Dose escalation to rash for erlotinib plus gemcitabine for metastatic pancreatic cancer: the phase II RACHEL study.

BACKGROUND: This phase II, open-label, randomised study evaluated whether patients with metastatic pancreatic cancer receiving erlotinib/gemcitabine derived survival benefits from increasing the erlotinib dose. METHODS: After a 4-week run-in period (gemcitabine 1000 mg m(-2) once weekly plus erlotinib 100 mg per day), patients with metastatic pancreatic cancer who developed grade 0/1 rash were randomised to receive gemcitabine plus erlotinib dose escalation (150 mg, increasing by 50 mg every 2 weeks (maximum 250 mg); n=71) or gemcitabine plus standard-dose erlotinib (100 mg per day; n=75). The primary end point was to determine whether overall survival (OS) was improved by increasing the erlotinib dose. Secondary end points included progression-free survival (PFS), incidence of grade ⩾2 rash, and safety. RESULTS: Erlotinib dose escalation induced grade ⩾2 rash in 29 out of 71 (41.4%) patients compared with 7 out of 75 (9.3%) patients on standard dose. Efficacy was not significantly different in the dose-escalation arm compared with the standard-dose arm (OS: median 7.0 vs 8.4 months, respectively, hazard ratio (HR), 1.26, 95% confidence interval (CI): 0.88-1.80; P=0.2026; PFS: median 3.5 vs 4.5 months, respectively, HR, 1.09, 95% CI: 0.77-1.54; P=0.6298). Incidence of adverse events was comparable between randomised arms. CONCLUSION: The erlotinib dose-escalation strategy induced rash in some patients; there was no evidence that the higher dose translated into increased benefit.

Phase II, randomized, biomarker identification trial (MARK) for erlotinib in patients with advanced pancreatic carcinoma.

BACKGROUND: A prospective, randomized phase II study, with mandatory tumor sampling at current disease stage, aimed to identify biomarkers predictive of improved progression-free survival (PFS) in patients with pancreatic cancer treated with erlotinib. PATIENTS AND METHODS: Patients with histologically/cytologically confirmed, unresectable, locally advanced/metastatic pancreatic cancer, who had failed on or were unsuitable for first-line chemotherapy, underwent a tumor biopsy and were then randomized to receive once-daily erlotinib 150 mg or placebo. The primary end point was identification of biomarkers predicting improved PFS with erlotinib. Secondary end points included PFS, overall survival, response and toxicity. RESULTS: At data cut-off, 207 patients were enrolled and analyzed. Prespecified biomarker analyses of EGFR protein expression, EGFR gene copy number/mutations/polymorphisms and KRAS mutations did not identify any subgroups with a detrimental effect or a strong benefit for PFS with erlotinib. In the primary analysis, the median PFS was 6.1 versus 5.9 weeks in the erlotinib and placebo arms, respectively [hazard ratio (HR) 0.83; 95% confidence interval (CI) 0.63-1.10; P = 0.1909]. However, observed baseline imbalances indicated worse prognosis in the erlotinib arm. After adjustment for baseline characteristics, a significant PFS benefit for erlotinib was observed (HR 0.68; 95% CI 0.50-0.91; P = 0.0102). Exploratory biomarker analyses showed patients with high baseline serum amphiregulin levels might benefit from erlotinib. CONCLUSION: This study in patients with inoperable pancreatic cancer did not identify any prespecified biomarkers predictive of PFS benefit with erlotinib. Exploratory analyses suggested high amphiregulin might predict PFS benefit from erlotinib. CLINICALTRIALSGOV NUMBER: NCT00674973.

A phase II study of erlotinib in combination with bevacizumab versus chemotherapy plus bevacizumab in the first-line treatment of advanced non-squamous non-small cell lung cancer.

BACKGROUND: Molecularly targeted agents for non-small cell lung cancer (NSCLC) can provide similar efficacy to chemotherapy without chemotherapy-associated toxicities. Combining two agents with different modes of action could further increase the efficacy of these therapies. The TASK study evaluated the efficacy and safety of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in combination with the anti-angiogenic agent bevacizumab as first-line therapy in unselected, advanced non-squamous NSCLC patients. METHODS: Patients were recruited from December 2007 to September 2008. Planned sample size was 200 patients, a total of 124 patients were randomized. Patients were randomized using a minimization algorithm 1:1 to receive bevacizumab (iv 15 mg/kg day 1 of each 21-day cycle) plus chemotherapy (gemcitabine/cisplatin or carboplatin/paclitaxel standard doses, 4-6 cycles) (BC arm) or bevacizumab plus erlotinib (p.o. 150 mg/day; BE arm) until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). If the hazard ratio (HR) of PFS for BE relative to BC was above 1.25 at the pre-planned interim analysis in favor of BC, the study would be re-evaluated. Secondary endpoints included overall survival, response rate and safety. RESULTS: All randomized patients (n = 63 BE; n = 61 BC) were evaluated for the efficacy analyses. At the updated interim analysis, median PFS was 18.4 weeks (95% confidence interval [CI] 17.0-25.1) versus 25.0 weeks (95% CI 20.6-[not reached]) for BE versus BC, respectively (HR for death or disease progression, BE relative to BC, 2.05, p = 0.0183). The incidence of death was 19% for BE treatment compared with 11.5% for BC treatment. The HR for PFS at the updated interim analysis was above 1.25, therefore patients on the BE arm were permitted to change arms or switch to another drug and the study was terminated. Adverse events reported were as expected. CONCLUSIONS: The TASK study did not show a benefit in terms of PFS for the combination of erlotinib with bevacizumab in unselected first-line advanced non-squamous NSCLC compared with chemotherapy plus bevacizumab.

A multicentre phase II gene expression profiling study of putative relationships between tumour biomarkers and clinical response with erlotinib in non-small-cell lung cancer.

BACKGROUND: Identification of appropriate markers for predicting clinical benefit with erlotinib in non-small-cell lung cancer (NSCLC) may be able to guide patient selection for treatment. This open-label, multicentre, phase II trial aimed to identify genes with potential use as biomarkers for clinical benefit from erlotinib therapy. METHODS: Adults with stage IIIb/IV NSCLC in whom one or more chemotherapy regimen had failed were treated with erlotinib (150 mg/day). Tumour biopsies were analysed using gene expression profiling with Affymetrix GeneChip microarrays. Differentially expressed genes were verified using quantitative RT-PCR (qRT-PCR). RESULTS: A total of 264 patients were enrolled in the study. Gene expression profiles found no statistically significant differentially expressed genes between patients with and without clinical benefit. In an exploratory analysis in responding versus nonresponding patients, three genes on chromosome 7 were expressed at higher levels in the responding group [epidermal growth factor receptor (EGFR), phosphoserine phosphatase (PSPH) and Rap guanine nucleotide exchange factor 5 (RAPGEF5)]. Independent quantification using qRT-PCR validated the association between EGFR and PSPH overexpression, but not RAPGEF5 overexpression, and clinical outcome. CONCLUSIONS: This study supports the use of erlotinib as an alternative to chemotherapy for patients with relapsed advanced NSCLC. Genetic amplification of the EGFR region of chromosome 7 may be associated with response to erlotinib therapy.

The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria.

Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2-concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6. 6 kb genomic region revealed the existence of five potential protein-coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH-1 complex), NdhD3 (subunit 4 of NDH-1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high-affinity CO2 uptake and were not fully capable of inducing high-affinity HCO3- transport. ndhD3 and ndhF3 mutants displayed P700 re-reduction rates identical to wild-type cells, suggesting that NdhD3 is part of a specific NDH-1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH-1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high-affinity CO2 transport. The possibility of multiple, functionally distinct NDH-1 complexes in cyanobacteria is discussed.

Deuteration effects on the in vivo EPR spectrum of the reduced secondary photosystem I electron acceptor A1 in cyanobacteria.

The photoreduction of the secondary PSI electron acceptor A1 in vivo has recently been detected via X-band EPR spectroscopy in intact spinach chloroplasts and in marine cyanobacteria Synechococcus PCC 7002 [Klughammer, C., and Pace, R. J. (1997) Biochim. Biophys. Acta 1318, 133-144]. A further study of the A1- EPR spectrum of Synechococcus PCC 7002 at room temperature with higher-field resolution revealed partially resolved hyperfine structure which was dominated by 0.4 mT splittings of three equivalent protons. The hyperfine splitting was not significantly affected by incubation of the cyanobacteria in 2H2O medium for 20 h, but was absent in fully deuterated cyanobacteria that were grown in 2H2O medium. Anisotropic g-factors consistent with a phylloquinone radical were derived by spectra simulation. Biosynthetic protonation of quinones via the CH3 donor L-methionine in deuterated cells maintained hyperfine structure in the A1- spectrum, indicating the incorporation of CH3 groups in 60% of the deuterated, photoactive A1 molecules. Conversely, biosynthetic quinone deuteration via L-[methyl-d3]methionine in protonated cells led to the loss of the 0. 4 mT splittings in 54% of the A1 molecules. These observations confirm the conclusion of Heathcote et al. [(1996) Biochemistry 35, 6644-6650] of the identity of EPR-detected, photoreduced A1- in vivo with a phylloquinone (vitamin K1) radical in PSI. The partially resolved hyperfine structure of the A1- spectrum indicates an altered spin distribution in the bound vitamin K1- radical in vivo compared to that of unbound vitamin K1- in vitro.

Solute transport across the tonoplast of barely mesophyll vacuoles: Mg2+ determines the specificity, and ATP lipophilic amino acids the activity of the amino acid carrier.

After stimulation with ATP and in the absence of divalent cations, isolated barely mesophyll vacuoles exhibited massive solute fluxes across the tonoplast, measured either as efflux of endogenous solutes or as uptake of radioactive-labeled compounds. Transported solutes were ions (particularly K+, NO3-, Cl-) and amino acids (for example, ala, arg, asp, gln, leu, met). Addition of Mg2+ in excess of added ATP inhibited fluxes of inorganic ions and of positively charged amino acids, but not, or to a smaller extent, those of neutral amino acids. Thus, Mg2+ increased the specificity of the carrier for amino acids such as alanine and glutamine. All ATP-stimulated transport processes were sensitive towards inhibition by lipophilic amino acids, for example by leucine and phenylalanine. After stimulation with sulfhydryl reagents, the inhibitory properties of Mg2+ and lipophilic amino acids were lost. These data concur with the hypothesis of a single transporter which exhibits a channel-like structure with a low degree of substrate selectivity in the absence of Mg2+, and which functions as a neutral amino acid carrier in the presence of Mg2+.

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel.

A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.