Targeted colonic release formulations of mesalazine - A clinical pharmaco-scintigraphic proof-of-concept study in healthy subjects and patients with mildly active ulcerative colitis.

Colonic targeting of orally applied therapeutic drugs remains a challenge. Tablet coatings relying on gastrointestinal pH and colonic bacterial enzymes as triggers in association with an inner alkaline layer are expected to improve targeting efficiency. Mesalazine release from three differently coated tablets labelled with 1 MBq 153Sm was characterised in a single centre, open-label, parallel group study in nineteen healthy subjects and seven patients with mildly active ulcerative colitis. Two semi-organic and one aqueous-based outer coating with different ratios of enteric polymer and resistant starch were tested. All coatings showed comparable release lagtimes in biorelevant dissolution media and were not affected by neutron-activation of the samarium tracer. Mesalazine pharmacokinetics and gamma scintigraphy were used to characterise drug release, anatomical site of tablet disintegration and gastrointestinal transit. Initial tablet disintegration occurred at the ileo-caecal junction or beyond in 92 % of the subjects. Time to initial tablet disintegration was inversely correlated with maximal plasma concentrations and systemic mesalazine exposure. Although high inter-subject variability precluded detection of differences between solvent types and different enteric polymer to polysaccharide ratios, the dual pH and enzymatic triggered release system in combination with an inner alkaline layer promoted mesalazine release at the target site with high accuracy.

Whole-genome sequencing reveals clinically relevant insights into the aetiology of familial breast cancers.

BACKGROUND: Whole-genome sequencing (WGS) is a powerful method for revealing the diversity and complexity of the somatic mutation burden of tumours. Here, we investigated the utility of tumour and matched germline WGS for understanding aetiology and treatment opportunities for high-risk individuals with familial breast cancer. PATIENTS AND METHODS: We carried out WGS on 78 paired germline and tumour DNA samples from individuals carrying pathogenic variants in BRCA1 (n = 26) or BRCA2 (n = 22) or from non-carriers (non-BRCA1/2; n = 30). RESULTS: Matched germline/tumour WGS and somatic mutational signature analysis revealed patients with unreported, dual pathogenic germline variants in cancer risk genes (BRCA1/BRCA2; BRCA1/MUTYH). The strategy identified that 100% of tumours from BRCA1 carriers and 91% of tumours from BRCA2 carriers exhibited biallelic inactivation of the respective gene, together with somatic mutational signatures suggestive of a functional deficiency in homologous recombination. A set of non-BRCA1/2 tumours also had somatic signatures indicative of BRCA-deficiency, including tumours with BRCA1 promoter methylation, and tumours from carriers of a PALB2 pathogenic germline variant and a BRCA2 variant of uncertain significance. A subset of 13 non-BRCA1/2 tumours from early onset cases were BRCA-proficient, yet displayed complex clustered structural rearrangements associated with the amplification of oncogenes and pathogenic germline variants in TP53, ATM and CHEK2. CONCLUSIONS: Our study highlights the role that WGS of matched germline/tumour DNA and the somatic mutational signatures can play in the discovery of pathogenic germline variants and for providing supporting evidence for variant pathogenicity. WGS-derived signatures were more robust than germline status and other genomic predictors of homologous recombination deficiency, thus impacting the selection of platinum-based or PARP inhibitor therapy. In this first examination of non-BRCA1/2 tumours by WGS, we illustrate the considerable heterogeneity of these tumour genomes and highlight that complex genomic rearrangements may drive tumourigenesis in a subset of cases.

BRCA1 tumours correlate with a HIF-1alpha phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression.

BACKGROUND: There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers. METHODS: Immunoperoxidase staining for HIF-1alpha, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes. RESULTS: BRCA1 tumours correlated with positivity for HIF-1alpha (P=0.008) and negativity for PHD3 (P=0.037). HIF-1alpha positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1alpha expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1alpha or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively). CONCLUSIONS: The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1alpha or its downstream targets.

Histopathological features of breast cancer in carriers of ATM gene variants.

AIMS: Germline variants in the ataxia telangiectasia mutated (ATM) gene have been implicated in increased breast cancer risk. The aim of this study was to determine whether the histopathology of breast cancers occurring in ATM variant carriers is distinctive or resembles the described BRCA1 mutation-associated phenotype. METHODS: The histopathological features of breast cancers occurring in ATM variant carriers from multiple-case breast cancer families were compared with matched controls. The test group included 21 cases of in situ and/or invasive cancer from carriers of either the IVS10-6T-->G, 2424V-->G or 1420L-->F ATM variants in the absence of BRCA1 or BRCA2 mutations. An additional four invasive cancers from carriers of a pathogenic BRCA1 mutation in the context of a familial ATM variant were also examined. RESULTS: The histopathology of breast cancers in ATM variant-only carriers was not significantly different from controls and known features of BRCA1 mutation-associated cancer were rarely seen. In contrast, these features were prominent in the small group of cases with a pathogenic BRCA1 mutation. CONCLUSIONS: Breast cancer occurring in carriers of ATM variants is not associated with distinctive histopathological features and does not resemble the tumour phenotype commonly observed in BRCA1 mutation carriers.

Clinical, pathological and genetic features of women at high familial risk of breast cancer undergoing prophylactic mastectomy.

Prophylactic mastectomy (PM) is a risk-management option for women at high familial risk of breast cancer (BC). This study describes the PM experience of women enrolled in a large observational cohort study involving families with a history of hereditary breast cancer. Within 357 multiple-case BC families [119 (33%) BRCA1 or BRCA2 mutation positive], identified via family cancer clinics, 49 cases of PM [21 (43%) BRCA1 or BRCA2 mutation positive] were identified and their clinical, pathological and genetic features reviewed. Families with at least one incidence of PM displayed stronger breast/ovarian cancer histories than did families without PM. Median age at time of PM was 45 years (range 28-58). Ten cases (21%) were bilateral PMs in unaffected women and 39 cases were contralateral PMs in women with prior invasive BC (71%) or ductal carcinoma in situ (DCIS) (8%). Most (88%) underwent total mastectomy. Unnecessary axillary surgery occurred in eight subjects (16%). Malignant histology was found in three PM specimens (6%). Prior to genetic testing, PM was performed in two women who were subsequently shown not to carry the mutation specific to their family. Optimal utilization of genetic testing to guide surgical decision making, appropriate surgical technique and careful pathology examination of PM specimens, are important issues to consider prior to PM in women at high familial risk of BC.

The intronic G13964C variant in p53 is not a high-risk mutation in familial breast cancer in Australia.

BACKGROUND: Mutations in BRCA1 and BRCA2 account for approximately 50% of breast cancer families with more than four affected cases, whereas exonic mutations in p53, PTEN, CHK2 and ATM may account for a very small proportion. It was recently reported that an intronic variant of p53--G13964C--occurred in three out of 42 (7.1%) 'hereditary' breast cancer patients, but not in any of 171 'sporadic' breast cancer control individuals (P = 0.0003). If this relatively frequent occurrence of G13964C in familial breast cancer and absence in control individuals were confirmed, then this would suggest that the G13964C variant plays a role in breast cancer susceptibility. METHOD: We genotyped 71 familial breast cancer patients and 143 control individuals for the G13964C variant using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. RESULTS: Three (4.2%; 95% confidence interval [CI] 0-8.9%) G13964C heterozygotes were identified. The variant was also identified in 5 out of 143 (3.5%; 95% CI 0.6-6.4%) control individuals without breast cancer or a family history of breast cancer, however, which is no different to the proportion found in familial cases (P = 0.9). CONCLUSION: The present study would have had 80% power to detect an odds ratio of 4.4, and we therefore conclude that the G13946C polymorphism is not a 'high-risk' mutation for familial breast cancer.

Diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets.

An asymmetrically-cleaving diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase (Ap4A-->ATP+AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap4A as an intra- and extracellular signalling molecule. Ap4A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap4A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M(r) 19,200. The erythrocyte and platelet enzymes had a Km for Ap4A of 0.70 +/- 0.05 microM (n = 3) while the Km for the leukocyte enzyme was 1.50 +/- 0.20 microM (n = 3). All three enzymes showed substrate inhibition above 10 microM Ap4A. The specific activity of the enzyme in erythrocytes was 0.067 U/10(6) cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap4A hydrolase activity. The last observation is of particular interest given the reported absence of Ap4A from enucleated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor stimulates the synthesis of cell-attachment proteins in the human breast cancer cell line PMC42.

The human breast cancer cell line PMC42 responds to the addition of epidermal growth factor (EGF) by proliferation and increased frequency of attachment of cell-organoid structures to the culture vessel. Antibodies to fibronectin and laminin reacted strongly, by immunoperoxidase, with the membranes of cells from organoids that became adherent following addition of EGF. This reaction was weak with membranes of cells of non-adherent organoids in cultures containing EGF and was negative with membranes of cells of free-floating organoids from cultures without EGF. An increase in biosynthetic labelling with 35S-methionine was found in cell lysates and supernatants of PMC42 cells cultured in the presence of EGF compared with control cells grown without EGF. One-dimensional SDS-polyacrylamide gel electrophoresis and 2-dimensional NEPHGE-PAGE of labelled cell proteins showed increased synthesis of several cellular proteins and the appearance in EGF-treated cells of 2 proteins which were not detected in cells from control cultures lacking EGF. Immunoprecipitation experiments using antibodies to fibronectin and laminin with lysates of 35S-methionine labelled PMC42 cells cultured with EGF showed strong immunoprecipitation at Mr 200 and 400 with anti-laminin, and at Mr 200 and 96 with anti-fibronectin. These immunoprecipitates were blocked specifically by purified laminin or fibronectin, respectively. No immunoprecipitates were detected with these antibodies in lysates from cells grown without EGF. EGF thus stimulates increased adherence of cultured PMC42 cell-organoid structures together with increased membrane expression of the cell-adhesive proteins laminin and fibronectin. These effects may play a role in normal development and neoplastic behaviour of breast epithelia.

Biochemical characterization of the monoclonal antibody-defined ovarian carcinoma-associated antigen SGA.

The molecular nature of SGA, the ovarian-carcinoma-associated antigen defined by the MAb OM-1, has been determined. The cell-surface form of the SGA molecule is a glycoprotein with p1 less than 4.2, which on PAGE analysis has an apparent MW of approximately 360 kDa. This was the only OM-1-reactive species found on the cell surface. The apparent MW was unaffected by reducing conditions. The predominant cytoplasmic form of SGA is a non-glycosylated 170-kDa molecule with p1 6.5. Pulse-chase experiments were complicated by the extremely slow rate of SGA synthesis. However, the data indicate that the SGA molecule is synthesized as a 190-kDa protein, cleaved to yield a 170-kDa non-glycosylated intracellular form which is slowly glycosylated to the 360-kDa cell-surface species. Western blotting experiments revealed the presence of the 360-kDa glycosylated molecule in human ovarian cell culture supernatants.

Monoclonal antibody CI-panHu defines a pan-human cell-surface antigen unique to higher primates.

The murine monoclonal antibody CI-panHu reacts strongly with the cell surface of all human cells, including erythrocytes, tumour cells and HLA-A,B,C-negative cell lines. As such, this antibody defines the first pan-human cell-surface antigen reported. The antigenic determinant detected is associated with a protein doublet of 16,000 MW whose expression is restricted to cells from humans, apes and some species of Old World monkeys. Antibody reactivity is not diminished by routine fixation procedures, nor by paraffin-embedding, and the antigenic determinant is relatively protease-resistant. The use of this antibody as a positive control in immunoassays of human cells is discussed.

The inactivation of herpes simplex virus by some Solanaceae glycoalkaloids.

The infectivity of herpes simplex virus Type I in tissue culture was inhibited by prior incubation with aqueous suspensions of glycoalkaloids in order of activity alpha-chaconine greater than alpha-tomatine greater than alpha-solasonine but not by the corresponding aglycones, solanidine, tomatidine and solasodine. However, inhibition was not only dependent on the presence of a sugar moiety since the glycone alpha-solanine was inactive under the conditions used. The glycones, but not the aglycones, showed cytopathic effects on cellular membranes of Vero cells and erythrocytes; therefore, it is suggested that inactivation of virus results from insertion of the glycones into the viral envelope.

The sebaceous gland antigen defined by the OM-1 monoclonal antibody is expressed at high density on the surface of ovarian carcinoma cells.

A monoclonal antibody, designated OM-1, was raised against ovarian serous papillary cystadenocarcinoma (stage IV) cells. This antibody was found to react strongly with primary and metastatic ovarian serous cystadenocarcinomas and endometrioid carcinomas but the antigen detected was either absent or at very low levels in ovarian mucinous adenocarcinomas, clear cell carcinomas, benign serous and mucinous cystadenomas and Brenner tumours. The OM-1 antibody gave no detectable reaction with 93 other human tumours, including examples of breast and colon adenocarcinomas. In normal tissues the OM-1 antibody reacted with normal sebaceous gland cells, lung type II pneumocytes and placental syncytial trophoblasts. In the normal ovary OM-1 reactivity was confined to extremely weak staining of the surface epithelium. No reaction with any other ovarian cell type could be detected. No evidence of reaction with other normal cell populations present in 24 adult and seven foetal tissues was found. The antigen detected is compared with other ovarian tumour-associated antigens. The OM-1 antibody is likely to prove of value in the detection and diagnosis of ovarian carcinoma.

Inactivation of measles and herpes simplex viruses by trypan blue.

The dye trypan blue inhibits infection of cells in tissue culture by measles and herpes simplex viruses at concentrations in excess of 0.01% (w/v) principally by direct inactivation of the virions. The inactivation is temperature-dependent (10(4) times greater at 37 degrees C than at 4 degrees C) and its mechanism appears to include inhibition of virus adsorption to cells.

Children's brain tumour cells produce RNA particles with incomplete retrovirus characteristics.

Short-term cultures of cells from human rain tumours have been reported to synthesise RNA particles of density in the range characteristic of C type RNA retroviruses, with associated DNA polymerase activity. Fresh tumour cells obtained from 6 children with astrocytoma and 7 children with medulloblastoma, together with one sample of normal brain tissue and normal leukocytes from brain tumour patients were assayed by several characteristics for the primate retrovirus. 1 or 6 (17%) astrocytomas and 4 of 7 (57%) medulloblastomas released RNA particles which banded in sucrose gradients at a density of 1.16-1.18 g/cm3 together with a short segment of DNA, which was eliminated by prior ribonuclease treatment and two proteins of 28k and 16k daltons. These findings were compatible with the presence of a primate retrovirus. Immune coprecipitation of 125I-labelled proteins from the 1.16-1.18 g/cm3 gradient region failed to show any reactivity with antisera to p28 core antigens or the p70 reverse transcriptase antigens of simian sarcoma virus, baboon endogenous virus or Mason Pfizer virus. Assays for DNA polymerase activity in culture supernatant fluid showed only a low amount of activity with template preferences not characteristic of the retroviral reverse transcriptase enzyme.