Targeting the cyclin dependent kinase and retinoblastoma axis overcomes standard of care resistance in BRAF V600E -mutant melanoma.

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAFV600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.

Novel patient-derived xenograft mouse model for pancreatic acinar cell carcinoma demonstrates single agent activity of oxaliplatin.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a rare malignancy, accounting for <1 % of all pancreatic neoplasms. Very few retrospective studies are available to help guide management. We previously reported the case of a patient with metastatic PACC who achieved prolonged survival following doxorubicin treatment. Personalized treatment was based on molecular and in vitro data collected from primary cells developed from their liver metastasis. We now report the characterization of a patient derived tumor xenograft (PDTX) mouse model that originated from this patient's PACC liver metastasis. METHODS: Fragments of biopsy tissue (5 mm(3)) from PACC liver metastasis were implanted into athymic nude mice. Tumors were grown and passaged from the host mice into new mice to be tested for therapeutic response. Immuno-histochemical (IHC) biomarkers were used to confirm that the PDTX model represents human PACC. The antitumor activities of multiple drugs (5-FU, irinotecan, oxaliplatin, gemcitabine, bevacizumab, erlotinib, doxorubicin and imatinib) were tested. Tumor size was measured over 74 days or until they reached an endpoint volume of ~800 mm(3). Tests to measure serum lipase levels and histological analyses of tumor tissues were also conducted to assess PACC progression and re-differentiation. RESULTS: The model presented here expresses the same IHC markers found in human PACC. In the chemotherapy study, oxaliplatin produced a prolonged durable growth response associated with increased apoptosis, decreased serum lipase levels and increased healthy acinar cells. Bevacizumab also produced a significant growth response, but the effect was not prolonged as demonstrated by oxaliplatin treatment. The other chemotherapies had moderate to little effect, particularly after treatment ceased. Mutations in DNA repair genes are common in PACC and increase tumor susceptibility to oxaliplatin. To explore this we performed IHC and found no nuclear expression of BRCA2 in our model, indicating a mutation affecting nuclear localization. Gene sequencing confirms BRCA2 has a homozygous gene deletion on Exon 10, which frequently causes a protein truncation. CONCLUSIONS: In summary, we report the development and characterization of the first and only preclinical PACC PDTX model. Here we show sustained anti-tumor activity of single agent oxaliplatin, a compound that is more effective in tumors that harbor mutations in DNA repair genes. Our data shows that BRCA2 is mutated in our PACC model, which could contribute to the oxaliplatin sensitivity observed. Further studies on this rare PACC model can serve to elucidate other novel therapies, biomarkers, and molecular mechanisms of signaling and drug resistance.

Preparation of the Drager Fabius CE and Drager Zeus anaesthetic machines for patients susceptible to malignant hyperthermia.

CONTEXT: Malignant hyperthermia may follow exposure to trace quantities of inhalational anaesthetics. In susceptible patients, the complete avoidance of these triggers is advised when possible; however, failing this, it is essential to washout or purge the anaesthesia machine of residual inhalational anaesthetics. OBJECTIVE: This study examined the washout profile of sevoflurane from the Drager Fabius CE and the Drager Zeus machines. DESIGN: The washout profile of sevoflurane was measured from the Fabius CE and Zeus anaesthesia machines following a standard period of exposure. The disposable tubing, CO2 absorber and other components of each machine were then replaced to examine their impact on the retention of sevoflurane. The effect of autoclaving the ventilator diaphragm and non-disposable ventilator tube or substituting for a new diaphragm and ventilation tube were examined in later parts of this study. SETTING: University teaching hospital. MAIN OUTCOME MEASURE: Time taken to reach 5 parts per million of sevoflurane when machines underwent standard washout with fresh gas flush. RESULTS: The concentration of sevoflurane reached 5 parts per million in the Fabius CE machines after an mean (SD) of 140 min (46) at a fresh gas flow (FGF) of 10 l min(-1). The time taken for sevoflurane to reach 5 parts per million was significantly reduced when the ventilator diaphragm and non-disposable tube were replaced with either new or autoclaved components [14 or 22 min, respectively (P = 0.017, P = 0.031)]. The concentration of sevoflurane reached 5 parts per million in the Zeus machines after an mean (SD) of 85 min (6) at a fresh gas flow of 10 l min(-1). When the fresh gas flow was increased to 18 l min(-1) (the maximum allowable), the time to reach 5 parts per million was reduced to 16 min. CONCLUSION: When preparing the Fabius CE for the malignant hyperthermia susceptible patient, remove the vaporiser, replace the disposable tubing, the reservoir bag and the CO2 absorber. Replace the ventilator diaphragm and non-disposable ventilator tube with new or autoclaved components and flush the machine at 10 l min(-1) for at least 36 min. When preparing the Zeus, remove the vaporiser, replace the disposable tubing, the reservoir bag and CO2 absorber and flush at a fresh gas flow of 10 l min(-1) for at least 90 min. In both the Fabius and Zeus, continue at a fresh gas flow of 10 l min(-1) for the duration of the operation.

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity.

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales. IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.

Isolation from sewage influent and characterization of novel Staphylococcus aureus bacteriophages with wide host ranges and potent lytic capabilities.

Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. PhiSA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while PhiSA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the PhiSA039 host range and showed that PhiSA012 had potent lytic capability. PhiSA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.

IgA response of BALB/c mice to orally administered Salmonella typhimurium flagellin-displaying T2 bacteriophages.

Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N-terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 x 10(3) PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti-FliCm and 0.22% for anti-FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80-465 times lower than the protein dose administered. The possibility that the immuno-stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines.