Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer.

BACKGROUND: Little is known about mechanisms of resistance to poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy in patients with metastatic breast cancer and BRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure. PATIENTS AND METHODS: We obtained tumor biopsies from metastatic breast cancer patients with BRCA1/2 deficiency before and after acquired resistance to PARPi or platinum chemotherapy. Whole exome sequencing was carried out on each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was carried out for functional assessment of intact homologous recombination (HR). RESULTS: Pre- and post-resistance tumor samples were sequenced from eight patients (four with BRCA1 and four with BRCA2 mutation; four treated with PARPi and four with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germline BRCA1 deficiency acquired genomic alterations anticipated to restore HR through increased DNA end resection: loss of TP53BP1 in one patient and amplification of MRE11A in another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of HR. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy. CONCLUSIONS: Genomic reversion in BRCA1/2 was the most commonly observed mechanism of resistance, occurring in four of eight patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

Crizotinib in ROS1-rearranged advanced non-small-cell lung cancer (NSCLC): updated results, including overall survival, from PROFILE 1001.

BACKGROUND: In the ongoing phase I PROFILE 1001 study, crizotinib showed antitumor activity in patients with ROS1-rearranged advanced non-small-cell lung cancer (NSCLC). Here, we present updated antitumor activity, overall survival (OS) and safety data (additional 46.2 months follow-up) for patients with ROS1-rearranged advanced NSCLC from PROFILE 1001. PATIENTS AND METHODS: ROS1 status was determined by FISH or reverse transcriptase-polymerase chain reaction. All patients received crizotinib at a starting dose of 250 mg twice daily. RESULTS: Fifty-three patients received crizotinib, with a median duration of treatment of 22.4 months. At data cut-off, treatment was ongoing in 12 patients (23%). The objective response rate (ORR) was 72% [95% confidence interval (CI), 58% to 83%], including six confirmed complete responses and 32 confirmed partial responses; 10 patients had stable disease. Responses were durable (median duration of response 24.7 months; 95% CI, 15.2-45.3). ORRs were consistent across different patient subgroups. Median progression-free survival was 19.3 months (95% CI, 15.2-39.1). A total of 26 deaths (49%) occurred (median follow-up period of 62.6 months), and of the remaining 27 patients (51%), 14 (26%) were in follow-up at data cut-off. Median OS was 51.4 months (95% CI, 29.3 to not reached) and survival probabilities at 12, 24, 36, and 48 months were 79%, 67%, 53%, and 51%, respectively. No correlation was observed between OS and specific ROS1 fusion partner. Treatment-related adverse events (TRAEs) were mainly grade 1 or 2, per CTCAE v3.0. There were no grade ≥4 TRAEs and no TRAEs associated with permanent discontinuation. No new safety signals were reported with long-term crizotinib treatment. CONCLUSIONS: These findings serve as a new benchmark for OS in ROS1-rearranged advanced NSCLC, and continue to show the clinically meaningful benefit and safety of crizotinib in this molecular subgroup. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier NCT00585195.

Phase I study of the checkpoint kinase 1 inhibitor GDC-0575 in combination with gemcitabine in patients with refractory solid tumors.

