CXCR4 interaction on processes involved in metastasis and chemoresistance in SCLC. We show here that STAT3 is expressed in both primary SCLC tumour tissues and SCLC cell lines. We investigated the function of STAT3 upon CXCL12 stimulation in SCLC cell lines. Small cell lung cancer cell lines present constitutive phosphorylation of STAT3, and in the reference cell lines NCI-H69 and NCI-H82 constitutive phosphorylation was further increased by CXCL12 stimulation. Further investigating this signalling cascade, we showed that it involves interactions between CXCR4 and JAK2 in both cell lines. However CXCL12-induced adhesion to VCAM-1 could be completely inhibited by the JAK2 inhibitor AG490 only in NCI-H82. Furthermore, CXCR4 antagonist but not AG490 inhibited cell adhesion whereas both antagonisms were shown to inhibit growth of the cells in soft agar, indicating the central involvement of this signalling in anchorage-independent growth of SCLC cells. Most interestingly, while using primary tumour material, we observed that in contrast to non-small-cell lung cancer samples from primary tumour tissues, all analysed samples from SCLC were strongly positive for tyrosine-phosphorylated STAT3. Taken together, these data indicate that STAT3 is constitutively phosphorylated in SCLC and is important in SCLC growth and spreading thus presenting an interesting target for therapy.
doi:10.1038/sj.bjc.6605068
PMCID: PMC2714241  PMID: 19455144
chemokine receptor; signal transduction; CXCR4; STAT3; lung cancer; SCLC
6.  In the absence of (early) invasive carcinoma, vulvar intraepithelial neoplasia associated with lichen sclerosus is mainly of undifferentiated type: new insights in histology and aetiology 
Journal of Clinical Pathology  2006;60(5):504-509.
Background
Differentiated vulvar intraepithelial neoplasia (VIN) is presumed to be the precursor of invasive squamous cell carcinoma (SCC) of the vulva. It is commonly assumed that differentiated VIN is related to lichen sclerosus (LS). However, evidence for this is limited to a small number of studies describing epithelial alterations adjacent to vulvar SCC.
Aim
To study the histology and human papillomavirus (HPV) status in patients with a history of both LS and VIN without coexistent SCC.
Methods
Original biopsy specimens and surgical specimens of patients retrieved from the pathology files were revised for the presence of LS, VIN and (early) invasive SCC, specifically focused on the two different types of VIN: differentiated and undifferentiated. Thereafter, VIN lesions were tested for the presence of HPV DNA.
Results
Twenty‐seven patients fulfilled the criteria for LS and VIN without SCC. In all 27 patients, LS was found to be related to undifferentiated VIN. Grading yielded the following results: VIN 1 (n = 10), VIN 2 (n = 11) and VIN 3 (n = 6). Additionally, VIN lesions from 26 patients could be tested for the presence of HPV DNA. HPV DNA, predominantly type 16, was present in 8 (31%) of them. Seven of these eight patients had VIN 2 or 3. During follow‐up, three patients progressed to (early) invasive carcinoma. In two of these patients, differentiated VIN was observed overlying early invasive SCC.
Conclusions
VIN related to LS without coexisting SCC is likely to be undifferentiated, in contrast to what was previously thought. HPV DNA was demonstrated in 31% of the lesions, and was strongly related to high‐grade VIN.
doi:10.1136/jcp.2005.031989
PMCID: PMC1994523  PMID: 16714399
7.  A tool to balance benefit and harm when deciding about adjuvant therapy 
British Journal of Cancer  2009;100(6):913-917.
