ICORG 10-14: NEOadjuvant trial in Adenocarcinoma of the oEsophagus and oesophagoGastric junction International Study (Neo-AEGIS).

BACKGROUND: Neoadjuvant therapy is increasingly the standard of care in the management of locally advanced adenocarcinoma of the oesophagus and junction (AEG). In randomised controlled trials (RCTs), the MAGIC regimen of pre- and postoperative chemotherapy, and the CROSS regimen of preoperative chemotherapy combined with radiation, were superior to surgery only in RCTs that included AEG but were not powered on this cohort. No completed RCT has directly compared neoadjuvant or perioperative chemotherapy and neoadjuvant chemoradiation. The Neo-AEGIS trial, uniquely powered on AEG, and including comprehensive modern staging, compares both these regimens. METHODS: This open label, multicentre, phase III RCT randomises patients (cT2-3, N0-3, M0) in a 1:1 fashion to receive CROSS protocol (Carboplatin and Paclitaxel with concurrent radiotherapy, 41.4Gy/23Fr, over 5 weeks). The power calculation is a 10% difference in favour of CROSS, powered at 80%, two-sided alpha level of 0.05, requiring 540 patients to be evaluable, 594 to be recruited if a 10% dropout is included (297 in each group). The primary endpoint is overall survival, with a minimum 3-year follow up. Secondary endpoints include: disease free survival, recurrence rates, clinical and pathological response rates, toxicities of induction regimens, post-operative pathology and tumour regression grade, operative in-hospital complications, and health-related quality of life. The trial also affords opportunities for establishing a bio-resource of pre-treatment and resected tumour, and translational research. DISCUSSION: This RCT directly compares two established treatment regimens, and addresses whether radiation therapy positively impacts on overall survival compared with a standard perioperative chemotherapy regimen Sponsor: Irish Clinical Research Group (ICORG). TRIAL REGISTRATION: NCT01726452 . Protocol 10-14. Date of registration 06/11/2012.

Fc-γ Receptor Polymorphisms, Cetuximab Therapy, and Survival in the NCIC CTG CO.17 Trial of Colorectal Cancer.

PURPOSE: Two germline Fc-γ receptor (FCGR) polymorphisms, rs1801274 [FCGR2A;His(H)131Arg(R)] and rs396991 [FCGR3A;Phe(F)158Val(V)] produce altered proteins through amino acid substitutions; both are reported to be associated with cetuximab-related outcomes. We performed a validation of these polymorphisms in NCIC CTG CO.17, a randomized trial of cetuximab monotherapy in refractory, metastatic colorectal cancer expressing EGFR. EXPERIMENTAL DESIGN: DNA extracted from formalin-fixed paraffin-embedded tissue was genotyped. In addition to log-rank tests, Cox proportional hazard models assessed their relationships with overall (OS) and progression-free survival (PFS), adjusting for clinically important prognostic factors, along with a polymorphism-treatment arm interaction term. RESULTS: Somatic KRAS status was wild-type for exon 2 in 153 (52%) of 293 patients, from whom tumor DNA was available. For FCGR2A H/H, a genotype-treatment interaction for KRAS wild-type patients was observed for OS (P = 0.03). In KRAS wild-type patients carrying FCGR2A H/H, cetuximab (vs. no cetuximab) improved survival substantially, with adjusted HRs (aHR) of 0.36 (OS) and 0.19 (PFS) and absolute benefits of 5.5 months (OS; P = 0.003) and 3.7 months (PFS; P = 0.02). In contrast, patients carrying FCGR2A R alleles (H/R or R/R) had aHRs of only 0.78 (OS; 2.8-month benefit) and 0.53 (PFS; 1.6-month benefit). No relationships were found for rs396991 (FCGR3A). CONCLUSIONS: In the CO.17 trial, cetuximab worked best for patients with KRAS wild-type colorectal cancers carrying FCGR2A H/H genotypes. Significantly lower benefits were observed in patients carrying germline FCGR2A R alleles. Clin Cancer Res; 22(10); 2435-44. ©2016 AACR.

Australian consensus guidelines for the safe handling of monoclonal antibodies for cancer treatment by healthcare personnel.

These consensus guidelines provide recommendations for the safe handling of monoclonal antibodies. Definitive recommendations are given for the minimum safe handling requirements to protect healthcare personnel. The seven recommendations cover: (i) appropriate determinants for evaluating occupational exposure risk; (ii) occupational risk level compared with other hazardous and non-hazardous drugs; (iii) stratification of risk based on healthcare personnel factors; (iv) waste products; (v) interventions and safeguards; (vi) operational and clinical factors and (vii) handling recommendations. The seventh recommendation includes a risk assessment model and flow chart for institutions to consider and evaluate clinical and operational factors unique to individual healthcare services. These guidelines specifically evaluated monoclonal antibodies used in the Australian cancer clinical practice setting; however, the principles may be applicable to monoclonal antibodies used in non-cancer settings. The guidelines are only applicable to parenterally administered agents.

Access to anticancer drugs: many evidence-based treatments are off-label and unfunded by the Pharmaceutical Benefits Scheme.

BACKGROUND: The off-label use of a drug refers to a use outside the terms of its approval by the Therapeutic Goods Administration (TGA). It is also possible to prescribe unlicensed drugs under the TGA's special access scheme. A high rate of off-label prescribing has previously been reported in cancer. Our study aimed to document the disparity between evidence-based clinical guidelines for anticancer therapy, product approval and funding status of these agents within an academic tertiary/quaternary cancer centre. METHODS: All chemotherapy protocols approved for use in our specialist oncology centre were assessed to determine if the drugs were off-label or unlicensed for that indication based on review of their current product information. The Pharmaceutical Benefits Scheme (PBS) funding status for each protocol was subsequently assessed. RESULTS: A total of 448 protocols containing 82 different drugs across 15 tumour groups was identified. Overall, 189 (42.2%) of protocols were off-label, and three (0.7%) were unlicensed. This resulted in all 192 protocols being unfunded by the PBS. Of the 189 off-label protocols, 132 (69.9%) were based on established evidence-based treatment guidelines, and a further 39 (20.6%) was based on phase II or III clinical trial data. CONCLUSION: Over 90% of off-label protocols are supported by established treatment guidelines or published peer-reviewed research even though the medications are not approved for that particular use by the TGA. However, these off-label protocols are unfunded by the PBS; this results in a marked inequality of access to appropriate medications for cancer patients across Australia.

Imitation switch complexes.

The imitation switch (ISWI) family of chromatin remodelling ATPases is found in organisms ranging from yeast to mammals. ISWI ATPases assemble chromatin and slide and space nucleosomes, making the chromatin template fluid and allowing appropriate regulation of events such as transcription, DNA replication, recombination and repair. The site of action of the ATPases is determined, in part by the tissue type in which the enzyme is expressed and in part by the nature of the proteins associated with the enzyme. The ISWI complexes are generally conserved in composition and function across species. Roles in gene expression and DNA replication in heterochromatin, gene activation and repression in euchromatin, and functions related to maintaining chromosome architecture are associated with different complexes. Defects in ISWI-associated proteins may be associated with neurodegenerative disease, anencephaly, William's syndrome and melanotic tumours. Finally, the mechanism by which yeast Isw Ib influences gene transcription is discussed.

In vivo chromatin remodeling by yeast ISWI homologs Isw1p and Isw2p.

Isw1p and Isw2p are budding yeast homologs of the Drosophila ISWI chromatin-remodeling ATPase. Using indirect-end-label and chromatin immunoprecipitation analysis, we show both independent and cooperative Isw1p- and Isw2p-mediated positioning of short nucleosome arrays in gene-regulatory elements at a variety of transcription units in vivo. We present evidence that both yeast ISWI complexes regulate developmental responses to starvation and that for Isw2p, recruitment by different DNA-binding proteins controls meiosis and haploid invasive growth.

Cadmium-inducible expression of the yeast GSH1 gene requires a functional sulfur-amino acid regulatory network.

Glutathione (gamma-l-glutamyl-l-cysteinylglycine) is an important antioxidant molecule, helping to buffer the cell against free radicals and toxic electrophiles. Expression of the yeast GSH1 gene, encoding the first enzyme involved in glutathione biosynthesis, gamma-glutamylcysteine synthetase, is regulated by oxidants and the heavy metal cadmium at the level of transcription. We present evidence that the transcription factors involved in controlling the network of sulfur amino acid metabolism genes are also responsible for regulating GSH1 expression in response to cadmium. In particular the transcription factors Met-4, Met-31, and Met-32 are essential for cadmium-mediated regulation of gene expression, whereas the DNA-binding protein Cbf1 appears to play a negative role in controlling GSH1 expression.

Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.

To investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 x 10(6) RNA copies per 10(6) cells and 3.7-4.2 x 10(3) copies per 10(6) cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7.3 x 10(3) per 10(6) cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4+, CD8+ and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 x 10(3) copies per 10(6) cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.

Three-dimensional computed tomography bronchoscopy using clinical datasets: a comparison with fibreoptic bronchoscopy.

OBJECTIVE: To assess three-dimensional computed tomography 'bronchoscopic' (3-DCTB) reconstruction of routine CT data as a non-invasive method of airway visualization, and compare it with fibreoptic bronchoscopy (FOB). METHODS: Fourteen datasets were acquired from 13 patients undergoing both FOB and CT examination of the chest. Standard continuous volume CT using 6 mm collimation and clinical FOB techniques were employed. Images were obtained from 3-DCTB reconstructions by segmentation and surface recognition algorithms generating surface rendered 'bronchoscopic views'. These were scored for technical quality and anatomical detail. The most distal bronchi seen in left upper and right lower lobes were recorded for FOB and 3-DCTB. RESULTS: On FOB, the subsegmental bronchi were seen in right lower and in left upper lobe in 10/14 cases and 4/14 cases, respectively. Visualization of the subsegmental airways was not achieved with 3-DCTB, as they could not be identified with confidence. 3-DCTB never achieved a more distal view than obtained by FOB. Using 3-DCT, the right, lower lobe segmental bronchi were seen in 10/14 cases, and lobar bronchus in 14/14 cases (two occluded). In the left upper lobe, 3-DCT showed segmental bronchi in 6/14 cases, lobar bronchus in 11/14 cases (one occluded) and the left main bronchus appeared occluded in 3/14 cases. Overall, technical quality and anatomical detail scores of the carina and proximal bronchi ranked significantly higher than views of segmental bronchi. CONCLUSIONS: 3-DCTB cannot routinely replace FOB for inspection of major and segmental bronchi. Subsegmental bronchi cannot be adequately demonstrated by 3-DCTB using 6 mm collimation datasets.

Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. The International HCV Collaborative Study Group.

Genotypes of hepatitis C virus (HCV) present within 104 samples from HCV-infected individuals from Africa, the Middle East, the Indian subcontinent and South-East Asia were identified by sequence comparisons in the core and NS-5 regions. Relatively short sequences (such as the 222 bp fragment of NS-5) provided effective discrimination of types, subtypes and isolates, and produced equivalent relationships between genotypes as were found upon comparison of longer sequences of NS-5, of the core region, and by comparison of the limited number of complete genomic sequences currently available. Measurement of evolutionary distances in the core and NS-5 regions allowed 79 of the 104 samples to be identified as examples of known genotypes, while 17 of the remainder could be provisionally classified as new subtypes of types 1 (Nigeria), 2 (Gambia), 3 (India, Pakistan and Bangladesh) and 4 (Middle East) on the basis of sequence comparison in core and NS-5 (n = 9) or provisionally using core alone (n = 8). The remaining sequences from Thailand made up two groups showing no close similarity to any of the six major genotypes classified to date, although one corresponded to an as yet unclassified variant of HCV also found in Thailand. However, phylogenetic analysis of the core and NS-5 regions indicated a distant relationship between these sequences with variants found in Vietnam and with type 6a, and collectively they formed a diverse single phylogenetic group. The existence of great diversity within a single genotype was also found amongst type 3 sequences in the Indian subcontinent, amongst type 4 variants in Central Africa and the Middle East, and amongst type variants in Nigeria. These findings may provide clues for understanding the origins and mechanisms of transmission of HCV.

Constrained peptide analogues of transforming growth factor-alpha residues cysteine 21-32 are mitogenically active. Use of proline mimetics to enhance biological potency.

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 microM; in the presence of low doses of TGF-alpha, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-alpha alone. These effects are consistent with the peptides acting via the same mechanism as TGF-alpha. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the beta-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.

Addressing the patient's agenda in the reorganization of antenatal and infant health care: experience in one general practice.

BACKGROUND: Antenatal care is outdated and largely unevaluated. The study practice is committed to involving patients in their own health care. It was decided to use the views of patients to guide a review of antenatal and infant health services. AIM: The study, carried out in 1993, was designed to find out what patients' priorities were, and what they thought were the strengths and weaknesses in the organization and delivery of antenatal and infant health care. METHOD: A total of 52 women attending antenatal appointments were interviewed at the practice using an interview topic guide within a qualitative research framework. Fourteen parents attending a day centre with their under five-year-olds participated in two focus groups. The findings of the interviews and focus group discussions were compared. RESULTS: The interviews revealed a high level of satisfaction with midwife care, although some changes in the organization of antenatal bookings and classes were indicated. Parents expected to be seen switfly at home or at the surgery when their infant was ill, and these high expectations were not always met. There was some degree of confusion and frustration over the role of the health visitor. The views expressed in the focus groups were broadly similar to those expressed in the interviews. CONCLUSION: The findings suggested that there was considerable potential for developing the role of both the midwife and the health visitor, and integrating their work more closely in the primary health care team. Examples are given of how these findings have altered the organization and delivery of antenatal and infant health care in the practice. The study showed that finding out and acting upon the views of patients is a practical way forward in the reshaping of services.

Extrahepatic immunologic manifestations in chronic hepatitis C and hepatitis C virus serotypes.

OBJECTIVE: To determine, using a serotyping assay, whether the occurrence of extrahepatic immunologic disorders in patients with chronic hepatitis C is dependent on hepatitis C virus serotype. DESIGN: Prospective study. SETTING: Liver unit and virology laboratory of a university hospital. PATIENTS: 59 consecutive patients with chronic hepatitis C. MEASUREMENTS: Hepatitis C virus serotype was determined using a recently developed immunoenzymatic assay that detects antibodies directed to serotype-specific immunodominant epitopes. Cryoglobulin, rheumatoid factor, and numerous antitissue antibodies were sought. Biopsies of labial salivary glands were done in 49 of the 59 patients. RESULTS: Prevalence was 59% for serotype 1, 10% for serotype 2, 12% for serotype 3, and 3% for mixed infection. Fifteen percent of patients could not be serotyped. Cryoglobulinemia was found in 36% of patients and rheumatoid factor was found in the serum of 71%. At least one antitissue antibody was found in the serum of 41% of patients; salivary gland biopsy showed lymphocytic capillaritis in 49% of patients. These immunologic abnormalities were seen in patients infected with any of the three serotypes, and prevalences of the abnormalities did not differ significantly among patients infected with different serotypes. CONCLUSIONS: We confirm that the prevalence of extrahepatic immunologic abnormalities is high in patients with chronic hepatitis C. These abnormalities may occur in patients infected with any of the three major hepatitis C virus serotypes now present in developed countries.

Pulmonary function testing in predicting complications from percutaneous lung biopsy.

Percutaneous needle biopsy is an accepted method of obtaining tissue for diagnosis of lung tumors. The depth of the lesion, size of the needle, operator experience, and the presence of emphysema have been identified as factors influencing the risk of postbiopsy pneumothorax, the most common complication. In this retrospective study of 308 patients, we enquired whether pulmonary function tests (available in 138 patients) and arterial PO2 (available in 103 patients) might predict the risk of pneumothorax following percutaneous needle biopsy. We found that as airway obstruction increases (FEV1.0/FVC less than 59% of predicted) or as arterial oxygenation decreases (PO2 less than 59 mm Hg), not only does the incidence of pneumothorax increase, but symptoms are more severe in that the number of pneumothoraces requiring chest tube drainage increases as well. We suggest that airway obstruction and arterial oxygenation are factors indicative of increased risk identifying patients who need close scrutiny after the procedure.

The organization and expression of the yeast retrotransposon, Ty.

The genome of the yeast Saccharomyces cerevisiae contains several copies of a mobile genetic element, Ty, which is representative of a group of eukaryotic transposable elements, retrotransposons. The organization and expression of Ty have several features in common with retroviral proviruses but they also show some important differences.

The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.

Adaptation of skeletal muscle to increased contractile activity. Expression nuclear genes encoding mitochondrial proteins.

An increase in mitochondrial biogenesis in mammalian cells requires a coordinated increase in the expression of a number of nuclear genes that encode mitochondrial proteins. To examine the regulatory mechanisms involved, we used specific anti-sense RNA probes to estimate the cellular concentrations of mRNA transcripts of two such nuclear genes in rabbit tibialis anterior muscles subjected in vivo to 10-21 days of indirect electrical stimulation. The unstimulated contralateral muscle in the same animals provided a base line for comparison. Change in expression of mitochondrial proteins was assessed in terms of the enzymatic capacity of citrate synthase and cytochrome oxidase, which increased 2.1-fold after 10 days and 5.5- and 4.1-fold, respectively, after 21 days of stimulation. As a proportion of total cellular RNA, messenger RNA encoding subunit beta of F1-ATPase increased 2.2-fold over control levels after 10 days and 2.3-fold after 21 days; mRNA encoding subunit VIC of cytochrome oxidase increased 1.3-fold and 1.9-fold over control levels after stimulation for 10 and 21 days, respectively. These changes were not attributable to nonspecific effects of stimulation on all mRNA transcripts, since aldolase A mRNA decreased to 26% of control levels after 21 days of stimulation. Furthermore, mRNA transcripts from these nuclear genes encoding mitochondrial proteins did not increase to the same extent as mRNA transcripts of mitochondrial genes such as cytochrome b, which increased 5.9-fold after 21 days of stimulation. We conclude that the increase in mitochondrial biogenesis induced by electrical stimulation of skeletal muscle is supported by pretranslational regulation of expression of nuclear genes encoding mitochondrial proteins. There are, however, indications that translational or post-translational regulatory events may also be involved.

The genetic organization of the yeast Ty element.

The genetic organization of the yeast transposon Ty resembles that of higher eukaryotic retroviruses and other elements such as the copia-like sequences of Drosophila. The Ty genome is 5.9 kb (10(3) bases) long. It has 340 bp (base pairs) terminal repeats known as delta sequences and it produces a terminally redundant 5.7 kb RNA that starts in the 5' delta and ends in the 3' delta. Ty transcription is directed by signals upstream and downstream of the major RNA start site and is regulated by the mating-type configuration of the cell. The 5.7 kb transcriptional unit is divided into two overlapping open reading frames, TYA and TYB. TYA occupies approximately the first quarter of the transcriptional unit while TYB occupies the rest. TYB overlaps TYA by either 38 or 44 nucleotides, depending on the element, and is in the plus one reading frame with respect to TYA. TYA is expressed to produce protein p1 (50 x 10(3) Mr) and TYB is expressed as a TYA:TYB fusion protein, p3 (190 x 10(3) Mr). Both of these proteins are subsequently cleaved to produce proteins p2, p4, p5, p6, reverse transcriptase and a protease that is responsible for some of these cleavage events. These proteins are assembled into virus-like particles (Ty-VLPs) that contain Ty RNA and reverse transcriptase activity. It is likely that the Ty-VLPs are units of transposition as Ty transposes via an RNA intermediate.