BACKGROUND: Checkpoint kinase 1 (Chk1) inhibition following chemotherapy-elicited DNA damage overrides cell cycle arrest and induces mitotic catastrophe and cell death. GDC-0575 is a highly-selective oral small-molecule Chk1 inhibitor that results in tumor shrinkage and growth delay in xenograft models. We evaluated the safety, tolerability, and pharmacokinetic properties of GDC-0575 alone and in combination with gemcitabine. Antitumor activity and Chk1 pathway modulation were assessed. PATIENTS AND METHODS: In this phase I open-label study, in the dose escalation stage, patients were enrolled in a GDC-0575 monotherapy Arm (1) or GDC-0575 combination with gemcitabine Arm (2) to determine the maximum tolerated dose. Patients in arm 2 received either i.v. gemcitabine 1000 mg/m2 (arm 2a) or 500 mg/m2 (arm 2b), followed by GDC-0575 (45 or 80 mg, respectively, as RP2D). Stage II enrolled disease-specific cohorts. RESULTS: Of 102 patients treated, 70% were female, the median age was 59 years (range 27-85), and 47% were Eastern Cooperative Oncology Group PS 0. The most common tumor type was breast (37%). The most frequent adverse events (all grades) related to GDC-0575 and/or gemcitabine were neutropenia (68%), anemia (48%), nausea (43%), fatigue (42%), and thrombocytopenia (35%). Maximum concentrations of GDC-0575 were achieved within 2 hours of dosing, and half-life was ∼23 hours. No pharmacokinetic drug-drug interaction was observed between GDC-0575 and gemcitabine. Among patients treated with GDC-0575 and gemcitabine, there were four confirmed partial responses, three occurring in patients with tumors harboring TP53 mutation. Pharmacodynamic data were consistent with GDC-0575 inhibition of gemcitabine-induced expression of pCDK1/2. CONCLUSION: GDC-0575 can be safely administered as a monotherapy and in combination with gemcitabine; however, overall tolerability with gemcitabine was modest. Hematological toxicities were frequent but manageable. Preliminary antitumor activity was observed but limited to a small number of patients with a variety of refractory solid tumors treated with GDC-0575 and gemcitabine. CLINICAL TRIAL NUMBER: NCT01564251.

Time to Detection with BacT/Alert FA Plus Compared to BacT/Alert FA Blood Culture Media.

Rapid identification of the causative pathogen in patients with bacteremia allows adjustment of antibiotic therapy and improves patient outcomes. We compared in vitro and real-life time to detection (TTD) of two blood culture media, BacT/Alert FA (FA) and BacT/Alert FA Plus (FA Plus), for the nine most common species of bacterial pathogens recovered from blood samples. Experimental data from simulated cultures was compared with microbiology records of TTD for both culture media with growth of the species of interest in clinical blood cultures. In the experimental conditions, median TTD was 3.8 hours (23.9 %) shorter using FA Plus media. The magnitude of reduction differed between species. Similarly, in real life data, FA Plus had shorter TTD than FA media; however, the difference between culture media was smaller, and median TTD was only 1 hour (8.5 %) less. We found shorter TTD with BacT/Alert FA Plus culture media, both experimentally and in real-life conditions and unrelated to antibiotic neutralization, highlighting the importance of appropriate blood culture media selection.

A randomized phase II study of ganetespib, a heat shock protein 90 inhibitor, in combination with docetaxel in second-line therapy of advanced non-small cell lung cancer (GALAXY-1).

BACKGROUND: This trial was designed to evaluate the activity and safety of ganetespib in combination with docetaxel in advanced non-small cell lung cancer (NSCLC) and to identify patient populations most likely to benefit from the combination. PATIENTS AND METHODS: Patients with one prior systemic therapy for advanced disease were eligible. Docetaxel (75 mg/m(2) on day 1) was administered alone or with ganetespib (150 mg/m(2) on days 1 and 15) every 3 weeks. The primary end points were progression-free survival (PFS) in two subgroups of the adenocarcinoma population: patients with elevated lactate dehydrogenase (eLDH) and mutated KRAS (mKRAS). RESULTS: Of 385 patients enrolled, 381 were treated. Early in the trial, increased hemoptysis and lack of efficacy were observed in nonadenocarcinoma patients (n = 71); therefore, only patients with adenocarcinoma histology were subsequently enrolled. Neutropenia was the most common grade ≥3 adverse event: 41% in the combination arm versus 42% in docetaxel alone. There was no improvement in PFS for the combination arm in the eLDH (N = 114, adjusted hazard ratio (HR) = 0.77, P = 0.1134) or mKRAS (N = 89, adjusted HR = 1.11, P = 0.3384) subgroups. In the intent-to-treat adenocarcinoma population, there was a trend in favor of the combination, with PFS (N = 253, adjusted HR = 0.82, P = 0.0784) and overall survival (OS) (adjusted HR = 0.84, P = 0.1139). Exploratory analyses showed significant benefit of the ganetespib combination in the prespecified subgroup of adenocarcinoma patients diagnosed with advanced disease >6 months before study entry (N = 177): PFS (adjusted HR = 0.74, P = 0.0417); OS (adjusted HR = 0.69, P = 0.0191). CONCLUSION: Advanced lung adenocarcinoma patients treated with ganetespib in combination with docetaxel had an acceptable safety profile. While the study's primary end points were not met, significant prolongation of PFS and OS was observed in patients >6 months from diagnosis of advanced disease, a subgroup chosen as the target population for the phase III study.

Co-responding Police-Mental Health Programs: A Review.

Co-responding police-mental health programs are increasingly used to respond to 'Emotionally Disturbed Persons' in the community; however, there is limited understanding of program effectiveness and the mechanisms that promote program success. The academic and gray literature on co-responding police-mental health programs was reviewed. This review synthesized evidence of outcomes along seven dimensions, and the available evidence was further reviewed to identify potential mechanisms of program success. Co-responding police-mental health programs were found to have strong linkages with community services and reduce pressure on the justice system, but there is limited evidence on other impacts. The relevance of these findings for practitioners and the major challenges of this program model are discussed, and future research directions are identified.

Sunitinib in combination with gemcitabine for advanced solid tumours: a phase I dose-finding study.

BACKGROUND: This phase I, dose-finding study determined the maximum tolerated dose (MTD), safety, and pharmacokinetics of sunitinib plus gemcitabine in patients with advanced solid tumours. METHODS: Two schedules with sunitinib (25-50 mg per day) and IV gemcitabine (750-1250 mg m(-2)) in escalating doses were studied. First, patients received sunitinib on a 4-weeks-on-2-weeks-off schedule (Schedule 4/2) plus gemcitabine on days 1, 8, 22, and 29. Second, patients received sunitinib on a 2-weeks-on-1-week-off schedule (Schedule 2/1) plus gemcitabine on days 1 and 8. The primary endpoint was determination of MTD and tolerability. RESULTS: Forty-four patients received the combination (Schedule 4/2, n=8; Schedule 2/1, n=36). With no dose-limiting toxicities (DLTs) at maximum dose levels on Schedule 2/1, MTD was not reached. Grade 4 treatment-related AEs and laboratory abnormalities included cerebrovascular accident, hypertension, and pulmonary embolism (n=1 each), and neutropenia (n=3), thrombocytopenia and increased uric acid (both n=2), and lymphopenia (n=1). There were no clinically significant drug-drug interactions. Antitumor activity occurred across dose levels and tumour types. In poor-risk and/or high-grade renal cell carcinoma patients (n=12), 5 had partial responses and 7 stable disease ≥ 6 weeks. CONCLUSION: Sunitinib plus gemcitabine on Schedule 2/1 with growth factor support was well tolerated and safely administered at maximum doses of each drug, without significant drug-drug interactions.

Phase I safety, pharmacokinetic, and pharmacodynamic study of the oral phosphatidylinositol-3-kinase and mTOR inhibitor BGT226 in patients with advanced solid tumors.

BACKGROUND: This phase I dose-escalation study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics (PDs), and preliminary antitumor activity of BGT226, a potent, oral dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin inhibitor. PATIENTS AND METHODS: Fifty-seven patients with advanced solid tumors received BGT226 2.5-125 mg/day three times weekly (TIW). Dose escalation was guided by an adaptive Bayesian logistic regression model with overdose control. Assessments included response per RECIST, [18F]-fluorodeoxyglucose uptake, and phosphorylated-S6 in skin and paired tumor samples. RESULTS: Three patients (125 mg cohort) had dose-limiting toxic effects (grade 3 nausea/vomiting, diarrhea). BGT226-related adverse events included nausea (68%), diarrhea (61%), vomiting (49%), and fatigue (19%). BGT226 demonstrated rapid absorption, variable systemic exposure, and a median half-life of 6-9 h. Seventeen patients (30%) had stable disease (SD) as best response. Nine patients had SD for ≥16 weeks. Thirty patients (53%) achieved stable metabolic disease as assessed by [18F]-fluorodeoxyglucose-positron emission tomography; however, no correlation between metabolic response and tumor shrinkage according to computed tomography was observed. PD changes suggested PI3K pathway inhibition but were inconsistent. CONCLUSIONS: The MTD of BGT226 was 125 mg/day TIW, and the clinically recommended dose was 100 mg/day TIW. Limited preliminary antitumor activity and inconsistent target inhibition were observed, potentially due to low systemic exposure.

Validation of self-reported epilepsy for purposes of community surveillance.

We evaluated the validity of questions designed to identify lifetime and active epilepsy, medication use, and seizure occurrence on population-based surveys. Subjects were interviewed by telephone, and responses were compared with information in their medical records. Prevalence, sensitivity, specificity, and positive predictive value (PPV) were calculated. The prevalence of ever having been diagnosed with epilepsy was 3.1% by self-report and 2.7% by medical record review. Sensitivity was 84.2%, specificity was 99.2%, and PPV was 73.5% for self-reported lifetime epilepsy, and values were similar for active epilepsy. By comparison, sensitivity was higher and specificity was lower for epilepsy medication use and seizure occurrence. The PPV for seizure occurrence was substantially higher for a recall period of 12 months than for 3 months. These results compare favorably with results for other chronic conditions, such as diabetes and arthritis, and indicate that questionnaires can be used to identify epilepsy at a population level.

Lurbinectedin, a selective inhibitor of oncogenic transcription, in patients with pretreated germline BRCA1/2 metastatic breast cancer: results from a phase II basket study.

BACKGROUND: Lurbinectedin, a selective inhibitor of oncogenic transcription, has shown preclinical antitumor activity against homologous recombination repair-deficient models and preliminary clinical activity in BRCA1/2 breast cancer. PATIENTS AND METHODS: This phase II basket multitumor trial (NCT02454972) evaluated lurbinectedin 3.2 mg/m2 1-h intravenous infusion every 3 weeks in a cohort of 21 patients with pretreated germline BRCA1/2 breast cancer. Patients with any hormone receptor and human epidermal growth factor receptor 2 status were enrolled. The primary efficacy endpoint was overall response rate (ORR) according to RECIST v1.1. Secondary endpoints included duration of response (DoR), progression-free survival (PFS), overall survival (OS) and safety. RESULTS: Confirmed partial response (PR) was observed in six patients [ORR = 28.6%; 95% confidence interval (CI) 11.3% to 52.2%] who had received a median of two prior advanced chemotherapy lines. Lurbinectedin was active in both BRCA mutations: four PRs in 11 patients (36.4%) with BRCA2 and two PRs in 10 patients (20.0%) with BRCA1. Median DoR was 8.6 months, median PFS was 4.1 months and median OS was 16.1 months. Stable disease (SD) was observed in 10 patients (47.6%), including 3 with unconfirmed response in a subsequent tumor assessment [ORR unconfirmed = 42.9% (95% CI 21.8% to 66.0%)]. Clinical benefit rate (PR + SD ≥ 4 months) was 76.2% (95% CI 52.8% to 91.8%). No objective response was observed among patients who had received prior poly (ADP-ribose) polymerase inhibitors. The most common treatment-related adverse events (AEs) were nausea (61.9%), fatigue (38.1%) and vomiting (23.8%). These AEs were mostly grade 1/2. The most common grade 3/4 toxicity was neutropenia (42.9%: grade 4, 23.8%: with no febrile neutropenia). CONCLUSIONS: This phase II study met its primary endpoint and showed activity of lurbinectedin in germline BRCA1/2 breast cancer. Lurbinectedin showed a predictable and manageable safety profile. Considering the exploratory aim of this trial as well as previous results in other phase II studies, further development of lurbinectedin in this indication is warranted.

Phase Ib study of eprenetapopt (APR-246) in combination with pembrolizumab in patients with advanced or metastatic solid tumors.

BACKGROUND: We conducted a phase I, multicenter, open-label, dose-finding, and expansion study to determine the safety and preliminary efficacy of eprenetapopt (APR-246) combined with pembrolizumab in patients with advanced/metastatic solid tumors (ClinicalTrials.gov NCT04383938). PATIENTS AND METHODS: For dose-finding, requirements were non-central nervous system primary solid tumor, intolerant to/progressed after ≥1 line of treatment, and eligible for pembrolizumab; for expansion: (i) gastric/gastroesophageal junction tumor, intolerant to/progressed after first-line treatment, and no prior anti-programmed cell death receptor-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy; (ii) bladder/urothelial tumor, intolerant to/progressed after first-line cisplatin-based chemotherapy, and no prior anti-PD-1/PD-L1 therapy; (iii) non-small-cell lung cancer (NSCLC) with previous anti-PD-1/PD-L1 therapy. Patients received eprenetapopt 4.5 g/day intravenously (IV) on days 1-4 with pembrolizumab 200 mg IV on day 3 in each 21-day cycle. Primary endpoints were dose-limiting toxicity (DLT), adverse events (AEs), and recommended phase II dose (RP2D) of eprenetapopt. RESULTS: Forty patients were enrolled (median age 66 years; range 27-85) and 37 received eprenetapopt plus pembrolizumab. No DLTs were reported and the RP2D for eprenetapopt in combination was 4.5 g/day IV on days 1-4. The most common eprenetapopt-related AEs were dizziness (35.1%), nausea (32.4%), and vomiting (29.7%). AEs leading to eprenetapopt discontinuation occurred in 2/37 patients (5.4%). In efficacy-assessable patients (n = 29), one achieved complete response (urothelial cancer), two achieved partial responses (NSCLC, urothelial cancer), and six patients had stable disease. CONCLUSIONS: The eprenetapopt plus pembrolizumab combination was well tolerated with an acceptable safety profile and showed clinical activity in patients with solid tumors.

A user-centered approach to developing a new tool measuring the behavioural and social drivers of vaccination.

BACKGROUND: Children around the world remain under-vaccinated for many reasons. To develop effective vaccine delivery programmes and monitor intervention impact, vaccine programme implementers need to understand reasons for under-vaccination within their local context. The World Health Organization (WHO) Working Group on the Behavioural and Social Drivers of Vaccination (BeSD) is developing standardised tools for assessing childhood vaccine acceptance and uptake that can be used across regions and countries. The tools will include: (1) a validated survey; (2) qualitative interview guides; and (3) corresponding user guidance. We report a user-centred needs assessment of key end-users of the BeSD tools. METHODS: Twenty qualitative interviews (Apr-Aug 2019) with purposively sampled vaccine programme managers, partners and stakeholders from UNICEF and WHO country and regional offices. The interviews assessed current systems, practices and challenges in data utilisation and reflections on how the BeSD tools might be optimised. Framework analysis was used to code the interviews. RESULTS: Regarding current practices, participants described a variety of settings, data systems, and frequencies of vaccination attitude measurement. They reported that the majority of data used is quantitative, and there is appetite for increased use of qualitative data. Capacity for conducting studies on social/behavioural drivers of vaccination was high in some jurisdictions and needed in others. Issues include barriers to collecting such data and variability in sources. Reflecting on the tools, participants described the need to explore the attitudes and practices of healthcare workers in addition to parents and caregivers. Participants were supportive of the proposed mixed-methods structure of the tools and training in their usage, and highlighted the need for balance between tool standardisation and flexibility to adapt locally. CONCLUSIONS: A user-centred approach in developing the BeSD tools has given valuable direction to their design, bringing the use of behavioural and social data to the heart of programme planning.

Pre-clinical evaluation of cyclin-dependent kinase 2 and 1 inhibition in anti-estrogen-sensitive and resistant breast cancer cells.

BACKGROUND: Cellular proliferation, driven by cyclin-dependent kinases (CDKs) and their cyclin partners, is deregulated in cancer. Anti-estrogens, such as tamoxifen, antagonise estrogen-induced ERalpha transactivation of cyclin D1, resulting in reduced CDK4/6 activity, p27(Kip1)-mediated inhibition of CDK2 and growth arrest. We hypothesised that direct inhibition of CDK2 and CDK1 may overcome the major clinical problem of anti-estrogen resistance. METHODS: The cellular effects of CDK2/1 siRNA knockdown and purine-based CDK2/1 inhibitors, NU2058 and NU6102, were measured in anti-estrogen-sensitive and resistant breast cancer cell lines. RESULTS: CDK2 knockdown caused G1 accumulation, whereas CDK1 depletion caused G2/M slowing, and dual CDK1/2 depletion resulted in further G2/M accumulation and cell death in both anti-estrogen-sensitive and resistant cells, confirming CDK2 and CDK1 as targets for breast cancer therapy. In contrast to tamoxifen, which only affected hormone-sensitive cells, NU2058 and NU6102 reduced CDK2-mediated phosphorylation of pRb, E2F transcriptional activity and proliferation, ultimately resulting in cell death, in both anti-estrogen-sensitive and resistant cells. Both drugs caused G2/M arrest, reflective of combined CDK2/1 knockdown, with a variable degree of G1 accumulation. CONCLUSION: These studies confirm the therapeutic potential of CDK2 and CDK1 inhibitors for cancer therapy, and support their use as an alternative treatment for endocrine-resistant breast cancer.

Safety, tolerability, pharmacokinetics and pharmacodynamics of the oral cyclin-dependent kinase inhibitor AZD5438 when administered at intermittent and continuous dosing schedules in patients with advanced solid tumours.

BACKGROUND: AZD5438 is an orally bioavailable inhibitor of cyclin E-cdk2, cyclin A-cdk2 and cyclin B-cdk1 complexes. Three phase I studies assessed the clinical safety, tolerability, pharmacokinetics and pharmacodynamics of AZD5438 when administered in different dosing schedules. PATIENTS AND METHODS: AZD5438 was administered four times daily, once every 7 days (study 1), for 14 consecutive days followed by 7 days of rest (study 2), or continuously (study 3), to patients with advanced solid tumours. Dose escalation proceeded until the emergence of dose-limiting toxic effects. RESULTS: Sixty-four patients were included across the three studies (19, 17 and 28, respectively). Nausea and vomiting were the most common adverse events. When dosed continuously, 40 mg four times daily was considered intolerable, and due to safety issues, all studies were terminated prematurely. Consequently, no intolerable dose was identified during the weekly schedule. Pharmacokinetics demonstrated dose-proportional exposure, high interpatient variability and accumulation after multiple doses. Skin biopsies indicated reduced retinoblastoma protein phosphorylation at cdk2 phospho-sites; other pharmacodynamic assessments did not reveal consistent trends. CONCLUSIONS: AZD5438 was generally well tolerated in a weekly dosing schedule, but not in continuous schedules. The clinical development programme for AZD5438 was discontinued owing to tolerability and exposure data from these studies.

The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272.

Mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.

The subunit structure of normal and hemophilic factor VIII.

Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by beta-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.

p16INK4A participates in a G1 arrest checkpoint in response to DNA damage.

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16INK4A in a G1 arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G1 following DNA damage. However, engineered expression of p16INK4A at levels compatible with cell proliferation restores a G1 arrest checkpoint in response to treatment with gamma-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53-/- fibroblasts that express p16INK4A. DNA damage-induced G1 arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16INK4A, or a cdk4 variant incapable of binding p16INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16INK4A or in amounts of p16INK4A found in complex with cdks 4 and 6. Nonetheless, p16INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16INK4A expression may be necessary for cells with wild-type Rb to bypass this G1 arrest checkpoint and attain a fully transformed phenotype.

Shipping noise in a dynamic sea: a case study of grey seals in the Celtic Sea.

Shipping noise is a threat to marine wildlife. Grey seals are benthic foragers, and thus experience acoustic noise throughout the water column, which makes them a good model species for a case study of the potential impacts of shipping noise. We used ship track data from the Celtic Sea, seal track data and a coupled ocean-acoustic modelling system to assess the noise exposure of grey seals along their tracks. It was found that the animals experience step changes in sound levels up to ~20dB at a frequency of 125Hz, and ~10dB on average over 10-1000Hz when they dive through the thermocline, particularly during summer. Our results showed large seasonal differences in the noise level experienced by the seals. These results reveal the actual noise exposure by the animals and could help in marine spatial planning.

A novel p16INK4A transcript.

p16INK4A and p15INK4B were initially identified as potent inhibitors of activated cyclin/cyclin-dependent kinase complexes. These genes were colocalized to chromosome 9p21, and p16 was subsequently found to be mutated in familial melanoma and deleted in a wide variety of sporadic cancers. We recently found that de novo methylation of a 5' CpG island led to transcriptional block of full-length p16 in many neoplasms. However, the presence of a truncated p16 transcript in methylated cell lines led us to investigate the presence of an alternative promoter or initiation site. We have now identified an abundant alternative p16 transcript in both methylated and unmethylated cell lines generated from a novel sequence (exon 1 beta) potentially involved in the complex regulation of these critical cell cycle genes.