Mark N. Kirstein

ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkins lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass ∼177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the ∼177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

Chromosomal assignment and genomic structure of Il15

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the beta and gamma chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.

Results of a Phase II Upfront Window of Pharmacokinetically Guided Topotecan in High-Risk Medulloblastoma and Supratentorial Primitive Neuroectodermal Tumor

PURPOSE To assess the antitumor efficacy of pharmacokinetically guided topotecan dosing in previously untreated patients with medulloblastoma and supratentorial primitive neuroectodermal tumors, and to evaluate plasma and CSF disposition of topotecan in these patients. PATIENTS AND METHODS After maximal surgical resection, 44 children with previously untreated high-risk medulloblastoma were enrolled, of which 36 were assessable for response. The topotecan window consisted of two cycles, administered initially as a 30-minute infusion daily for 5 days, lasting 6 weeks. Pharmacokinetic studies were conducted on day 1 to attain a topotecan lactone area under the plasma concentration-time curve (AUC) of 120 to 160 ng/mL.h. After 10 patients were enrolled, the infusion was modified to 4 hours, with dosage individualization. RESULTS Of 36 assessable patients, four patients (11.1%) had a complete response and six (16.6%) showed a partial response, and disease was stable in 17 patients (47.2%). Toxicity was mostly hematologic, with only one patient experiencing treatment delay. The target plasma AUC was achieved in 24 of 32 studies (75%) in the 30-minute infusion group, and in 58 of 93 studies (62%) in the 4-hour infusion group. The desired CSF topotecan exposure was achieved in seven of eight pharmacokinetic studies when the topotecan plasma AUC was within target range. CONCLUSION Topotecan is an effective agent against pediatric medulloblastoma in patients who have received no therapy other than surgery. Pharmacokinetically guided dosing achieved the target plasma AUC in the majority of patients. This drug warrants testing as part of standard postradiation chemotherapeutic regimens. Furthermore, these results emphasize the importance of translational research in drug development, which in this case identified an effective drug.

Relationship between tumor extracellular fluid exposure to topotecan and tumor response in human neuroblastoma xenograft and cell lines

Purpose: We have reported a 6-fold difference in the topotecan (TPT) lactone systemic exposure achieving a complete response in the human neuroblastoma xenografts NB-1691 and NB-1643. However, the relationship between tumor extracellular fluid (ECF) exposure to TPT and the antitumor activity in xenograft and in vitro models has not been established. Methods: TPT was given i.v. to mice bearing NB-1691 and NB-1643 tumors. Prior to dosing, microdialysis probes were placed in tumors of mice bearing NB-1691 and NB-1643 tumors. Plasma and tumor ECF concentrations of TPT lactone were assayed by high performance liquid chromatography. The inhibitory concentration (IC50) was determined for NB-1691 and NB-1643 cell lines in vitro. Results: The TPT AUCECF values determined for NB-1691 (n=10) and NB-1643 (n=11) were 7.3 ± 0.84 and 25.6 ± 0.76 ng h ml−1, respectively (P < 0.05). TPT tumor ECF penetration in NB-1691 and NB-1643 was 0.04 ± 0.04 and 0.15 ± 0.11 (P < 0.05), respectively. The IC50 values recorded after 6 h of TPT exposure daily for 5 consecutive days for NB-1691 and NB-1643 were 2.7 ± 1.1  and 0.53 ± 0.19 ng/ml, respectively (P < 0.05). Conclusions: NB-1643 was more sensitive in vitro than NB-1691, and at similar plasma TPT exposures, NB-1643 had a greater degree of TPT tumor ECF exposure and penetration as compared with NB-1691. Potential factors affecting tumor TPT ECF disposition include tumor vascularity, capillary permeability, and interstitial pressure. The clinical importance of this study is underscored by the need to select anticancer agents with a high capacity for tumor penetration and to optimize drug administration to increase tumor penetration.

Protracted Intermittent Schedule of Topotecan in Children With Refractory Acute Leukemia: A Pediatric Oncology Group Study

PURPOSE To determine dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD) of a protracted, intermittent schedule of daily 30-minute infusions of topotecan (TPT) for up to 12 consecutive days, every 3 weeks, in children with refractory leukemia. PATIENTS AND METHODS Forty-nine children were enrolled onto this phase I trial (24 with acute nonlymphoblastic leukemia [ANLL] and 25 with acute lymphoblastic leukemia [ALL]). TPT dosage was escalated from 2.0 to 5.2 mg/m(2)/d for 5 days and 2.4 mg/m(2)/d from 7 days to the same dose for 9 and 12 days in cohorts of three to six patients when no DLT was identified. TPT pharmacokinetics were studied in 33 children once or twice (first and last doses in patients who received TPT for > 7 days). RESULTS Seventy assessable courses of TPT were administered to 49 children who had refractory leukemia. DLTs were typhlitis, diarrhea, and mucositis, and the MTD was 2.4 mg/m(2)/d for 9 days in this group of heavily pretreated children. In 33 patients, the median TPT lactone clearance after the first dose was 19.2 L/h/m(2) (range, 9.4 to 45.9 L/h/m(2)) and did not change during the course. There were significant responses (one complete response [CR] and four partial responses [PR] in patients with ANLL and one CR and two PRs in patients with ALL), and all but one were at dosages of TPT given for at least 9 days. CONCLUSION The MTD was 2.4 mg/m(2)/d for 9 days. Further testing is warranted of TPTs schedule dependence in children with leukemia.

Cytotoxic effect of zoledronic acid-loaded bone cement on giant cell tumor, multiple myeloma, and renal cell carcinoma cell lines.

BACKGROUND Local recurrence with subsequent osteolysis is a problem after intralesional curettage of giant cell tumor of bone, myeloma, and metastatic carcinoma. The bisphosphonate zoledronic acid (zoledronate) has been shown to reduce osteoclast activity, and its local administration is a potentially attractive therapy, especially for the osteoclast-rich giant cell tumor. The aim of this study was to analyze the elution dynamics of zoledronic acid release from acrylic bone cement and its in vitro antitumor efficacy. METHODS Various concentrations of zoledronic acid were mixed with bone cement and placed in distilled water. The concentration in the water was measured daily for fourteen days. The cytotoxic effects of the dissolved zoledronic acid on cultures of multiple myeloma, giant cell tumor, and renal cell carcinoma cells were tested with use of the MTT assay (tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye) and analyzed according to the zoledronic acid concentration and the elapsed time. RESULTS The release of zoledronic acid was greatest during the first twenty-four hours for all concentrations and decreased rapidly during the next forty-eight hours to reach a plateau after four days. The proliferation assay (MTT) showed zoledronic acid to have significant cytotoxicity in cultures of stromal giant cell tumor, multiple myeloma, and renal cell carcinoma cells. In addition, zoledronic acid decreased the number of viable tumor cells in a dose-dependent manner. Renal cell carcinoma from bone (RBM1-IT4) and stromal giant cell tumor of bone were more susceptible to zoledronic acid than was multiple myeloma. CONCLUSIONS The method presented in our study is a reproducible technique for evaluating zoledronic acid elution from bone cement and determining its impact on tumor growth. Zoledronic acid is released from bone cement, remains biologically active despite the polymerization of cement, and inhibits the in vitro growth of cell lines from giant cell tumor of bone, myeloma, and renal cell carcinoma.

Determination of plasma topotecan and its metabolite N-desmethyl topotecan as both lactone and total form by reversed-phase liquid chromatography with fluorescence detection.

Topotecan (TPT) undergoes hepatic N-demethylation forming N-desmethyl topotecan (NDS). To evaluate the effect of drug-drug interactions on NDS disposition in children receiving TPT we developed and validated a sensitive and specific HPLC-fluorescence detection method for lactone and total (lactone plus carboxylate) TPT and NDS. Deproteinized plasma is vortexed, centrifuged, and the methanolic extract diluted with water for the lactone form of NDS and TPT or diluted with 1.5% phosphoric acid for NDS and TPT total. A 100 microL sample is injected onto a Varian ChromGuard RP column attached to an Agilent SB-C(18) reversed-phase analytical column held at 50 degrees C. The mobile phase (flow-rate, 0.8 mL/min) consists of methanol-aqueous buffer (27:73, v/v) (75 mM potassium phosphate and 0.2% triethylamine, pH 6.5). TPT and NDS were detected with excitation and emission wavelengths set at 376 and 530 nm, respectively. The standard curves for both forms of TPT ranged from 0.25 to 80 ng/mL, and for NDS ranged from 0.10 to 8.0 ng/mL. Within-day and between-day precision (% RSD) was </=4% for TPT and </=6.2% for NDS, respectively. Within-day and between-day percentage error ranged from 1.4 to 6.3% and from 1.4 to 2.4% for TPT, and from 1.6 to 3.1% and from 0.0 to 3.7% for NDS, respectively. No significant on-column conversion from TPT or NDS lactone to carboxylate was observed. With one method we can measure lactone and total TPT and NDS with adequate sensitivity to allow for evaluation of the disposition of these compounds in children receiving TPT.

Pathway-based pharmacogenomics of gemcitabine pharmacokinetics in patients with solid tumors

AIM The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy. MATERIALS & METHODS SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics. RESULTS Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1. CONCLUSION Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

Pharmacodynamic Modeling of Sequence-Dependent Antitumor Activity of Insulin-like Growth Factor Blockade and Gemcitabine

Agents that block insulin-like growth factor (IGF) signaling are under investigation in clinical trials. Antitumor effects are likely to be enhanced when combined with other agents, but administration sequence effects on activity are not well-described. Three breast cancer cell lines (MCF-7, MDA-MB-231, and Hs-578T) were treated with Gemcitabine and small molecule receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine (PQIP) as single agents and then in combination in the forward (Gemcitabine followed by PQIP) and reverse (PQIP followed by Gemcitabine) sequences. Antitumor effects were assessed longitudinally by Bayesian analysis using WinBUGS. The pharmacodynamic model adequately predicted the observed data. The differences in the cell-kill rate constants for the forward vs. reverse sequence ranged from 0.11 to 0.64 (day−1), and statistical significance was generally dependent on cell line and PQIP concentration. These data indicate that treatment with Gemcitabine first, followed by PQIP is superior to the reverse sequence in vitro.

Molecular targets in the inhibition of angiogenesis

Angiogenesis, the process of blood vessel formation, is crucial for malignant tumour growth and metastases; therefore, it has become an attractive target for anticancer therapy. Theoretically applicable to most solid tumours, this therapy may be advantageous over existing cytotoxic therapy, since it is directed at genetically stable endothelium growing within tumours rather than at malignant cells, which acquire resistance to treatment. Many promising angiogenesis inhibitors have been developed, although their activity has yet to be demonstrated in human clinical trials. To improve therapeutic benefit, this may require further insight into tumour angiogenesis, development of appropriate surrogate markers of activity, treatment of early stage neoplastic disease and probably a combination of different classes of antiangiogenesis agents to overcome redundant mechanisms of angiogenesis control.