A new approach to integrate toxicity grade and repeated treatment cycles in the analysis and reporting of phase I dose-finding trials.

BACKGROUND: Safety assessment beyond the dose-limiting toxicity evaluation period provides relevant information to define the recommended phase II dose (RP2D) of a new treatment. We retrospectively analyzed three phase I trials to illustrate two indicators: per-cycle probability of graded toxicity and cumulative probability of severe toxicity over the treatment period. PATIENTS AND METHODS: Data were collected from two continual reassessment method (CRM) trials (T1: aviscumine in solid tumors with short time on treatment; T2: erlotinib + radiotherapy in brainstem gliomas with longer time on treatment) and one 3 + 3 design (T3: liposomal doxorubicin + cyclophosphamide combination in ovarian carcinoma). The probability of severe and moderate or severe toxicity per cycle was estimated at each dose level with mixed proportional odds model. The cumulative probability of severe toxicity was also estimated with the time-to-event CRM. RESULTS: Eighty-three patients were included in the three trials; 94, 96 and 72 treatment cycles were administered, in T1, T2 and T3, respectively. Moderate toxicities were at least twice as frequent as severe toxicities. An increased probability of toxicity over time was detected in T3 [P = 0.04; per-cycle probability of severe toxicity: 27% (cycle 1) to 59% (cycle 6) at the RP2D]. At the RP2D, 37% of patients experienced at least one severe toxicity over the first six cycles in T2, and 78% in T3. CONCLUSIONS: Dedicated methods can be used to analyze toxicities from all cycles of treatment. They do not delay accrual and should be integrated in the analysis and reporting of phase I dose-finding trials.

[Potential role of antiangiogenic treatment in neuroblastoma]. / Neuroblastome : intérêt des traitements anti-angiogéniques.

Focus on new drug development over the last few years has yielded new agents that differ from unspecific classical chemotherapeutics and ionizing radiation, while still targeting the cancer cell itself. Antiangiogenesis is a totally distinct approach targeting the tumor's blood vessels. This concept has now found its eligibility for the treatment of several adult solid tumors: the human antivascular endothelial growth factor (VEGF) antibody bevacizumab, as well as the VEGF receptor tyrosine kinase inhibitors, sunitinib and sorafinib, have recently been licensed by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for the treatment of colorectal, renal, and lung cancer. Other antiangiogenic drugs are under preclinical and early clinical evaluation. However, what do we know of the use of these drugs in pediatric solid tumors, such as sarcomas and embryonal and neuronal tumors? For some time now, neuroblastoma has been shown to be dependent on angiogenesis. However, the first preclinical data on antiangiogenic drugs in neuroblastoma have not been published until recently, and clinical trials with antiangiogenic agents in neuroblastoma treatment protocols are scarce. This review adresses current knowledge on the important role and mechanisms of angiogenesis in neuroblastoma and summarizes available preclinical and clinical results of antiangiogenic agents used to treat neuroblastoma. Our review clearly demonstrates that clinical trials are urgently needed to bring forward promising antiangiogenesis concepts in neuroblastoma therapy.

Pleiotrophin, a candidate gene for poor tumor vasculature and in vivo neuroblastoma sensitivity to irinotecan.

In vivo neuroblastoma (NB) xenograft model, resistant to the DNA-topoisomerase I inhibitor irinotecan (CPT-11), has been established to study resistance mechanisms acquired in a therapeutic setting. Common mechanisms of resistance were not involved in this resistance. Thus, we compared the gene expression profiles of sensitive, resistant, and reverted tumors using cDNA expression arrays. Expression of selected transcripts was confirmed by quantitative real-time PCR. We found that pleiotrophin (PTN), a heparin-binding growth factor, was the only gene significantly affected: PTN gene expression was downregulated in all resistant tumors (8-14-fold) as compared to sensitive tumors, and was increased (2-4-fold) in all reverted tumors as compared to resistant tumors. PTN thus appeared to be a likely candidate gene associated with resistance to CPT-11 in this in vivo model. To investigate the direct implication of PTN in NB, we transfected two NB cell lines with RNA interferences in order to silence PTN. PTN failed to demonstrate implication in resistance to CPT-11 in vitro but could influence sensitivity to CPT-11 exclusively through an in vivo mechanism. Indeed, vasculature was significantly enhanced in resistant NB xenografts compared to sensitive and reverted xenografts, and we suggest that PTN is acting in our resistant in vivo NB model as an angiostatic factor.

[Targeted therapies in pediatric oncology: a new therapeutic approach?]. / Thérapies ciblées en oncologie pédiatrique: nouvelle approche thérapeutique.

A multidisciplinary therapeutic approach has led to significant increase in survival of children with cancer, however often with a high rate of severe sequela. Better understanding in tumor cell biology and transformation process allowed to describe active tyrosine kinases (mainly growth factor receptors) as a new target for cancer treatment. This review presents 2 approaches to target receptor tyrosine kinase activity: on one hand, antibodies that target the extracellular domain, the natural ligand binding site, and on the other hand, small inhibiting molecules, such as imatinib, targeted against the activated intracellular receptor tyrosine kinase. We focus on their clinical development and current application in the treatment of childhood cancer. Targeted therapies are in full rise and new perspectives are explored, such as their association to other treatment modalities and the targeting of microenvironment. This new therapeutic approach necessitates well designed clinical trials that include relevant biomarkers to evaluate its real therapeutic potential.

Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy.

We examined the cytotoxicity of the immunosuppressant agent rapamycin and its analogue CCI-779 in human brain tumor cell lines in vitro and in vivo as single agents and in combination with standard chemotherapeutic drugs. In the rapamycin-sensitive PNET/MB cell line DAOY, rapamycin exhibited additive cytotoxicity with cisplatin and with camptothecin. In vivo, CCI-779 delayed DAOY xenograft growth by 160% after 1 week and 240% after 2 weeks of systemic treatment, compared with controls. Single high-dose treatment induced 37% regression of tumor solume. Growth inhibition of DAOY xenografts was 1.3 times greater after simultaneous treatment with CCI-779 and cisplatin than after cisplatin alone. Interestingly, CCI-779 also produced growth inhibition of xenografts derived from U251 malignant glioma cells, a human cell line resistant to rapamycin in vitro. These studies suggest that the rapamycin analogue CCI-779 is an important new agent to investigate in the treatment of human brain tumors, particularly PNET/MB.

Antitumor activity of a human cytotoxic T-cell line (TALL-104) in brain tumor xenografts.

Malignant glioma in adults and primitive neuroectodermal tumors/medulloblastomas in children are the most common malignant primary brain tumors that either respond poorly to current treatment or tend to recur. Adoptive therapy with TALL-104 cells-an IL-2-dependent, major histocompatibility complex nonrestricted, cytotoxic T-cell line-has demonstrated significant antitumor activity against a broad range of implanted or spontaneously arising tumors. This study investigates distribution of systemically and locally administered TALL-104 cells and their efficacy in effecting survival of a rat model of human brain tumor. In vitro, TALL-104 cells showed significant cytotoxic activity when added to human glioblastoma cell lines U-87 MG, U-251 MG, and A1690; the medulloblastoma cell lines DAOY, D283 Med, and D341 Med; and the epidermoid cancer cell line A431. In brain tumor-bearing rats, the amount of fluorescent dye-labeled TALL-104 cells in brain increased after they were given by intracarotid injection as compared with i.v. cell administration. However, TALL-104 cells rapidly decreased to low levels within 1 h after intracarotid injection. This finding suggests that TALL-104 cells given systemically may not invade brain or tumor tissues, but rather may remain in the vascular system, making this approach less efficient for brain tumor treatment. In a model of athymic rats engrafted with human A431 carcinoma brain tumor, repetitive local administration of TALL-104 cells directly into the tumor bed resulted in a significant increase in survival time compared with control animals. Therefore, local therapy with TALL-104 cells may be a novel and highly effective treatment approach for malignant brain tumors.

Autocrine secreted insulin-like growth factor-I stimulates MAP kinase-dependent mitogenic effects in human primitive neuroectodermal tumor/medulloblastoma.

Primitive neuroectodermal tumors/medulloblastoma (PNET/MB) have similarities to neuroectodermal progenitor cells of the developing CNS. Since insulin-like growth factor I (IGF-I) exerts pleiotrophic effects on cells in the developing CNS, we evaluated the production, mitogenic effects and signaling pathways of IGF-I in PNET/MB cells and found that IGF-I is an autocrine growth factor in human PNET/MB cell lines tested. Stimulation of DAOY cells by IGF-I led to phosphorylation of its cognate receptor (IGF-IR) and resulted in cell proliferation. These effects of IGF-I were suppressed by IGF-IR blocking antibodies and by PD 98059, MAP kinase pathway inhibitor. The results demonstrate the existence of an autocrine IGF-I/IGF-IR loop and indicate that IGF-I promotes proliferation via MAP kinase pathway.

Dose-finding designs in pediatric phase I clinical trials: comparison by simulations in a realistic timeline framework.