Adjuvant therapy aims to prevent outgrowth of residual disease but can induce serious side effects. Weighing conflicting treatment effects and communicating this information with patients is not elementary. This study presents a scheme balancing benefit and harm of adjuvant therapy vs no adjuvant therapy. It is illustrated by the available evidence on adjuvant pelvic external beam radiotherapy (RT) for intermediate-risk stage I endometrial carcinoma patients. The scheme comprises five outcome possibilities of adjuvant therapy: patients who benefit from adjuvant therapy (some at the cost of complications) vs those who neither benefit nor contract complications, those who do not benefit but contract severe complications, or those who die. Using absolute risk differences, a fictive cohort of 1000 patients receiving adjuvant RT is categorised. Three large randomised clinical trials were included. Recurrences will be prevented by adjuvant RT in 60 patients, a majority of 908 patients will neither benefit nor suffer severe radiation-induced harm but 28 patients will suffer severe complications due to adjuvant RT and an expected four patients will die. This scheme readily summarises the different possible treatment outcomes and can be of practical value for clinicians and patients in decision making about adjuvant therapies.
doi:10.1038/sj.bjc.6604962
PMCID: PMC2661793  PMID: 19293808
absolute risk increase; absolute risk reduction; adjuvant radiotherapy; decision making; endometrial carcinoma
8.  Identification of claudin-4 as a marker highly overexpressed in both primary and metastatic prostate cancer 
British Journal of Cancer  2008;99(3):491-501.
In the quest for markers of expression and progression for prostate cancer (PCa), the majority of studies have focussed on molecular data exclusively from primary tumours. Although expression in metastases is inferred, a lack of correlation with secondary tumours potentially limits their applicability diagnostically and therapeutically. Molecular targets were identified by examining expression profiles of prostate cell lines using cDNA microarrays. Those genes identified were verified on PCa cell lines and tumour samples from both primary and secondary tumours using real-time RT–PCR, western blotting and immunohistochemistry. Claudin-4, coding for an integral membrane cell-junction protein, was the most significantly (P<0.00001) upregulated marker in both primary and metastatic tumour specimens compared with benign prostatic hyperplasia at both RNA and protein levels. In primary tumours, claudin-4 was more highly expressed in lower grade (Gleason 6) lesions than in higher grade (Gleason ⩾7) cancers. Expression was prominent throughout metastases from a variety of secondary sites in fresh-frozen and formalin-fixed specimens from both androgen-intact and androgen-suppressed patients. As a result of its prominent expression in both primary and secondary PCas, together with its established role as a receptor for Clostridium perfringens enterotoxin, claudin-4 may be useful as a potential marker and therapeutic target for PCa metastases.
doi:10.1038/sj.bjc.6604486
PMCID: PMC2527792  PMID: 18648369
prostate cancer; marker expression; RNA and protein; claudin-4
9.  Mdm2-SNP309 polymorphism in prostate cancer: no evidence for association with increased risk or histopathological tumour characteristics 
British Journal of Cancer  2008;99(1):78-82.
The search for inherited cancer susceptibility factors is a major focus of epidemiologic cancer studies. Analyses of single-nucleotide polymorphisms (SNP) in a variety of genes revealed a correlation between a specific allele variant and cancer predisposition. Human mouse double-minute 2 protein (Mdm2) is a cellular E3 ligase capable of ubiquitination and degradation of p53. Therefore, Mdm2 is a crucial factor of cell cycle control and cell survival. The Mdm2 promoter SNP309 was shown to increase Mdm2 expression and can, thereby, inhibit the p53 pathway. This SNP was found to be associated with increased risk and early onset of various malignancies. For prostate cancer no studies are reported to date. In a case–control study we determined the distribution of the Mdm2 SNP309 in 145 male subjects with prostate cancer and in 124 male controls without any malignancy using RFLP analysis. Cases and controls showed a similar distribution of the SNP (P=0.299). Genotype distribution showed neither an association with histopathological characteristics of the tumours nor with prognosis. Age at disease onset was also not modified by the SNP. This first study of the Mdm2 SNP309 in prostate cancer patients suggests no correlation between a certain allelic variant and an increased cancer risk.
doi:10.1038/sj.bjc.6604441
PMCID: PMC2453021  PMID: 18577987
prostate cancer; Mdm2 SNP309; RFLP; p53 pathway
10.  A nationwide survey to measure practice variation of catheterisation management in patients undergoing vaginal prolapse surgery 
Introduction and hypothesis
Urinary catheterisation following vaginal prolapse surgery causes inconvenience for patients, risk of urinary tract infections and potentially longer hospitalisation. Possibly, practice variation exists concerning diagnosis and management of abnormal postvoid residual (PVR) volume implying suboptimal treatment for certain subgroups.
Methods
Nationwide questionnaire-based survey.
Results
Post-operatively, 77% performed transurethral indwelling catheterisation, 12% suprapubic catheterisation and 11% intermittent catheterisation. Catheterisation was applied 3 days (1–7 days) following anterior repair and 1 day (1–3 days) following all other procedures. The median cut-off point for abnormal PVR was 150 mL (range 50–250 mL). Treatment of abnormal PVR consisted mostly of prolonging transurethral indwelling catheterisation for 2 days (range 1–5 days; 57%), 29% by intermittent and 12% by suprapubic catheterisation. Antibiotics were administered by 21% either routinely or based on symptoms only.
Conclusions
Due to insufficient evidence and suboptimal implementation of available evidence, practice variation in catheterisation regimens is high.
doi:10.1007/s00192-009-0847-4
PMCID: PMC2690835  PMID: 19495543
Pelvic floor repair; Pelvic organ prolapse; Survey; Urinary catheterisation
11.  Telomerase and its potential for therapeutic intervention 
British Journal of Pharmacology  2007;152(7):1003-1011.
Telomerase and telomeres are attractive targets for anticancer therapy. This is supported by the fact that the majority of human cancers express the enzyme telomerase which is essential to maintain their telomere length and thus, to ensure indefinite cell proliferation – a hallmark of cancer. Tumours have relatively shorter telomeres compared to normal cell types, opening the possibility that human cancers may be considerably more susceptible to killing by agents that inhibit telomere replication than normal cells. Advances in the understanding of the regulation of telomerase activity and the telomere structure, as well as the identification of telomerase and telomere associated binding proteins have opened new avenues for therapeutic intervention. Here, we review telomere and telomerase biology and the various approaches which have been developed to inhibit the telomere/telomerase complex over the past decade. They include inhibitors of the enzyme catalytic subunit and RNA component, agents that target telomeres, telomerase vaccines and drugs targeting binding proteins. The emerging role of telomerase in cancer stem cells and the implications for cancer therapy are also discussed.
doi:10.1038/sj.bjp.0707374
PMCID: PMC2095101  PMID: 17603541
telomerase; telomeres; telomere targeting agents; G-quadruplex; telomere uncapping; hTERT; hTERC
12.  Mcm2 predicts recurrence hazard in stage Ta/T1 bladder cancer more accurately than CK20, Ki67 and histological grade 
British Journal of Cancer  2007;96(11):1711-1715.
Stage Ta/T1 urothelial carcinoma of the bladder (Ta/T1 BC) has a marked tendency to recur. Besides histopathology, markers such as CK20 expression and proliferation index (Ki67) have been shown to predict its clinical course. The replication-licensing factor minichromosome maintenance protein 2 (Mcm2) is a marker of proliferative potential shown to be a promising prognostic marker in various malignancies. The aim of the present study was to evaluate the prognostic value of Mcm2 in comparison to stage, grade, CK20 and Ki67. Initial sporadic Ta/T1 BC (n=71) were evaluated for their expression of CK20, Ki67 and Mcm2 by immunohistochemistry and tissue microarray technology. Prognostic power was analysed by univariate and multivariate Cox regression model for tumour recurrence rate. Median follow-up period was 39 months. A total of 35% patients experienced recurrence. While CK20 was not predictive, grade, Ki67 and Mcm2 were significantly related to recurrence rate in univariate Cox regression model. Only grade (HR 2.37; 95% CI 1.24–4.51; P=0.009) and Mcm2 expression with a cutoff ⩾40% (HR 5.81; 95% CI 2.41–14.00; P<0.001) were independent predictors of recurrence rate in multivariate Cox regression analysis. In addition to grade, expression of Mcm2 is an independent predictor of recurrence in Ta/T1 BC.
doi:10.1038/sj.bjc.6603784
PMCID: PMC2359908  PMID: 17505513
Mcm2; Ki67; CK20; bladder cancer
13.  Telomere uncapping by the G-quadruplex ligand RHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism 
British Journal of Cancer  2007;96(8):1223-1233.
The pentacyclic acridinium methosulfate salt RHPS4 induces the 3′single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase to the telomere and thus G-quadruplex ligands can effectively inhibit both the catalytic and capping functions of telomerase. In this study, we examined mechanisms underlying telomere uncapping by RHPS4 in uterus carcinoma cells (UXF1138L) with short telomeres and compared the susceptibility of bulk and clonogenic cancer cells to the G-quadruplex ligand. We show that treatment of UXF1138L cells with RHPS4 leads to the displacement of the telomerase catalytic subunit (hTERT) from the nucleus, induction of telomere-initiated DNA-damage signalling and chromosome fusions. We further report that RHPS4 is more potent against cancer cells that grow as colonies in soft agar than cells growing as monolayers. Human cord blood and HEK293T embryonic kidney cell colony forming units, however, were more resistant to RHPS4. RHPS4-treated UXF1138L xenografts had a decreased clonogenicity, showed loss of nuclear hTERT expression and an induction of mitotic abnormalities compared with controls. Although single-agent RHPS4 had limited in vivo efficacy, a combination of RHPS4 with the mitotic spindle poison Taxol caused tumour remissions and further enhancement of telomere dysfunction.
doi:10.1038/sj.bjc.6603691
PMCID: PMC2360152  PMID: 17406367
telomere; telomerase; stem cells; G-quadruplex; RHPS4; Taxol
14.  The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase 
British Journal of Cancer  2003;89(8):1538-1544.
doi:10.1038/sj.bjc.6601301
PMCID: PMC2394340  PMID: 14562029
RNF11; ubiquitination; Smurf2; TGFβ signalling
15.  No association of anti-Chlamydia trachomatis antibodies and severity of cervical neoplasia 
Sexually Transmitted Infections  2001;77(2):101-102.
Objective: To explore whether the presence of Chlamydia trachomatis antibodies is associated with the severity of neoplastic lesions in women with cervical dyskaryosis.
Methods: In a cross sectional study in two groups of women referred for an abnormal Papanicolaou smear (group A: 296, group B: 331 women) blood samples were analysed for antichlamydial antibodies by enzyme immunoassay. Cervical neoplasia was graded histologically.
Results: In group A no association was found between increasing grade of CIN and the presence of antichlamydial antibodies. The proportion (93%) of women with antichlamydial antibodies was higher in 14 women with (micro)invasive carcinoma than in women with CIN (35%). As the high prevalence of antichlamydial antibodies in women with cervical carcinoma is not consistent with prevalences reported in recent literature, we analysed a second group of women in which indeed the high prevalence was not confirmed
Conclusion: Our results suggest that the presence of circulating antichlamydial antibodies is not associated with the severity of neoplastic lesions and it seems unlikely that C trachomatis has a role in the progression of cervical neoplasia.
Key Words: cervical neoplasia; Chlamydia trachomatis
doi:10.1136/sti.77.2.101
PMCID: PMC1744283  PMID: 11287686
16.  Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer. 
Journal of Clinical Pathology  1997;50(11):960-961.
The prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by polymerase chain reaction in 12 of 35 patients with histologically cancer free lymph nodes. Of these 12 patients, only one developed a recurrence, suggesting HPV-16 DNA detection in cancer free lymph nodes has no prognostic value.
PMCID: PMC500325  PMID: 9462250
17.  Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311 
British Journal of Cancer  1999;81(2):367-375.
C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690702
PMCID: PMC2362878  PMID: 10496367
C1311; HT29 cells; colorectal cancer; cytotoxicity; DNA intercalation; lysosomes
18.  Antibodies to human papillomavirus type 16 E7 related to clinicopathological data in patients with cervical carcinoma. 
Journal of Clinical Pathology  1995;48(5):410-414.
AIMS--To investigate the correlation between antibodies to the transforming protein E7 of human papillomavirus (HPV) type 16 and clinicopathological indices in women with cervical squamous carcinoma. METHODS--A synthetic peptide of the HPV type 16 E7 protein (amino acids 6 to 35) was used to screen sera from 29 children, 130 women with cervical intraepithelial neoplasia, 443 women with cervical cancer, and 222 controls, for antibodies against this viral antigen. Bivariate and multivariate analyses were used to investigate the correlation between the serological status in the pretreatment sera and clinicopathological indices (size of the lesions, histological grade, stomal infiltration, vascular invasion, and nodal spread). Survival analysis was done using the Cox regression model for all FIGO stages and stages IB and ILA. RESULTS--Cervical carcinoma patients had a significantly higher prevalence of antibodies to synthetic peptide E7/6-35 than women with cervical intraepithelial neoplasia (17.7% v 7%, p < 0.005) or controls (17.7% v 11%, p < 0.05). Bivariate analysis of the data on the presence of anti-E7/6-35 antibodies in the pretreatment sera from these patients and clinicopathological indices showed a significant correlation between the presence of anti-E7/6-35 antibodies and the size of the lesion (p = 0.0009), histological grade (p = 0.0031), and lymph node metastasis (p = 0.01). 0.011). In addition, the Cox regression model, analysing four risk factors which can be determined before treatment, showed a significant correlation between the presence of anti-E7/6-35 antibodies and a worse prognosis (p = 0.003). Survival analysis revealed that both for all FIGO stages (p = 0.0005) and for stages IB and IIA alone (p = 0.0021), anti-E7/6-35 positive patients before treatment had a significantly shorter life expectancy. CONCLUSIONS--The presence of antibodies against E7/6-35 in pretreatment sera from patients with cervical carcinoma correlates with the size of the lesions, lymph node involvement, and a worse prognosis.
PMCID: PMC502614  PMID: 7629285
19.  Effects of selective phosphodiesterase 3 inhibition in the perfused liver of the rat after endotoxin treatment. 
British Journal of Pharmacology  1996;118(3):790-796.
1. This study was designed to investigate the role of rat phosphodiesterase 3 (RPDE3) in regulation of liver metabolism in sepsis. We studied the effects of the phosphodiesterase 3 inhibitor (PDI), enoximone, alone and in combination with regulating factors of hepatic carbohydrate metabolism and bile secretion in the perfused liver of rats treated 4 h earlier with endotoxin. In addition, cyclic AMP and cyclic GMP levels were determined in the effluate and bile by radio immunoassay methods. 2. After endotoxin treatment, infusion of enoximone at three concentrations (1 microM, 10 microM) resulted in an increased glucose output from -1.4 +/- 0.9 to 7.8 +/- 2.5 mumol l-1 20 min-1. Bile acid-independent bile flow increased also, in a dose-dependent manner. 3. In untreated livers, cyclic AMP release increased in the effluate from 1000 +/- 73 fmol g-1 min-1 to 1710 +/- 143 fmol g-1 min-1 when enoximone (10 microM) was administered. In bile from untreated livers, the level of cyclic AMP was also significantly increased by enoximone. After endotoxin treatment, the enoximone (10 microM) effect on cyclic AMP levels in effluate and bile was greatly reduced. Levels of cyclic GMP in the effluate and bile appeared unchanged in the presence of enoximone. 4. During co-infusion of glucagon (1 nM) and enoximone (10 microM), cyclic nucleotide levels in the effluate and bile of livers after endotoxin treatment were determined. In the effluate, cyclic AMP release increased from 827 +/- 144 fmol g-1 min-1 to 17802 +/- 2821 fmol g-1 min-1 when glucagon was administered. The presence of enoximone enhanced cyclic AMP further to 41696 +/- 920 fmol g-1 min-1. The same changes in cyclic AMP release were found in bile. Levels of cyclic GMP in the effluate and bile were not significantly affected by the administration of glucagon and the PDI. 5. Glucose release was determined during glucagon, sympathetic nerves stimulation and phenylephrine administration in the presence and absence of enoximone. The addition of enoximone to glucagon increased glucose release by 8.2 +/- 2.8 mumol g-1 20 min-1, without alteration of lactate balance. The PDI enhanced the glycogenolytic effects of nerve stimulation and of phenylephrine, accompanied by a reduction in lactate production. 6. Enoximone significantly enhanced the bile acid independent bile flow after glucagon, nerves stimulation and after administration of phenylephrine. Bile acid secretion was unaffected by the PDI. The vasoconstrictor effect of nerve stimulation was reduced by the PDI. 7. We conclude that endotoxin treatment reduces the ability of the PDI, enoximone, to increase cyclic AMP release in the perfused liver. The significant increase in cyclic AMP release after stimulation with glucagon and enoximone favours the view that RPDE3 is involved in the degradation of cyclic AMP in the liver after exposure to endotoxin. Additionally, the inhibition of the RPDE3 results in glucose release, vasodilatation and choleresis in endotoxin pretreated livers.
PMCID: PMC1909729  PMID: 8762109
20.  Telomerase activity in normal and malignant mammalian tissues: feasibility of telomerase as a target for cancer chemotherapy. 
British Journal of Cancer  1997;75(4):516-522.
Telomerase, a ribonucleoprotein enzyme, has been found in immortalized but not in most somatic adult human tissues, and thus emerged as a novel target for cancer chemotherapy. However, its usefulness could still be limited by normal tissue toxicity. This study compares enzyme activity in tissues and tumours in conventional in vivo models and human biopsy material, specifically normal human liver, with a view to determining the therapeutic potential of anti-telomerase therapy. The telomeric repeat amplification protocol (TRAP assay) was used to measure enzyme activity and levels were semiquantified by assaying equal concentrations of cellular protein. Telomerase activity was high in the murine embryonic stem cell line CGR8.8, WRL 68 human embryo liver cells, testis, ovary and liver of adult mouse and rat. Low activity was detected in normal human liver, marmoset and pig liver. Very low enzyme activity was seen in mouse, rat and marmoset bone marrow, brain or skin; no activity could be detected in mammalian lung and heart. On the contrary, all 30 human and murine malignant tissues studied showed high to moderate enzyme levels. However, activity found in murine liver was often higher than in tumour, e.g. in the transplantable adenocarcinoma of the colon MAC16. Our findings indicate that telomerase is present not only in murine but also in other normal mammalian tissues such as liver, and that this activity might result from the presence of somatic stem cells. In view of this, the role of telomerase as a potential selective target for therapy needs further investigation. Furthermore, the understanding of regulatory pathways of this enzyme and the selection of screening models will be critical.
Images
PMCID: PMC2063310  PMID: 9052403
21.  Preclinical evaluation of novel imidazoacridinone derivatives with potent activity against experimental colorectal cancer. 
British Journal of Cancer  1996;74(9):1369-1374.
Novel imidazoacridinone derivatives, C1310 and C1311, have been evaluated for their potential to inhibit tumour cell growth in vitro and in vivo. A cell line panel, including seven human and murine colon carcinoma cell lines and three in vivo models, was used. The compounds were found to be potent inhibitors of tumour cell growth with IC50 values ranging between 10 nM and 2 microM in human colon cancer cell lines. Statistically significant tumour growth delay (P < 0.01) was observed after a single intraperitoneal (i.p.) dose of C1311 (100 mg kg-1 body weight) in MAC15A, MAC29 murine and HT29 human adenocarcinomas of the colon. Rapid accumulation of fluorescence of both C1310 and C1311 was seen in the nuclei of HT29 human colon tumour cells in culture. C1311 was also found to bind into calf thymus DNA as shown by spectrophotometric titration and thermal denaturation and to cause early inhibition of thymidine incorporation in HT29 cells in vitro. The results of this study suggest that C1311 should be considered as a candidate for clinical development.
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PMCID: PMC2074789  PMID: 8912531
22.  Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. 
Journal of Clinical Microbiology  1996;34(3):745-747.
We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined, 72.8%). The efficacy of each primer pair seemed to be inversely correlated to the length of the amplimer produced. By using newly developed type-specific primer pairs (amplimer length, approximately 100 bp), an increase in HPV DNA detection (87.6%) was found.
PMCID: PMC228883  PMID: 8904451
23.  Epidemiological evidence of cervical intraepithelial neoplasia without the presence of human papillomavirus. 
British Journal of Cancer  1996;73(6):831-836.
The aim of this paper was to provide epidemiological evidence to support the notion that cervical intraepithelial neoplasia (CIN) without human papillomavirus (HPV) is a true entity. If a diagnosis of HPV-negative cervical neoplasia is erroneous, one would not expect there to be any differences in risk factors between HPV-positive and HPV-negative patients. Patients at a gynaecological outpatient clinic of a university hospital [a total of 265 consecutive women with dyskaryotic cervical smears who were subsequently diagnosed with CIN I (n=37), CIN II (n=48) or CIN III (n=180)] completed a structured questionnaire regarding smoking habits and sexual history. Analysis of an endocervical swab for Chlamydia trachomatis, analysis of a cervical scrape for HPV, and morphological examination of cervical biopsy specimens were also performed. HPV was found in 205 (77.4%) out of the 265 women. Univariate analysis showed that current age (P=0.02), current smoking behaviour (P=0.002) and the number of sexual partners (P=0.02) were significantly associated with the presence of HPV. Age at first sexual intercourse, a past history of venereal disease or genital warts, and current infection with Chlamydia trachomatis were not associated with the presence of HPV. Using multivariate logistic regression analysis, the number of sexual partners and current smoking behaviour showed an independent significant association with HPV. HPV-negative and HPV-positive CIN patients differ with respect to the risk factors for HPV. These findings suggest that HPV-negative CIN is a separate true entity.
PMCID: PMC2074377  PMID: 8611390
24.  Genotyping human papillomavirus type 16 isolates from persistently infected promiscuous individuals and cervical neoplasia patients. 
Journal of Clinical Microbiology  1995;33(11):2957-2962.
Nucleotide sequence variation in the noncoding region of the genome of human papillomavirus type 16 (HPV16) was determined by direct sequencing and single-strand conformation polymorphism analysis of DNA fragments amplified by PCR. Individuals of diverse sexual promiscuity and/or cervicopathology were studied. In a group of 14 healthy, monogamous HPV16-positive females, only two HPV16 sequence variants could be documented. Among 17 females and 3 males with multiple sex partners and living in the same geographical region, nine sequence variants were found, whereas among 7 patients with cervical neoplasia from another region, five variants were detected. Although numbers are limited, in the group of individuals at high risk of acquiring a sexually transmitted disease or with cervical neoplasia, a larger number of HPV16 sequence variants was encountered (two types among 14 individuals versus nine types among 20; Fisher's exact test, P = 0.07). Seven of the individuals were sampled repeatedly over time. For these persistently infected women, no differences in HPV16 sequences were detected, irrespective of promiscuity, and persistence of a single viral variant, spread over multiple anatomic sites, for more than 2 years could be demonstrated. This indicates that viral persistence may be a common feature and that successful superinfection with a new variant may be rare, despite a potentially high frequency of viral reinoculation.
PMCID: PMC228614  PMID: 8576353

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