This study provides the largest preclinical evaluation to date of topotecan as a single agent against both in vitro
and in vivo
models of pediatric malignancy. The cell lines tested in vitro
were all inhibited by topotecan with IC50
values within three orders of magnitude. The neuroblastoma cell lines showed the widest range of sensitivity to this drug. The IC50
values of the group formed by the rhabdoid and rhabdomyosarcoma cell lines (all above the general median) are higher than those of the Ewing and leukemias grouped together (nine out of twelve under the median), although with a p
value close to 0.05. The relative differences in sensitivity could point to biological differences among the histotypes, but since this distribution of susceptibility to growth inhibition in vitro
found no correspondence for the solid tumors in the in vivo
panels it is more likely to be due to particular features of the cell lines used, particularly in light of the proliferation dependence of topotecan-induced cytotoxicity. Similarly, Jonsson et al [37
] reported differences in the activity of topotecan, irinotecan and SN-38 between cell lines and primary tumor cells.
The efficacy results obtained by the PPTP for topotecan as a single agent in vivo
are similar to those that have been reported in children with the respective clinical diagnoses. In the experiments reported here the dose of topotecan was adjusted from the mouse MTD of 2.0 mg/kg on the daily × 5 for 2 weeks schedule to a dose of 0.6 mg/kg, based on pharmacokinetic data suggesting that this lower dose produced systemic exposures that are comparable to those achieved in humans for 21 day courses of topotecan [38
]. Whenever possible, this adjustment to clinically relevant doses for preclinical testing should be made to produce more meaningful results. At this dose none of the xenografts was excluded from reporting because of excessive toxicity.
Among the solid tumors the Wilms tumor panel was the only one with two xenografts reaching high activity for the EFS T/C measure, while the rhabdoid, the medulloblastoma and the ependymoma panels did not record any high activity for this parameter.
Topotecan induced significant differences in tumor volume between treated and control groups in 33 of 37 solid tumor xenografts (89%). High activity for this measure among the solid tumor panels (defined as T/C < 15%) was observed in 4 xenografts, including one xenograft each from the Wilms tumor, neuroblastoma, glioblastoma, and rhabdomyosarcoma panels ().
In terms of objective response measure, two of three Wilms tumor xenografts achieved maintained complete responses as did a rhabdomyosarcoma xenograft (1 of 5) and a Ewing sarcoma xenograft (1 of 5). Among the 6 xenografts in the neuroblastoma panel, 3 achieved a partial response and 1 had stable disease while one glioblastoma (out of 4) achieved a partial response (see heat map in ).
In a phase II trial for children with newly diagnosed neuroblastoma, topotecan was administered daily for 5 days over 2 consecutive weeks for two cycles, with doses individualized to attain a single-day topotecan lactone area under the plasma concentration-time curve (AUC) of 80 to 120 ng/mL, which was comparable to that associated with responses in neuroblastoma xenografts [11
]. The response rate achieved was 60% (95% CI, 41% to 77%); with one complete and 17 partial responses. Single agent activity was also observed for newly diagnosed neuroblastoma using the daily × 5 every 3 week schedule [13
Clinical activity for topotecan was reported for patients with recurrent Wilms tumor using the same 10-day treatment schedule described above for neuroblastoma, with dosages adjusted to achieve a target AUC of 80 ng/mL*h [16
]. Out of 25 assessable patients with favorable histology Wilms tumor, 12 had partial response (PR), six had stable disease (SD), and seven had progressive disease (PD), for an overall response rate of 48% (95% CI, 27.8% to 68.7%). Of 11 assessable patients with anaplastic histology WiIms tumor, two had PR, one had SD, and eight had PD.
Topotecan demonstrated activity in children with previously untreated high-risk medulloblastoma when administered daily × 5 days, with a second course administered at day 21 and targeting a plasma AUC of 120 to 160 ng/mL*h [15
]. 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%). Topotecan was also effective in the treatment of newly diagnosed rhabdomyosarcoma using the daily × 5 every three week schedule, with a reported objective response rate of 46% [14
The ALL panel achieved the highest frequency of objective responses (seven out of eight), with three complete responses and two maintained complete responses. These results confirm previous reports and point to topotecan as a potentially relevant drug for the treatment of ALL refractory to conventional drugs. Topotecan has been studied in a phase I trial of children with leukemia, which included children with relapsed or refractory ALL. Within this heavily pretreated population, evidence of anti-leukemia activity was observed when the drug was administered for 9 to 12 days, with one CR and two PRs observed among 25 patients in this phase I study [17
]. A recent report on a phase II study for relapsed ALL described the use of topotecan in combination with the conventional platform dexamethasone, L-asparaginase and vincristine. The administration of topotecan at 2.4 mg/m2 daily for 5 days preceded the regular induction schedule [19
]. With 28 evaluable patients, 89.3% had responses (defined as a ≥ 25% decrease in circulating blast count on day 6 or 7) to the treatment with topotecan alone, while at the end of induction 74.2% had a complete response, 3.2% a partial response and 16.1% had no response.
It is worth noting that the only xenograft that did not to achieve an objective response in the ALL panel was ALL-4, which was derived from a patient with Bcr-Abl expressing Ph+ ALL. A more detailed analysis of this and other BCR-ABL ALL xenografts could lead to identifying underlining mechanisms of resistance to this drug in leukemia with this genetic alteration.
Our results also indicate that gene expression levels of topo I do not give an indication of the response/resistance to topotecan (data not shown), although a recent report suggests that protein levels and enzyme activity may prove more relevant parameters for relating to topotecan activity [39
]. Resistance to topo I inhibitors has been associated with a variety of factors [40
] including reduction of topo I enzyme activity and consequent reduced generation of drug-induced cleavable complexes, with mutations of topo I, with changes in drug transport and efflux (mainly ABCG2 and Pgp) [41
], and with changes in DNA repair mechanisms and apoptosis regulation in cell lines (reviewed in [42
]). Also events downstream from DNA damage sensors could influence resistance since both p53-dependent and -independent mechanisms may operate to induce death as a consequence of topotecan exposure.
Cross-resistance with other chemotherapeutic agents to topo I inhibitors is limited, making topotecan and other members of this family of drugs appealing candidates for the treatment of resistant forms of cancer. Combination of topotecan with other agents has led to the establishment of effective treatment regimens, particularly with cyclophosphamide [43
], and platinum based drugs [46
]. Exposure to topotecan also potentiates the anti-cancer effects of radiotherapy [47
] extending the potential range of applications of this drug.
The reported activities of cyclophosphamide [36
] and cisplatin [48
] against the PPTP panel differ quite clearly from those reported here for topotecan. Although all three drugs cause death through DNA damage, the patterns of activity delineated from the xenografts for the different histotypes are quite divergent pointing to different mechanisms of action.
In summary, the reported activities of topotecan against a broad range of cell lines in vitro and xenografts in vivo at a clinically relevant dose from multiple PPTP panels situate this drug among the most effective anticancer agents available. The activities observed using the PPTP's models have generally been replicated in clinical trials of topotecan against a range of childhood cancer histotypes further validating the results obtained by the PPTP and confirming the usefulness of this strategy to prioritize drugs for clinical trials.