OBJECTIVE: Usual dose-finding methods in oncology are sequential. Accrual is suspended after each group of patients to assess toxicity before increasing the dose. An adapted Continual Reassessment Method (CRM) and Rolling 6 (R6) method, designed to avoid this suspension of accrual in pediatric oncology, are compared with the traditional 3+3 design. STUDY DESIGN AND SETTING: The competing performances were evaluated in a simulation study integrating the temporal dimension, and a phase I trial was reanalyzed. We compared methods for various interpatient arrival times and dose-toxicity relations, in terms of distribution of final recommendations, number of skipped children and duration of trials. RESULTS: R6 and CRM can be safely implemented to limit trial suspensions, especially when mean interpatient arrival time is short. CRM was found to be more efficient than algorithm-based methods (44% of good recommendations vs. 38%) but moderately increased the risk of overtreatment. The R6 design included more patients at suboptimal doses. The design with the shortest study duration depended on the number of dose to escalate before the target. CONCLUSION: These new methods can reduce the number of skipped patients, but only provide limited gain in terms of ability to select the right dose. New designs are needed.

Ethical issues in pediatric oncology phase I-II trials based on a mother's point of view.

Phase I-II trials are developing in Pediatrics and raise many complex relational, psychological and ethical issues. We present and discuss these based on an interview in a pediatric oncology setting, with a mother who accepted that her daughter be included in such trials and who expressed why she accepted with great sensitivity and profoundness. She explained that after many years of inefficient treatments she had lost all her landmarks and was ready to accept any proposition, even those she would have considered unacceptable earlier. She did not know whether there is a limit to what is acceptable. Her only objective was to gain any time possible in order to continue living with her daughter. She found it important that the research doctor be different from the doctor involved in patient care, and that the latter remains the major decision-maker and correspondent: thus the child's best interests take precedence over that of research. Interviews with the psycho-oncologist can help the parents and the doctors gain a better insight into the various aspects, rational and irrational, conscious and unconscious, involved in the proposition to participate in a clinical trial and in the parents' or the child's acceptance or refusal.

Dose finding and O6-alkylguanine-DNA alkyltransferase study of cisplatin combined with temozolomide in paediatric solid malignancies.

Cisplatin may have additive activity with temozolomide due to ablation of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (MGMT). This phase I/II study determined recommended combination doses using the Continual Reassessment Method, toxicities and antitumour activity in paediatric patients, and evaluated MGMT in peripheral blood mononuclear cells (PBMCs) in order to correlate with haematological toxicity. In total, 39 patients with refractory or recurrent solid tumours (median age approximately 13 years; 14 pretreated with high-dose chemotherapy, craniospinal irradiation, or having bone marrow involvement) were treated with cisplatin, followed the next day by oral temozolomide for 5 days every 4 weeks at dose levels 80 mg m(-2)/150 mg m(-2) day(-1), 80/200, and 100/200, respectively. A total of 38 patients receiving 113 cycles (median 2, range 1-7) were evaluable for toxicity. Dose-limiting toxicity was haematological in all but one case. Treatment-related toxicities were thrombocytopenia, neutropenia, nausea-vomiting, asthenia. Hearing loss was experienced in five patients with prior irradiation to the brain stem or posterior fossa. Partial responses were observed in two malignant glioma, one brain stem glioma, and two neuroblastoma. Median MGMT activity in PBMCs decreased after 5 days of temozolomide treatment: low MGMT activity correlated with increased severity of thrombocytopenia. Cisplatin-temozolomide combinations are well tolerated without additional toxicity to single-agent treatments; the recommended phase II dosage is 80 mg m(-2) cisplatin and 150 mg m(-2) x 5 temozolomide in heavily treated, and 200 mg m(-2) x 5 temozolomide in less-heavily pretreated children.

Metabolic activity and clinical features of primary ganglioneuromas.

BACKGROUND: Ganglioneuroma (GN) is considered by most to be a benign tumor of neural crest origin. It may evolve from differentiating neuroblastoma or may be diagnosed as primary ganglioneuroma. The rarity of this tumor and the lack of understanding of its biology often lead to inaccurate diagnosis and treatment. METHODS: The authors analyzed clinical features and biologic behavior of primary ganglioneuroma in 49 patients who were registered with but were not part of the national neuroblastoma trials. Data included age and symptoms at diagnosis, gender, tumor localization and size, (123)I-metaiodobenzylguanidine (mIBG) scintigraphy, secretion of catecholamines, histology, treatment, and outcome, whenever available. RESULTS: Patients with primary ganglioneuroma were significantly older than patients with neuroblastoma. Median age at diagnosis was 79 months compared with 16 months (P < 0.0001). Ganglioneuroma were equally distributed between males and females (1.13:1). A preference of thoracic (41.5%) and abdominal, nonadrenal tumors (37.5%) was observed compared with adrenal GN (21%). At diagnosis, thoracic tumors appeared larger than nonthoracic ones. Local lymph node metastases occurred in two patients. One ganglioneuroma had metastasized to soft tissues. (123)I-mIBG scintigraphy detected mIBG uptake at tumor site in 57% of the GN tumors. Levels of catecholamines in plasma and/or urine were increased in 39%. Slight immaturity of ganglion cells was observed in 93% of all ganglioneuroma tumors. None of the 22 tumors analyzed exhibited MYCN gene alterations. Although 12 patients had macroscopic residuals, no tumor progression or recurrence was observed in a median follow-up of 25 months. CONCLUSIONS: Ganglioneuroma may present with metabolic activity such as increased secretion of catecholamines and/or mIBG uptake. There are no specific diagnostic signs or symptoms discriminating ganglioneuroma and neuroblastoma tumors. Therefore, ganglioneuroma requires tissue investigation for diagnosis. Prognosis after surgical resection without further therapy seems to be excellent.

Prognostic significance of Ki-67 (MIB-1) proliferation index in childhood primitive neuroectodermal tumors of the central nervous system.

BACKGROUND: Primitive neuroectodermal tumors (PNET) of the central nervous system, including medulloblastomas, are the most common malignant brain tumors of childhood. Whereas some patients experience prolonged disease control after surgery and adjuvant therapy, others with tumors that appear comparable will relapse and eventually die from progressive disease. PROCEDURE: Because proliferative activity may provide a potential correlate of biologic aggressiveness, PNETs of 78 well-characterized patients were evaluated by Ki-67 (MIB-1) immunohistochemistry. Proliferation indices (PI) were determined by counting Ki-67 (MIB-1) positive tumor cells either in the highest staining region (hot spot PI), or in at least 15 randomly chosen fields (random PI). RESULTS: Twenty-five of 78 PNETs showed amore than twofold higher value of hot spot PI (median 9.3%; range 0.6-56%), compared to random PI (median 5.6%; range 0.2-41.3%), Univariate Cox regression analysis revealed that PNETs with a high hot spot PI had a significantly greater risk of progression and death than PNETs with a low hot spot PI (hazard ratio 1.58, P = 0.04). The hazard ratio remained significant after adjusting for M-stage in multivariate analysis. In contrast to hot spot PI, random PI proved not to be a significant prognostic predictor. CONCLUSIONS: Hot spot PI is a significant and independent prognostic factor in PNETs. Its assessment is uncomplicated, reliable, and may supplement routine histologic examination as a means for improving the accuracy of predicting the biologic behavior of childhood PNETs.

Biological stability of RNA isolated from RNAlater-treated brain tumor and neuroblastoma xenografts.

BACKGROUND: Advances in molecular biological research have led to identification of prognostic factors such as Trk mRNA expression in primitive neuroectodermal tumors of the CNS and neuroblastoma. To study prospectively the importance of these prognostic factors in large groups of homogeneously treated patients, tumor specimens of good quality must be acquired, preserved, and stored at multiple institutions. Immediate freezing of tumor biopsy samples in liquid nitrogen and storage at -70 degrees C are the most commonly used method of tissue preservation for future RNA analysis. PROCEDURE: To evaluate alternative methods of preserving tissue samples for subsequent RNA analysis, we tested a new RNA stabilization solution. Using tumor tissue of two CNS tumor and one neuroblastoma human xenografts, we compared total RNA isolated from tumor tissue stored for 7 days at room temperature in stabilization solution to that of snap-frozen tissue. The quality of the RNA was studied by spectrophotometry, gel electrophoresis, RT-PCR, and gene expression profiling. RESULTS: No major differences were observed in the quality of RNA isolated from tumor samples stored at room temperature in the RNA stabilization solution compared to snap-frozen tumor samples stored at -70 degrees C. CONCLUSIONS: High-quality RNA can be prepared from tumor tissue stored at room temperature. Whenever snap freezing is not feasible, pieces of tumor tissue can be treated with RNAlater for subsequent RNA analysis. Short-term storage and shipment of well-preserved tumor tissue are clearly feasible for all institutions, thereby facilitating large multiinstitutional studies of biological prognostic factors.

Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts.

In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015 (dl1520, CI-1042; ONYX Pharmaceuticals) in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 x 10(8) PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a 'subtherapeutic' dose of 1 x 10(7) PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours.