James F. Eliason

Relationship between molecular mass and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein

Proteins conjugated with polyethylene glycol (PEG) have increased in vivo activity compared to native proteins. We examined the activity of a variety of PEG conjugates prepared with a recombinant mutein of granulocyte colony-stimulating factor (nartograstim [NTG], KW-2228). The total PEG mass was varied by the number and size of the PEG molecules conjugated. In vitro activity, determined using a proliferation assay with G-NFS-60 cells, demonstrated an inverse relationship between PEG mass and concentration required for half-maximal proliferation. In vivo activity was examined by injecting compounds subcutaneously into normal mice and determining neutrophil counts at various times. Initial experiments in C57BL/6J mice indicated that neutrophil levels were significantly elevated 5 days after a single injection of 25 micrograms/mouse of each PEG-NTG preparation. More detailed experiments were performed with several of the preparations in C3H/HeJ mice lacking endotoxin receptors. The results demonstrated that the time after injection at which neutrophil numbers reached a maximum increased with increasing size of PEG. Similar results were obtained with purified preparations containing 1, 2, or 3 units of 20-kDa PEG per molecule of NTG, showing that increasing the extent of PEGylation also increases in vivo activity. Dose-response studies with the 20-kDa PEG-NTG demonstrated a plateau at doses > 2.7 micrograms/mouse at day 3. The plateau dose increased to 8.4 micrograms/mouse at day 5, and no plateau was evident at the highest dose tested (50 micrograms/mL) at days 7 and 10. These results demonstrate that elevated neutrophil levels can be maintained for extended periods following single administration of high-molecular-weight PEG-NTG.

Common EGFR mutations conferring sensitivity to gefitinib in lung adenocarcinoma are not prevalent in human malignant mesothelioma

Dear Sir, Gefitinib (Iressa) is a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) that produces markedly favorable responses in a subset of patients with nonsmall cell lung cancer (NSCLC). Activating somatic mutations in the EGFR tyrosine-kinase domain (TKD) are strongly predictive of both clinical and in vitro sensitivity to gefitinib. To date, gefitinib-sensitizing EGFR mutations have been detected almost exclusively in lung adenocarcinomas, with only 1 missense TKD mutation identified in a colorectal carcinoma. The paucity of gefitinib-sensitizing mutations in cancers exclusive of the lung is consistent with the modest therapeutic responses gefitinib has generally shown in early clinical studies of nonlung cancers. However, not all cancers have been clinically evaluated for gefitinib responsiveness, and screening for gefitinib-sensitizing mutations may be an efficient means of detecting additional responsive cancers. Patients with mesothelioma have a notoriously poor prognosis, with few therapeutic options beyond palliative surgery. Although mesotheliomas have been shown to overexpress EGFR and have been considered as targets for EGFR inhibitors, a screen for EGFR TKD mutations in this cancer has not been reported. In this letter, we describe our screening of human malignant mesothelioma for the most common EGFR TKD mutations identified in gefitinib-responsive NSCLC. A point mutation at EGFR nucleotide 2573 (TfiG) in exon 21, conferring an arginine for leucine substitution at position 858 (L858R), and a series of in-frame deletions in exon 19 have together comprised 70–100% of the TKD mutations detected in gefitinib-responsive NSCLC in the 4 relevant studies published to date. Three additional point mutations, at nucleotide 2582 (TfiA) in exon 21 for L861Q and 2 exon 19 substitutions at nucleotide 2155 (GfiT or A) for G719S and G719C, respectively, have been identified with a 0–20% combined frequency. We developed PCR-based assays to rapidly screen tumor DNA for these 5 somatic mutations One primer in each PCR was labeled with a cyanine-based fluorescent dye (D3-PA or D4-PA) for subsequent fragment analysis using the CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). For detection of deletions in the EGFR exon 19, primers 50-D3-PA-GCTGGTAACATCCACCCAGA-30 and 50-GAGAAAAGGTGGGCCTGAG-30 were designed to amplify a 247 bp region encompassing the entire EGFR exon 19, which was directly sized by fragment analysis. The EGFR point mutations were identified by endonuclease digestion and restriction fragment analysis of their respective amplicons. Primers utilized for genotyping the 858 and 861 codons were 50-D3-PA-GCAGAGCTTCTTCCCATGAT-30 and 50-CTGACCTAAAGCCACCTCCTT-30 and for the 760 codon, 50-D4-PA-GCTGAGGTGACCCTTGTCTC-30 and 50-CCTGTGCCAGGGACCTTA-30. For codon 858, Fau I specifically cleaves the TfiG mutation, such that the 235 bp 858/ 861 amplicon yields a 154 bp labeled restriction fragment. For codon 861, the mutant TfiA is cut by Pvu II, reducing the 858/861 amplicon to a 170 bp labeled fragment. The 182 bp codon 760 amplicon was digested with BsiHKA1, which cleaves at 135 bp both GfiT and GfiA mutants, can be subsequently discriminated by Sac I and cuts exclusively the GfiA. PCRs were performed in 5 ll or 10 ll reactions using PCR Master Mix (Promega, Madison, WI) and 5 ng of tumor DNA purified with the DNeasy 96 Tissue Kit (Qiagen, Valencia, CA), both according to manufacturer’s recommendations. Cycling parameters for all PCRs were as follows: primary denaturation at 95 C for 5 min, then 25 to 30 cycles of 95 C denaturation for 30 sec, 62 C annealing for 30 sec and 72 C extension for 30 sec, followed by a final extension for 5 min. All endonucleases were purchased from New England Biolabs (Beverly, MA) and used with appropriate controls according to manufacturer’s recommendations. One microliter of unpurified PCR was digested for 4–6 hours in a 10 ll reaction volume, 5 ll of which was subsequently analyzed in the CEQ8000 using the standard fragment analysis parameters. Sixty-six patients underwent resection for mesothelioma at the Karmanos Cancer Institute at Wayne State University in Detroit, Michigan, from 2001–2004. The case series is an expansion of that described elsewhere and includes 56 males and 10 females, all Caucasian and with a mean age of 66 years (range 41–87). Mesothelioma DNA from each subject was successfully extracted and screened for EGFR mutations. No size polymorphisms in EGFR exon 19 were detected, nor were point mutations found in any of the evaluated codons: 858, 861 and 719. In a concurrent screen of 99 lung adenocarcinoma specimens, 19 (19%) were found to harbor either a heterozygous inframe deletion in exon 19 (9 samples) or a heterozygous L858R (10 samples). Direct DNA sequencing of the amplicons derived from the lung cancer mutants and an equal number of wild-type specimens confirmed the results of our screening assays.

Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells

BackgroundBoth Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer.MethodsThe total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data.ResultsTaxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents.ConclusionsTaxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer.

Expression of uridine and thymidine phosphorylase genes in human breast carcinoma

Uridine phosphorylase (UPase) and an angiogenic enzyme, thymidine phosphorylase (dThdPase) are involved in degradation of the pyrimidine nucleosides through phosphorolysis. The expression levels of UPase and dThdPase are higher in human solid tumors including breast carcinomas than in normal tissues. To clarify the correlation between the expression levels of UPase and dThdPase genes and the clinicopathological factors, mRNA levels of these enzymes were examined by RT‐PCR in 43 breast carcinomas. UPase gene expression was not correlated with dThdPase gene expression (regression coefficient R = 0.032). Although the expression level of the dThdPase gene was correlated with angiogenesis, detected by immunostaining endothelial cells (R = 0.66), that of UPase gene was not (R = 0.044). These results suggest that UPase does not have a strong angiogenic activity. The UPase gene expression levels in tumors of patients who relapsed were significantly higher than in those from patients who did not (p = 0.039). Although the expression levels of neither UPase or dThdPase were associated with age, pT, pN, pM, estrogen or progesterone receptor positivity, the patients with the higher levels of UPase gene expression had worse survival (p = 0.0038) than those with lower levels. In contrast, the expression of dThdPase gene was not related to relapse or survival of these patients with breast carcinoma. Our findings suggest that the expression level of UPase gene may be an independent prognostic marker in human breast carcinoma.

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy

BackgroundTraditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets.MethodsWe evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp.ResultsThe DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens.ConclusionThe DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

Extended Activity in Cynomolgus Monkeys of a Granulocyte Colony‐Stimulating Factor Mutein Conjugated With High Molecular Weight Polyethylene Glycol

The activity of a granulocyte colony‐stimulating factor (G‐CSF) mutein (nartograstim; [NTG]) conjugated with an average of two polyethylene glycol (PEG) chains per protein molecule was examined in cynomolgus monkeys following a single s.c. injection. Groups of monkeys were given 10 μg/kg, 30 μg/kg, or 100 μg/kg. For comparison, one group of monkeys was given 5 μg/kg of recombinant human G‐CSF (rHuG‐CSF) daily for six days. In monkeys given 100 μg/kg of PEG‐NTG, neutrophil levels reached a peak one day after injection approximately 20‐fold higher than baseline levels. Neutrophil numbers in these animals were still significantly elevated six days after injection. In contrast, peak neutrophil levels in monkeys given six injections of rHuG‐CSF reached a peak only on day 6 and were approximately the same as that in monkeys given a single dose of PEG‐NTG six days before. Pharmacokinetics of PEG‐NTG in these monkeys indicated that the area under the plasma concentration time curve (AUC) increased with increasing the dose from 497 ng•h/ml at 10 μg/kg, 6,140 ng•h/ml at 30 μg/kg to 27,900 ng•h/ml at 100 μg/kg. In a separate study, the effects of single doses of 100 μg/kg of PEG‐NTG, rHuG‐CSF, and unmodified NTG were compared. In this experiment, peak numbers of neutrophils were reached two days after injection in animals receiving PEG‐NTG and one day after in animals given unmodified proteins. The pharmacokinetic parameters demonstrated increased exposure for PEG‐NTG relative to the unmodified proteins with an AUC0‐∞ of 21,012 ng•h/ml compared with 5,492 ng•h/ml for rHuG‐CSF and 5,153 ng•h/ml for NTG. These results demonstrate that conjugation of a G‐CSF mutein with high molecular weight PEG results in a preparation that can induce prolonged elevation of neutrophils in normal nonhuman primates following a single injection.

Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry

The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor‐selective pre‐prodrug of 5‐fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections.

Phase II Trial of Capecitabine and Weekly Docetaxel for Metastatic Castrate Resistant Prostate Cancer

PURPOSE Synergy is observed with the combination of capecitabine and docetaxel due to docetaxel mediated up-regulation of thymidine phosphorylase. A phase II trial was performed with the combination for metastatic, castrate resistant prostate cancer. MATERIALS AND METHODS Eligible patients had metastatic, castrate resistant prostate cancer, no prior chemotherapy for metastatic disease and normal organ function. Docetaxel (36 mg/m(2) per week intravenously) on days 1, 8 and 15, and capecitabine (1,250 mg/m(2) per day in 2 divided doses) on days 5 to 18 were administered in 28-day cycles. The response was assessed every 2 cycles. Biomarker correlative studies were performed on blood dihydropyrimidine dehydrogenase, and the thymidine phosphorylase-to-dihydropyrimidine dehydrogenase and thymidine synthase-to-dihydropyrimidine dehydrogenase ratios in available prostate tumor tissue. RESULTS A total of 30 patients with a median age of 69 years were enrolled in the study. We noted bone pain in 21 patients (70%), Gleason score 8 or higher in 18 (60%), measurable disease progression in 9, bone scan progression in 18 and prostate specific antigen progression in 22. Grade 3 or 4 neutropenia was seen in 3 patients and grade 3 hand-foot syndrome was found in 2. No treatment related deaths occurred. A prostate specific antigen response of 50% or greater decrease was observed in 22 patients (73%), of whom 9 (30%) had 90% or greater decrease. A partial response was noted in 5 of 9 patients (56%) with measurable disease. Median time to progression was 6.7 months (90% CI 4.2-7.7) and median overall survival was 22.0 months (90% CI 18.4-25.3). CONCLUSIONS The combination was well tolerated and it demonstrated favorable response rates with durable remission and survival outcomes.

Potential for Predicting Toxicity and Response of Fluoropyrimidines in Patients

The efficacy of cancer therapy is compromised by the fact that there are currently no good ways to predict which patients will benefit from treatment. This long standing goal is closer to becoming a reality as more is learned about the molecules that affect the activities of various therapeutic agents. The fluoropyrimidine antimetabolites drugs have been in clinical use for over 4 decades and the cellular proteins important for their activities have been studied in detail. The most important are the major target enzyme, thymidylate synthase (TS) and the rate limiting enzyme in the degradation pathway, dihydropyrimidine dehydrogenase (DPD), equally important for the analogue capecitabine is thymidine phosphorylase (TP), which is rate limiting for activation of this prodrug. A number of assays are available for these enzymes, including enzyme activity measurements. quantitative PCR for RNA expression and immunological methods for protein expression. With each of these methods, more clinical studies are required to validate their clinical usefulness.

INDUCTION OF APOPTOSIS BY A SHORT-CHAIN NEUROPEPTIDE ANALOG IN SMALL CELL LUNG CANCER

Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 = 2.8-3.7 microM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 = 3.5-11.2 microM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 microM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents.