Mutant p53: a novel target for the treatment of patients with triple-negative breast cancer?

The identification and validation of a targeted therapy for patients with triple-negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti-cancer activity of PRIMA-1 and PRIMA-1MET (APR-246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild-type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA-1 and PRIMA-1MET was found to be cell-line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA-1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=-0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA-1MET induced apoptosis and inhibited migration in a p53 mutant-dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA-1MET is a potential new approach for treating p53-mutated breast cancer, including the subgroup with triple-negative (TN) disease.

Population pharmacokinetics and pharmacodynamics of teicoplanin in neonates: making better use of C-reactive protein to deliver individualized therapy.

OBJECTIVES: There is uncertainty about the optimal teicoplanin regimens for neonates. The study aim was to determine the population pharmacokinetics (PK) of teicoplanin in neonates, evaluate currently recommended regimens and explore the exposure-effect relationships. METHODS: An open-label PK study was conducted. Neonates from 26 to 44 weeks post-menstrual age were recruited (n = 18). The teicoplanin regimen was a 16 mg/kg loading dose, followed by 8 mg/kg once daily. Therapeutic drug monitoring and dose adjustment were not conducted. A standard two-compartment PK model was developed, followed by models that incorporated weight. A PK/pharmacodynamic (PD) model with C-reactive protein serial measurements as the PD input was fitted to the data. Monte Carlo simulations (n = 5000) were performed using Pmetrics. The AUCs at steady state and the proportion of patients achieving the recommended drug exposures (i.e. Cmin >15 mg/L) were determined. The study was registered in the European Clinical Trials Database Registry (EudraCT: 2012-005738-12). RESULTS: The PK allometric model best accounted for the observed data. The PK parameters medians were: clearance = 0.435 × (weight/70)0.75 (L/h); volume = 0.765 (L); Kcp = 1.3 (h-1); and Kpc = 0.629 (h-1). The individual time-course of C-reactive protein was well described using the Bayesian posterior estimates for each patient. The simulated median AUC96-120 was 302.3 mg·h/L and the median Cmin at 120 h was 12.9 mg/L; 38.8% of patients attained a Cmin >15 mg/L by 120 h. CONCLUSIONS: Teicoplanin population PK is highly variable in neonates, weight being the best descriptor of PK variability. A low percentage of neonates were able to achieve Cmin >15 mg/L. The routine use of therapeutic drug monitoring and improved knowledge on the PD of teicoplanin is required.

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells.

BACKGROUND: Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC. METHODS: We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines. RESULTS: D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells. CONCLUSION: Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.

ADAM-17: a novel therapeutic target for triple negative breast cancer.

BACKGROUND: Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS: Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS: In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION: It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.

Trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in HER-2-non-amplified breast cancer cell lines.

BACKGROUND: Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD56+natural killer (NK) cells may contribute to the activity of trastuzumab in HER-2-amplified tumours. In this study, we investigated the possibility that trastuzumab might induce ADCC against HER-2-non-amplified breast cancer cells. METHODS: Induction of NK cell-mediated ADCC was examined in trastuzumab-treated HER-2-non-amplified breast cancer cell lines. HER-2 protein levels were also determined in tumour and autologous normal tissue samples from patients with HER-2 negative breast cancer. RESULTS: Trastuzumab induced a significant ADCC response in the HER-2-amplified HCC1954 and SKBR3 cell lines, and in all five of the non-amplified cell lines, which had low levels of detectable HER-2 by western blot (CAL-51, CAMA-1, MCF-7, T47D, and EFM19). Trastuzumab did not induce ADCC in the K562 control cell line or MDA-MB-468, which has very low levels of HER-2 detectable by enzyme-linked immunosorbent assay (ELISA) only. HER-2 protein was detected by ELISA in 14/15 patient tumour samples classified as HER-2-non-amplified. Significantly lower levels of HER-2 were detected in normal autologous tissue compared with tumour samples from the same patients. CONCLUSION: Our results suggest that HER-2-non-amplified breast cancer cells, with low but detectable levels of HER-2 protein, can bind trastuzumab and initiate ADCC.

Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity.

A costimulatory signal through B7 to its counter-receptor CD28 on T cells enhances T cell activation. We have generated recombinant retroviruses containing cDNA for murine B7 and transduced a panel of murine tumor lines with varying immunogenicity to study the effect of B7 costimulation on antitumor immunity. In contrast to the progressive outgrowth of all wild-type (B7-) tumors in unimmunized syngeneic mice, four immunogenic tumors, lymphoma RMA, EL4, mastocytoma P815, and melanoma E6B2, regressed completely when transduced with the B7 gene. In contrast, four nonimmunogenic tumors, sarcomas MCA101, MCA102, and Ag104, and melanoma B16, remained tumorigenic after transduction of the B7 gene. Immunization with B7-transduced immunogenic tumors enhanced protective immunity and increased specific cytotoxic T lymphocyte (CTL) activity against the respective wild-type tumors as compared to immunization with nontransduced or mock-transduced tumors. Moreover, cocultivation of CTL with B7-transduced EL4 cells augmented the specificity of tumor-reactive CTL in long-term cultures. Treatment by injection of B7-transduced tumor cells cured 60% of mice with established wild-type EL4 lymphoma. In contrast, immunization with nonimmunogenic tumors transduced with B7 did not provide protective immunity and did not increase specific CTL activity. Our results show that tumor immunogenicity is critical to the outcome of costimulation of T cell-mediated tumor immunity by B7.

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes.

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.

Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database.

Traditionally, matrix metalloproteinases (MMPs) have been implicated in cancer invasion and metastasis. Because of their role in these processes, several MMPs have been investigated for potential prognostic value as well as targets for antimetastatic therapy. In this investigation, we used a publically available database to relate messenger RNA expression levels for 17 different MMPs to tumor characteristics and outcome in patients with breast cancer. Of the MMPs investigated, only MMP-1 was significantly increased in tumors >2 cm in size compared with those

ADAM-17 predicts adverse outcome in patients with breast cancer.

ADAM-17 is a matrix metalloproteinase-like enzyme involved in the release of several ligands that have been shown to promote both cancer formation and progression. These ligands include transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor, epiregulin and tumor necrosis factor-alpha. In this investigation, we measured the expression of total ADAM-17 by enzyme-linked immunosorbent assay in 153 invasive breast cancers. We also measured the precursor and active forms by western blotting in 140 invasive breast cancers. Expression of ADAM-17 was significantly increased in high-grade compared with low-grade tumors and was independent of tumor size, lymph node metastasis and estrogen receptor status. Patients with high expression of ADAM-17 had a significantly shorter overall survival compared with those with low expression. Significantly, the prognostic impact of ADAM-17 was independent of conventional prognostic factors for breast cancer. Our results are further evidence that ADAM-17 is involved in breast cancer progression and thus provides further impetus for exploiting ADAM-17 as new target for cancer treatment.

B7-1/CD80-transduced tumor cells elicit better systemic immunity than wild-type tumor cells admixed with Corynebacterium parvum.

Tumor cells genetically modified by transduction of B7 (B7-1/CD80), a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, can elicit potent tumor immunity, and they can be effective for treatment of established cancers in animal models. In this study, three tumor lines, the EL4 lymphoma, the P815 mastocytoma, and the MCA102 sarcoma were transduced with recombinant retrovirus containing the murine B7 gene, and their potency to induce systemic immunity protective against challenge with wild-type tumor was compared to that of the same tumor cells admixed with the commonly used adjuvant Corynebacterium parvum. While admixture of tumor cells with C. parvum resulted in complete regression of tumors in syngeneic mice, it did not induce protective immunity against a subsequent challenge of wild-type cells from any of the 3 tumors tested. In contrast, B7-transduced EL4 and P815 tumors regressed locally and induced a potent systemic immunity to wild-type tumors and a higher level of cytotoxic T-cell activity than did tumor cells admixed with C. parvum. No systemic immunity was induced by B7-transduced nonimmunogenic MCA102 sarcoma cells. Our results demonstrate that immunogenic tumor cells transduced with the B7 gene are superior to tumor cells mixed with C. parvum for the induction of systemic tumor immunity.

