Genz644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development.
Genz644282 was tested against the PPTP in vitro panel (0.1 nM–1 μM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz644282 was tested at 4, 3, 2 and 1 mg/kg in 3 models to assess the dose response relationship. mRNA gene signatures predictive for Genz644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively.
In vitro, Genz644282 demonstrated potent cytotoxic activity with a median IC50 of 1.2 nM (range 0.2–21.9 nM). In vivo, Genz644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz644282.
Genz644282 was highly active within a narrow dose range (2–4 mg/kg), typical of other topoisomerase I poisons.. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent.
Preclinical Testing; Developmental Therapeutics; Genz644282
Bulk allograft reconstruction plays an important role in limb-salvage surgery; however, non-union has been reported in up to 27% of cases. The purpose of this study is to quantify average surface contact areas across simulated intraoperative osteotomies using both free-hand and computer-assisted navigation techniques. Pressure-sensitive paper was positioned between two cut ends of a validated composite sawbone and compression was applied using an eight-hole large fragment dynamic compression plate. Thirty-two samples were analyzed for surface area contact to determine osteotomy congruity. Mean contact area using the free-hand osteotomy technique was equal to 0.21 square inches. Compared with a control of 0.69 square inches, average contact area was found to be 30.5% of optimal surface contact. Mean contact area using computer-assisted navigation was equal to 0.33 square inches. Compared with a control of 0.76 square inches, average contact area was found to be 43.7% of optimal surface contact. Limited contact achieved using standard techniques may play a role in the high rate of observed non-union, and an increase in contact area using computer-assisted navigation may improve rates of bone healing. The development of an oncology software package and navigation hardware may serve an important role in decreasing non-union rates in limb salvage surgery.
Although osteosarcoma is the most common primary malignant bone tumor in children and adolescents, its cell of origin and the genetic alterations are unclear. Previous studies have shown that serially introducing hTERT, SV40 large TAg, and H-Ras transforms human mesenchymal stem cells into two distinct sarcomas cell populations, but they do not form osteoid. In this study, β-catenin was introduced into mesenchymal stem cells already containing hTERT and SV40 large TAg to analyze if this resulted in a model which more closely recapitulated osteosarcoma. Results. Regardless of the level of induced β-catenin expression in the stable transfectants, there were no marked differences induced in their phenotype or invasion and migration capacity. Perhaps more importantly, none of them formed tumors when injected into immunocompromised mice. Moreover, the resulting transformed cells could be induced to osteogenic and chondrogenic differentiation but not to adipogenic differentiation. Conclusions. β-catenin, although fostering osteogenic differentiation, does not induce the malignant features and tumorigenicity conveyed by oncogenic H-RAS when introduced into partly transformed mesenchymal stem cells. This may have implications for the role of β-catenin in osteosarcoma pathogenesis. It also may suggest that adipogenesis is an earlier branch point than osteogenesis and chondrogenesis in normal mesenchymal differentiation.
The ability to define osteosarcoma (OS) patients at greatest risk for metastatic progression and non-responsiveness to conventional therapy is currently not possible. Such biomarkers are needed to predict overall prognosis, probability of metastases at diagnosis, and response to chemotherapy. The tissue microarray (TMA) serves as a powerful tool for detecting and validating protein biomarkers across a variety of patients. We constructed a novel outcome-linked TMA to add to and address shortcomings of currently available osteosarcoma tissue resources. To test the utility of our TMA we surveyed the expression of eukaryotic initiation factor 4E (eIF4E) in osteosarcoma patients using immunohistochemistry. Aberrant regulation of translation initiation is a feature of many cancers. eIF4E is central to initiation of protein synthesis. Its expression and activity have been implicated in tumor formation and potentially malignant and/or metastatic progression in some carcinomas. We found that eIF4E was uniformly expressed in osteosarcoma patient samples. No association was found between eIF4E and outcome in osteosarcoma patients. This novel osteosarcoma TMA provided a facile mechanism to assess the role of a relevant protein biomarker in osteosarcoma.
Tissue microarray; osteosarcoma; eIF4E; immunohistochemistry
PG11047 is a novel conformationally restricted analog of the natural polyamine spermine that lowers cellular endogenous polyamine levels and competitively inhibits natural polyamine functions leading to cancer cell growth inhibition. The activity of PG11047 was evaluated against the PPTP’s in vitro and in vivo panels.
PG11047 was evaluated against the PPTP in vitro panel using 96 hour exposure at concentrations ranging from 10 nM to 100 μM. It was tested against the PPTP in vivo panels at a dose of 100 mg/kg administered by the intraperitoneal (IP) route weekly for 6 weeks.
In vitro PG11047 demonstrated a concentration-response pattern consistent with cytostatic activity. The median relative IC50 for PG11047 was 71 nM. Cell lines of the Ewing sarcoma panel had a lower median relative IC50 value compared to the remaining cell lines in the panel, while cell lines of the neuroblastoma panel had a higher median relative IC50 value. In vivo PG11047 induced significant differences in EFS distribution compared to control in 5 of 32 (15.6%) of the evaluable solid tumor xenografts and in 0 of 7 (0%) of the evaluable ALL xenografts. The single case of tumor regression occurred in an ependymoma xenograft.
Further pediatric development of PG11047 will require better defining a target population and identifying combinations for which there is a tumor-selective cytotoxic effect. The regression observed for an ependymoma xenograft and the exquisite sensitivity of some Ewing sarcoma cell lines to the antiproliferative effects of PG11047 provide leads for further preclinical investigations.
Preclinical Testing; Developmental Therapeutics; polyamine
GSK690693 is a small molecule ATP-competitive inhibitor of the pro-survival kinase Akt. Since Akt regulates multiple downstream targets including transcription factors, glycogen synthase 3, the pro-apoptotic protein Bad, as well as MDM2 and mTORC1, it was tested against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP).
GSK690693 was tested in vitro at concentrations from 1 nM to 10 μM, and against the in vivo panel of xenografts at a dose of 30 mg/kg daily x 5 for 6 consecutive weeks. Three measures of in vivo antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event measure based on the median event-free survival (EFS) of treated and control animals for each xenograft.
GSK690693 inhibited cell growth in vitro with IC50 values between 6.5 nM and >10 μM. In vivo, GSK690693 significantly increased EFS in 11 of 34 (32%) solid tumor xenografts, most notably in all 6 osteosarcoma models, but not in any of the 8 ALL xenografts tested. No objective responses were observed and only one solid tumor met EFS T/C criteria for intermediate activity.
GSK690693 demonstrated broad activity in vitro, however our results against both the solid tumor and ALL PPTP in vivo panels demonstrate that, as single agent at the dose and schedule used, GSK690693 has only modest antitumor activity.
Preclinical Testing; Developmental Therapeutics; Akt inhibitor
AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.
AZD6244 was tested against the PPTP in vitro panel (1 nM-10μM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily five days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting.
At the highest concentration used in vitro (10 μM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment.
At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.
Preclinical Testing; Developmental Therapeutics; AZD6244
Seneca Valley virus (NTX-010) is a non-recombinant, replication competent RNA virus that is undergoing phase 1 clinical trials in adults for tumors with neuroendocrine characteristics. Here we have evaluated the antitumor activity of NTX-010 administered systemically.
In vitro NTX-010 was tested against 23 cell lines exposed for 96 hours at 1 × 10−4 to 104 viral particles (vp)/cell. In vivo NTX-010 was administered intravenously once at 3 × 1012 vp/kg. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume for solid tumor models), measure based on the median event-free survival (EFS) of treated and control animals for each xenograft.
In vitro NTX-010 demonstrated a marked cytotoxic effect in a subset of the cell lines from the neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma panels. In vivo the most consistent activity was observed for the rhabdomyosarcoma and the neuroblastoma panels, with all four of the alveolar rhabdomyosarcoma xenografts and 4 of 5 neuroblastoma xenografts achieving CR or maintained CR. Objective responses were also observed in the rhabdoid tumor, Wilms tumor, and glioblastoma panels.
NTX-010 demonstrated a high level of activity both in vitro and in vivo. Further analysis of existing testing and molecular characterization data may help define the biological characteristics of cancer cells that are associated with response to NTX-010.
Preclinical Testing; Developmental Therapeutics; Seneca Valley virus (NTX-010)
MLN8237 is a small molecule inhibitor of Aurora Kinase A (AURKA) that is currently in early phase clinical testing. AURKA plays a pivotal role in centrosome maturation and spindle formation during mitosis.
MLN8237 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel at concentrations ranging from 1.0 nM to 10 μM and was tested against the PPTP in vivo panels at a dose of 20 mg/kg administered orally twice daily × 5 days. Treatment duration was 6 weeks for solid tumor xenografts and 3 weeks for ALL xenografts.
MLN8237 had a median IC50 of 61 nM against the PPTP in vitro panel. The ALL cell lines were more sensitive and the rhabdomyosarcoma cell lines less sensitive than the remaining PPTP cell lines. In vivo, MLN8237 induced significant differences in event-free survival (EFS) distributions compared to controls in 32/40 (80%) solid tumor models and all (6/6) ALL models. Maintained complete responses (CRs) were observed in 3 of 7 neuroblastoma xenografts, and all 6 evaluable ALL xenografts achieved CR (n=4) or maintained CR (n=2) status. Maintained CRs were observed among single xenografts in other panels, including the Wilms tumor, rhabdoid tumor, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, and medulloblastoma.
The in vivo activity observed against the neuroblastoma panel far exceeds that observed for standard agents evaluated against the panel by the PPTP. High levels of in vivo activity were also observed against the ALL xenograft panel. These data support expedited clinical development of MLN8237 in childhood cancer.
Preclinical Testing; Developmental Therapeutics; MLN8237
Many childhood malignancies including sarcomas, neuroblastoma and Wilms tumor show the presence of both, active, type-1-insulin-like growth factor receptor (IGF-1R), and the autocrine production of its ligands IGF-1/IGF-2. IMC-A12 is a fully human IgG1 antibody that prevents ligand binding to the IGF-1R.
IMC-A12 was evaluated against the 23 cell lines of the Pediatric Preclinical Testing Program (PPTP) in vitro panel using 96 hour exposure at concentrations ranging from 0.01 nM to 0.1 μM. IMC-A12 was tested in vivo at a dose of 1 mg/mouse administered intraperitoneally twice weekly for six weeks.
In vitro, IMC-A12 induced T/C values less than 50% in only three cell lines, a rhabdomyosarcoma cell line (Rh41) and two Ewing sarcoma cell lines (TC-71 and CHLA-9). In vivo, IMC-A12 induced significant differences in EFS distribution compared to control in 24 of 34 (71%) evaluable solid tumor xenografts. Using the PPTP “time to event” activity measure, IMC-A12 induced intermediate (n=13) or high (n=1) activity in 33 xenografts evaluable for this activity measure, including 6 of 6 rhabdomyosarcoma xenografts, 3 of 5 osteosarcoma xenografts, 2 of 5 neuroblastoma xenografts, and 1 of 5 Ewing sarcoma xenografts. The only objective response observed was observed in a rhabdomyosarcoma xenograft (Rh28) that achieved a maintained complete response.
IMC-A12 demonstrated broad antitumor activity against the PPTP’s in vivo solid tumor panels, with the activity primarily being tumor growth inhibition rather than tumor regression. IMC-A12 showed its greatest activity in vivo against the PPTP’s rhabdomyosarcoma xenografts.
Preclinical Testing; Developmental Therapeutics; IMC-A12
Topotecan is a small molecule DNA topoisomerase I poison, that has been successful in clinical trials against pediatric solid tumors and leukemias. Topotecan was evaluated against the PPTP tumor panels as part of a validation process for these preclinical models.
In vivo three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume for solid tumor models, or ≥25% human CD45+ cells in the peripheral blood for ALL models) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft.