Concentrated parenteral nutrition solutions and central venous catheter complications in preterm infants.

UNLABELLED: Standardised, concentrated neonatal parenteral nutrition (PN) regimens can overcome early nutritional deficits in very preterm infants. A PN regimen with increased macronutrient content (standardised, concentrated, added macronutrients parenteral (SCAMP)) has been shown to improve early head growth in a randomised controlled trial. Line complications including late onset sepsis were secondary outcomes of this study. Infants were started on standardised, concentrated PN at birth and randomised at 2-5 days to either switch to SCAMP or remain on control PN. Central venous catheter (CVC), blood culture (BC) and inflammatory marker data were collected for the 28-day intervention period. 150 infants were randomised with mean (SD) birth weight (g) of 900 (158) versus 884 (183) in SCAMP (n=74) and control (n=76) groups, respectively. There were no differences in CVC use/type or duration or in positive/negative BC with/without associated C reactive protein rise in SCAMP versus control groups. Increasing the macronutrient content of a standardised, concentrated neonatal PN regimen does not increase CVC complication rates. TRIAL REGISTRATION NUMBER: ISRCTN 76597892.

Synthesis and anticancer activity evaluation of η(5)-C5(CH3)4R ruthenium complexes bearing chelating diphosphine ligands.

The complexes [RuCp*(PP)Cl] (Cp* = C5Me5; [], PP = dppm; [], PP = Xantphos), [RuCp(#)(PP)Cl] (Cp(#) = C5Me4(CH2)5OH; [], PP = dppm; [], PP = Xantphos) and [RuCp*(dppm)(CH3CN)][SbF6] [] were synthesized and evaluated in vitro as anticancer agents. Compounds gave nanomolar IC50 values against normoxic A2780 and HT-29 cell lines, and were also tested against hypoxic HT-29 cells, maintaining their high activity. Complex yielded an IC50 value of 0.55 ± 0.03 µM under a 0.1% O2 concentration.

Studies of oxidative stress in cellular systems. The interaction of monocytes and erythrocytes.

1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti-oxidant defence.

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells.

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.

Antibiotic-resistant oral streptococci in dental patients susceptible to infective endocarditis.

The aim of this study was to determine the incidence of amoxycillin and erythromycin resistance in oral streptococci in patients at risk from infective endocarditis. Samples of gingival crevicular flora were taken from 65 patients at the site of dental treatment, prior to the prophylactic administration of amoxycillin (54 patients) or erythromycin (11 patients). Samples were also taken from 65 dental patients who were not considered to be at risk from infective endocarditis. No isolate had a minimum inhibitory concentration (MIC) of amoxycillin greater than 24 mg/L. However, erythromycin-resistant oral streptococci with MIC values greater than 3.5 mg/L were isolated from 22% of patients receiving amoxycillin prophylaxis, 9% of patients receiving amoxycillin prophylaxis, 9% of patients given erythromycin prophylaxis and 9% of patients not at risk from infective endocarditis. The antibiotic-resistant streptococci comprised mainly Streptococcus sanguis biotype II, although S. sanguis biotype I, S. mitis and S. salivarius were also frequently recovered.

Adhesion and reliability of interfaces in cemented total joint arthroplasties.

Debonding of the prosthetic/polymethylmethacrylate interface has been implicated in the initial failure process of cemented total hip arthroplasties. However, little quantitative understanding of the debonding process, as well as of the optimum interface morphology for enhanced resistance to debonding, exists. Accordingly, a fracture-mechanics approach has been used in which adhesion at the interface is characterized in terms of the interface fracture energy, G (J/m2), and shown to be a strong function of the morphology, debonding length, and loading mode of the interface. Double-cantilever-beam and four-point-flexure fracture-mechanics samples containing four clinically relevant prosthetic surface preparations were prepared to survey a range of interface roughness and loading modes. Adhesion at the interface could not be characterized with a single-valued material property but was found to exhibit resistance-curve behavior in which resistance to debonding increased with both the initial debond extension and the roughness of the interface. Values of debonding initiation, Go, were relatively insensitive to the roughness of the surface and the loading mode, whereas steady-state fracture resistance of the interface, Gss, increased significantly with the roughness and shear loading of the interface. These quantitative results suggest that debonding of the prosthetic/polymethylmethacrylate interface may be primarily attributed to surface interactions such as interlocking and the pullout of rough asperities that occur behind the debond tip. A simple mechanics analysis of such interactions was performed and revealed increases in the fracture resistance of the interface that were consistent with experimentally measured values.

Early detection of colorectal cancer by quantitative fluorescence image analysis of exfoliated cells.

Early-stage colorectal cancer is potentially curable. In the present study, we applied quantitative fluorescence image analysis (QFIA) cytology to the detection of experimental colorectal cancer in a rodent model. QFIA cytology combines visual cytologic examination with quantitation of DNA content in single exfoliated cells. Cancer was induced by treating 110 rats with subcutaneous 1,2-dimethylhydrazine. Sequential colon washes were obtained weekly from each animal for 20 weeks. Control animals were treated identically except for the administration of carcinogen. Cells that were cytologically abnormal or had increased DNA content were found starting in the second week. By the eighth week, roughly 50 percent of animals had positive results, and this level remained approximately constant for the duration of the study. Tissue pathologic results were normal during weeks 1 to 7. Dysplasias became common during weeks 8 to 15 whereas most cancers appeared during weeks 16 to 21. These results indicate that QFIA cytology is a highly sensitive method for detecting even preneoplastic changes resulting from carcinogen administration and may prove useful in detecting human colorectal cancer.

Moving beyond information: evaluation of a nutrition education tool based on a theoretical model.

OBJECTIVE: This study investigated the relative effectiveness of a nutrition education brochure based on a theoretical model versus a more traditional information-based brochure in getting subjects to accurately assess daily calcium intake, make a plan to increase intake if needed, and to implement the plan. DESIGN: A randomized trial involving 216 women between the ages of 19-49y. Subjects were randomly assigned to a group which received educational materials containing an interactive brochure designed using the Motivation Generating model (Calcium CalculatorS), or to a group which received a calcium information brochure (An Appetite for Good Health). Within a two week period the women were contacted by telephone to assess use of materials, calcium intake assessment information, and plans for dietary change. SETTING: Subjects were recruited at five fitness centres in the Vancouver area. The research was conducted by the Institute of Health Promotion Research at the University of British Columbia. RESULTS: Results indicated significantly greater numbers of subjects conducting self- assessment and increased group accuracy for calcium intake assessment in subjects using the interactive brochure. CONCLUSION: Use of a theoretical model designed to create behaviour change such as the Motivation Generating Model can increase specific behaviours which may lead to improvements in dietary consumption.

Quantitative 1H-NMR analysis of amniotic fluid.

Ten amniotic fluid samples (36-38 weeks gestation) are analysed by NMR spectroscopy. Of the species identified in the spectra, valine (mean 198 microM: SEM 57 microM), lactate (9.73 mM; 2.05 mM), alanine (689 microM: 115 microM), acetate (6.87 mM: 1.54 mM), citrate (363 microM: 59 microM), glucose (4.54 mM: 1.28 mM) indoxyl-sulphate (n = 4,270 microM), histidine (n = 6, 125 microM: 31 microM) and formate (n = 4, 92 microM) are quantified using standard addition. The factors governing the detection limits and lowest quantifiable amounts are discussed as are the extension of the work into in vivo magnetic resonance spectroscopy (MRS) in the clinic.