Topotecan inhibited cell growth in vitro with IC50 values between 0.71 nM and 489 nM. Topotecan significantly increased EFS in 32 of 37 (87%) solid tumor xenografts and in all 8 of the ALL xenografts. Seventy five percent of solid tumors met EFS T/C activity criteria for intermediate (n=17) or high activity (n=7). Objective responses were noted in 8 solid tumor xenografts (Wilms, rhabdomyosarcoma, Ewing sarcoma, neuroblastoma). Among the 6 neuroblastomas, three achieved a PR. For the ALL panel, two maintained CRs, three CRs, and two PRs were observed.
Topotecan demonstrated broad activity in vitro and in vivo against both the solid tumor and ALL panels, with significant tumor growth delay generated in all the panels. These results further demonstrate the validity of the PPTP panel for preclinical testing of new drugs.
Preclinical Testing; Developmental Therapeutics; topotecan
To gain a greater understanding of the potential of the Aurora kinase A inhibitor MLN8237 in the treatment of pediatric malignancies.
The activity of MLN8237 was evaluated against 28 neuroblastoma and Ewing sarcoma cell lines, and its in vivo efficacy was studied over a range of doses against 12 pediatric tumor xenograft models. Pharmacokinetic, pharmacodynamic, and genomic studies were undertaken.
In vitro neuroblastoma cell lines were generally more sensitive to MLN8237 than Ewing sarcoma lines. MLN8237 demonstrated significant activity in vivo against solid tumor models at the maximum tolerated dose (MTD); however, only 2 of 6 neuroblastoma models had objective responses at 0.25MTD. In contrast, MLN8237 induced objective responses at its MTD and at 0.5MTD in three ALL models and in two out of three at 0.25MTD. Pharmacokinetic studies at 0.5MTD demonstrated a Tmax of 0.5 h, Cmax of 24.8 μM, AUC(0–24) of 60.3 μM h, and 12 h trough level of 1.2 μM. Mitotic indices increased 6–12 h after MLN8237 administration. AURKA copy number variation was frequent in xenografts, and expression was highly correlated with copy number.
Objective responses were more frequent in tumors with decreased AURKA copy number (5/8) compared to those with increased gene copy number (2/14). This report confirms the significant activity against both solid tumor and ALL xenografts at the MTD, with a steep dose response. These data support clinical development of MLN8237 in childhood cancer. Because of the steep dose–response relationship, such studies should target achieving trough levels of 1 μM or higher for sustained periods of treatment.
Electronic supplementary material
The online version of this article (doi:10.1007/s00280-011-1618-8) contains supplementary material, which is available to authorized users.
Preclinical testing; Developmental therapeutics; MLN8237; Pediatric cancer
Mapatumumab (HGS-ETR1) is a fully human IgG1 agonistic monoclonal antibody that exclusively targets and activates tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1). It was tested in vitro at concentrations from 0.01 to 100 μg/ml and in vivo at a dose of 10 mg/kg administered intraperitoneally using a twice-weekly schedule. Mapatumumab demonstrated limited activity against the 23 cell lines of the PPTP in vitro panel with no lines achieving 50% growth inhibition. Mapatumumab induced significant differences in event-free survival distribution compared to controls in 9 of 37 evaluable solid tumor xenografts tested, but in none of the 8 ALL xenografts.
Preclinical Testing; Developmental Therapeutics; Mapatumumab
Rapamycin demonstrated broad-spectrum tumor growth inhibition activity against the in vivo panels of childhood tumors used in the Pediatric Preclinical Testing Program (PPTP). Here we have evaluated rapamycin combined with agents used frequently in the treatment of childhood malignancies.
Rapamycin was tested in vitro against 23 cell lines alone or in combination with melphalan, cisplatin, vincristine, or dexamethasone (leukemic models only). In vivo, the impact of combining rapamycin with a cytotoxic agent was evaluated using two measures: 1) the “therapeutic enhancement” measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supra-additivity for the combination compared to the agents used alone.
Combining rapamycin with cytotoxic agents in vitro gave predominantly sub-additive or additive effects, except for dexamethasone in leukemia models for which supra-additive activity was observed. In vivo testing demonstrated that therapeutic enhancement was common for rapamycin in combination with cyclophosphamide and occurred for 4 of 11 evaluable xenografts for the rapamycin and vincristine combination. The combinations of rapamycin with either cyclophosphamide or vincristine were significantly more effective than the respective standard agents used alone at their MTDs for most evaluable xenografts. The combination of rapamycin and cisplatin produced excessive toxicity requiring cisplatin dose reductions, and therapeutic enhancement was not observed for this combination.
Addition of rapamycin to either cyclophosphamide or vincristine at their respective MTDs appears promising, as these combinations are relatively well tolerated and as many of the pediatric preclinical models evaluated demonstrated therapeutic enhancement for these combinations.
Preclinical Testing; Developmental Therapeutics; Rapamycin; cyclophosphamide; cisplatin; vincristine
Osteosarcoma does not respond well to conventional dose methotrexate but does respond to high-dose methotrexate. Previous work has indicated that this resistance may be due to impaired transport of methotrexate across the cell membrane. In this study, the PT430 competitive displacement assay was adapted to evaluate methotrexate transport in 69 high-grade osteosarcoma tumor samples. All samples studied were shown to have relatively impaired methotrexate transport by PT430 assay. Ninety-nine percent of the samples had less than 20% PT430 displacement by methotrexate. Eighty-eight percent exhibited displacement by methotrexate at less than 50% of the displacement by trimetrexate. The high frequency of impaired transport suggests the presence of decreased functionality of the reduced folate carrier protein. The overwhelming presence of impaired transport may explain why methotrexate needs to be given in high doses to be effective in osteosarcoma therapy and suggests that reduced folate carrier-independent antifolates should be explored.
Ispinesib is a highly specific inhibitor of kinesin spindle protein (KSP, HsEg5), a mitotic kinesin required for separation of the spindle poles. Here we report the activity of ispinesib against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP).
Ispinesib was tested against the PPTP in vitro panel cell lines at concentrations from 0.1 nM to 1 μM and against the in vivo tumor panel xenografts by intraperitoneal administration (5 or 10 mg/kg) every 4 days for 3 doses repeated at day 21.
Ispinesib was highly potent against the PPTP’s in vitro cell lines with a median IC50 of 4.1 nM. Ispinesib (10 mg/kg) induced unexplained toxicity in mice bearing osteosarcoma xenografts and exceeded the MTD in 12 of 40 non-osteosarcoma xenografts. Ispinesib induced significant tumor growth delay in 88% (23/26) of evaluable xenografts. Using a time to event measure of efficacy, ispinesib had intermediate and high levels of activity against 4 (21%) and 5 (26%) of the 19 evaluable solid tumor xenografts, respectively. Ispinesib induced maintained complete responses (CR) in a rhabdoid tumor, a Wilms tumor and a Ewing sarcoma xenograft. Ispinesib (5 mg/kg) produced 2 complete and 2 partial responses among 6 evaluable xenografts in the ALL panel. The in vivo pattern of activity was distinctive from that previously reported for vincristine.
Ispinesib demonstrated broad in vivo antitumor activity, including maintained complete responses for several xenografts, although with high toxicity rates at the doses studied.
Preclinical Testing; Developmental Therapeutics; Ispinesib
The cell of origin of sarcoma is still unclear. High grade osteosarcomas frequently demonstrate the potential for multipotent differentiation and along with several other lines of evidence suggest that human mesenchymal stem cells (hMSC) might be the cell of origin.
The hMSCs were transformed with retrovirus containing human telomerase reverse transcriptase (hTERT), simian virus 40 large t antigen (SV40 TAg) and lentivirus containing oncogenic H-Ras serially. The changes of cellular phenotypes and multilineage differentiation capacity were observed and compared with the standard osteosarcoma cell lines.
Two distinct genotypic and phenotypic sarcoma cell lines resulted from the same genetic events. The gene expression profiles became more complicated and the karyotype became more chaotic during hMSCs' tumorigenesis. The motility of transformed hMSC was promoted. hMSC and its derivatives could be induced to osteogenic, adipogenic and chondrogenic differentiation except that MSC-TSR4 lost osteogenic differentiation capacity.
Multilineage differentiation potential was retained during tumorigenesis of hMSCs and distinct sarcoma cell lines could arise with the same genetic events, providing good models in better understanding the concept of hMSC and in further investigation of the relationship of hMSCs and osteosarcomas.
human mesenchymal stem cell; tumorigenesis; osteosarcoma; multilineage differentiation potential
Lapatinib is a small molecule reversible tyrosine kinase inhibitor of EGFR and ErbB2 that shows in vitro and in vivo activity against a range of EGFR and ErbB2-dependent adult cancer cell lines and that has clinical efficacy against ErbB2-overexpressing breast cancer.
Lapatinib was tested against the cell lines of the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10.0 μM. Lapatinib was tested against the xenografts of the PPTP in vivo panels using a twice-daily oral administration schedule for six weeks (5-days on, 2-days off) at a dose of 160 mg/kg (320 mg/kg/day). Lapatinib pharmacokinetic parameters were determined in scid−/− mice.
The median IC50 value for lapatinib against the entire PPTP cell line panel was 6.84 μM (range, 2.08 μM to > 10.0 μM). Lapatinib was well tolerated in vivo, with toxicity in only 1.5% of the treated animals. Lapatinib induced significant differences in EFS distribution compared to controls in 1 of 41 xenografts tested. No objective responses were observed in any of the solid tumor panels or in the ALL panel. Lapatinib systemic exposure was consistent with previously observed values.
Lapatinib has little activity against the xenografts of the PPTP's in vivo panel, and its in vitro activity occurs at concentrations above those associated with specific EGFR/ErbB2 inhibition. These results likely reflect lack of ErbB2 overexpression in the models studied and suggest that adult and pediatric cancers may fundamentally differ in the applicability of EGFR family members as therapeutic targets.
Preclinical Testing; Developmental Therapeutics; Lapatinib
Aplidin was tested in vitro at concentrations ranging from from 0.1 nM to 1.0 μM and in vivo at a dose of 0.6 mg/kg administered intraperitoneally on an every 4 days × 3 schedule that was repeated at day 21. In vitro, Aplidin was most active against acute lymphoblastic leukemia (ALL) cell lines. In vivo, Aplidin induced significant differences in EFS distribution in 12 of 28 (43%) solid tumor models and 2 of 6 evaluable ALL models. Aplidin showed potent in vitro activity and induced significant in vivo tumor growth inhibition in some xenografts, but did not induce tumor regressions.
Preclinical Testing; Developmental Therapeutics; Aplidin
Vorinostat, a histone deacetylase inhibitor, was evaluated against the in vitro and in vivo childhood solid tumor and leukemia models in the Pediatric Preclinical Testing Program (PPTP). In vitro testing was performed by the DIMSCAN cytotoxicity assay. In vivo, vorinostat was administered intraperitoneally to mice bearing xenografts. Vorinostat demonstrated 2-log cell growth inhibitory activity in vitro, but generally at concentrations not sustainable in the clinic. No objective responses were observed for any of the solid tumor or acute lymphoblastic leukemia xenografts. Preclinical studies with appropriate drug combinations may provide direction for further clinical evaluations of vorinostat against selected pediatric cancers.
Preclinical Testing; Developmental Therapeutics; Vorinostat
High-dose methotrexate is a standard component in the treatment of osteogenic sarcoma. Impaired methotrexate uptake associated with decreased reduced folate carrier expression is a common mechanism of methotrexate resistance in osteogenic sarcoma samples. We investigated whether promoter methylation and polymorphisms in the 3′ untranslated region are involved in regulating reduced folate carrier expression. In a cohort of 66 osteogenic sarcoma specimens, quantitative methylation-specific polymerase chain reaction and single-strand conformation polymorphism were performed. We found detectable levels of promoter methylation in 84.3% of samples. When related to the reduced folate carrier mRNA levels, a trend was observed that reduced folate carrier expression is lower in samples (median, 0.7) with greater than 10% DNA methylation as compared with those (median, 2.3) with less than 10% DNA methylation. The heterozygous polymorphisms of 2582 T/G and 2617C/T in the 3′ untranslated region showed reduced folate carrier expression (median, 0.9) as compared with the wild-type 2582T and 2617C (median, 4.2). The data suggest promoter methylation and polymorphisms in the 3′ untranslated region of the reduced folate carrier may be involved in its transcriptional regulation in osteogenic sarcoma. Further study is required to confirm this finding.
Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1α) and vascular endothelial growth factor (VEGF). The roles of these markers have been studied previously in several cancer lines. We ascertained the frequency of HIF-1α expression, VEGF expression, the degree of neovascularization, and cell proliferation in osteosarcoma samples. Samples from osteosarcoma patients were assessed for HIF-1α and VEGF protein expression using immunohistochemistry, neovascularization using antibodies for Factor VIII, and cell proliferation using the Ki-67 labeling index. Associations between these parameters and clinical features were examined. HIF-1α staining was positive in 35% of patients and metastases were present in 61% of these HIF-1α-positive patients. VEGF protein expression was detected in 69% of patients, 92% of whom were female. We observed an insignificant trend for a higher frequency of VEGF expression in the high-grade as compared to low-grade osteosarcoma. We observed no association between vascular density and proliferation index and any clinical parameters. We found an association between HIF-1α expression and metastatic disease and between VEGF expression and female gender.
Osteosarcoma is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and accounts for ∼60% of malignant bone tumors. Our comparative genomic hybridization (CGH) studies have identified frequent amplification at 6p12-p21, 12q13-q15, and 17p11.2 in osteosarcoma. Of these amplified regions, 6p12-p21 is particularly interesting because of its association with progression and poor prognosis in patients with osteosarcoma. In an attempt to identify aberrantly expressed gene(s) mapping to the 6p12-p21 amplicon, a region-specific array was generated using 108 overlapping BAC and P1 clones covering a 28.8-Mb region at 0.26-Mb intervals. Based on array CGH analysis, the 6p amplicon was refined to 7.9 Mb between the clones RP11-91E11 and RP1-244F2 and 10 amplified clones, with possible target genes, were identified. To study the expression pattern of the target genes from the hotspot amplicon and known candidate genes from 6p12-21, we did quantitative reverse transcription-PCR analysis of MAPK14, MAPK13, CDKN1A, PIM1, MDGA1, BTB9, DNAH8, CCND3, PTK7, CDC5L, and RUNX2 on osteosarcoma patient samples and seven cell lines. The combined array CGH and quantitative reverse transcription-PCR analysis identified amplification and overexpression of CDC5L, CCND3, and RUNX2. We screened these three genes for protein expression by Western blotting and immunohistochemistry and detected overexpression of CDC5L. Furthermore, we used an in vivo assay to show that CDC5L possesses potential oncogenic activity. These results indicate that CDC5L, a cell cycle regulator important for the G2-M transition, is the most likely candidate oncogene for the 6p12-p21 amplicon found in osteosarcoma.
Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed member of the polycomb gene family and part of the human polycomb complex hPRC-H. We found that in normal cells PHC3 associated with both hPRC-H complex components and with the transcription factor E2F6. In differentiating and confluent cells, PHC3 and E2F6 showed nuclear co-localization in a punctate pattern that resembled the binding of polycomb bodies to heterochromatin. This punctate pattern was not seen in proliferating cells suggesting that PHC3 may be part of a E2F6-polycomb complex that has been shown to occupy and silence target promoters in G0. Previous loss of heterozygosity (LoH) analyses had shown that the region containing PHC3 underwent frequent LoH in primary human osteosarcoma tumors. When we examined normal bone and human osteosarcoma tumors we found loss of PHC3 expression in 36 of 56 osteosarcoma tumors. Sequence analysis revealed that PHC3 was mutated in 9 of 15 primary osteosarcoma tumors. These findings suggest that loss of PHC3 may favor tumorigenesis by potentially disrupting the ability of cells to remain in G0.
tumor suppressor gene; osteosarcoma; polycomb gene; polyhomeotic-like; cell cycle regulation
Methotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied.
In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines.
A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines.
This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.