As described before, recruited patients were over 3 years of age and had radiologic evidence of brainstem glioma by gadolinium-enhanced MRI performed within 14 days before initiating the study [13] . The diagnosis made by an MRI without the necessity of a biopsy is generally accepted when the following criteria for DIPG are used. Based on MRI only, DIPG can be diagnosed if the tumor has an epicenter in the pons and involves more than 50% of the pons. Patients with neurofibromatosis are not covered by this definition and were not included. The tumors involving less than 50% of the pons or exophytic infiltrating the pons were classified as DIPG if they had anaplastic or GBM histology [3] [20] [21] . The remaining types of brainstem gliomas included focal pontine, exophytic, cervicomedullary, and midbrain tumors [22] [23] .

Eligibility criteria included a Karnofsky Performance Status (KPS) of 60 - 100. The details of the eligibility criteria and the removal from the study were previously published [13] .

All study subjects and/or guardians read, understood, and signed written informed consent prior to enrollment. This study was conducted in accordance with the U.S. Code of Federal Regulations, Title 21, Parts 11, 50, 56, and 312; the Declaration of Helsinki (1964) including all amendments and revisions; the Good Clinical Practices: Consolidated Guideline (E6); International Conference on Harmonization; and the Food and Drug Administration (FDA) Guidance for Industry. The study was sponsored by the Burzynski Research Institute, Inc. (BRI) and conducted by the Burzynski Clinic (BC) in Houston, Texas. The patients did not pay for the investigational agents.

The study was designed as a single-arm, two-stage, interventional Phase II trial of ANP as monotherapy in a high-risk, poor-prognosis study population. The study was listed by the National Cancer Institute (NCT000034 59). It was supervised by the independent Institutional Review Board (BRI-IRB, BC-BT-11).

The study was performed according to Protocol BT-11 which was submitted to the FDA under the IND 43,742. The study commenced on March 13, 1996, and closed to accrual on January 10, 2007. Subsequently, the protocol was amended by BRI several times; however, none of the amendments altered the aim or design of the original study objectives/outcomes. Initial protocol excluded adult patients, but based on the FDA’s communication on May 15, 1996, the admission criteria were amended to also accept adults.

The study design called for initial 20 patients to be accrued followed by an additional 20 patients. A total of 40 patients were enrolled to the study, as required and statistically significant results were reported to the FDA. Subgroups RPDIPG and NDIPG were analyzed separately because these were the only homogenous groups in the entire brainstem glioma population of the study.

The sample size was calculated based upon the previously used method described by Chang et al. [24] . Overall survival (OS) was measured from the first day of ANP administration until death from any cause. The distributions of survival were estimated by Kaplan-Meier analysis.

ANP A10 and AS2-1 were delivered via a dual-channel infusion pump and single-lumen subclavian catheter (Broviac or Groshong) every 4 hours. The effective dose was defined as the universal dosage which in previous studies was associated with an objective response (OR); (CR + partial response (PR)). The subject continued the daily administration of 6 doses of A10 and AS2-1 (every 4 hours via automated pump) until OR was determined. The subject was then advised to continue treatment for at least eight months after an OR was documented. ANP treatment was stopped at the patient’s request or if their clinical condition worsened.

The details of the administration of ANP and additional medications, including corticosteroids, were published before [12] [13] .

The products of the two greatest perpendicular diameters of all lesions measured on MRIs were calculated and totaled, providing a baseline evaluation for each study subject. The overall tumor size was also measured including T2 and FLAIR images [21] [25] . The evaluation criteria were described before [13] .

During the study the generally accepted criteria for evaluation of responses in glioma changed toward reliance on OS rather than tumor responses. As a result, the protocol was amended to include also survival analysis.

Blood, urine, and radiological tests were conducted as described before [13] . Toxicity was evaluated in all patients enrolled (NDIPG) and graded according to the Common Terminology Criteria for Adverse Events, Version 3.0 (CTCAE v3.0).

Subject enrollment started on March 13, 1996, and continued through January 16, 2007. The total of 40 patients was accrued in the study, but one of them did not meet eligibility criteria. This patient was initially thought to have midbrain and thalamic tumor, but was later confirmed as thalamic anaplastic astrocytoma.

The 39 BSG candidates who met eligibility criteria had a median age of 11.7 years old. The distribution between the genders was 44% female and 56% male, and the majority of subjects (84.7%) were Caucasian.

Patient demographics did not change during the study and were comparable to the other studies conducted on brainstem glioma.

The most important patient subgroup within the safety or efficacy study involved 17 children with RPDIPG. The results of treatment of these patients were published before [13] .

Among the second group of patients who were diagnosed with NDIPG, there were four cases of focal pontine and four cases of cervicomedullary tumors, two cases of midbrain and one exophytic BSG. The results of treatment of this group are described in detail in this paper.

The remaining patients were subdivided into two groups: DIPG Non-recurrent Pediatric Patients (N = 7) and DIPG in Adult Patients (N = 4). These two groups were too small for statistical evaluation and were not described in this paper.

Table 1 shows characteristics of 28 patients divided into the two main patient subgroups: RPDIPG and NDIPG. Table 2 summarizes confirmation of diagnosis, recurrence and responses. The results of the treatment of the RPDIPG patients were published before, but are shown here again for the reader’s convenience [13] .

Note: This group of BSG patients includes only RPDIPG and NDIPG. A―African American; AT―at the end of treatment; BSG―brainstem glioma; BT―at treatment start; C―Caucasian; CH―Chemotherapy; DIPG―diffuse intrinsic pontine glioma; H―Hispanic; HO―Hormonal; NDIPG―non-DIPG; O―Oriental; RPDIPG―recurrent pediatric DIPG; RT―radiation therapy; SU―surgery.

Note: Pediatric recurrent DIPG patients 1 - 17 were previously reported in the article entitled “The response and survival of children with recurrent diffuse intrinsic pontine glioma based on phase II study of antineoplastons A10 and AS2-1 in patients with brainstem glioma” in Child’s Nervous System 2014; DOI 10.1007-S00381-014-2401-Z. Note: AA?anaplastic astrocytoma; A2?astrocytoma Grade 2; ANP?antineoplastons; C?chemotherapy,; CR?complete response; CRR?central radiology review; DIPG?diffuse intrinsic pontine glioma; HO?hormonal; MRI?magnetic resonance imaging; NA?not applicable; NE?not evaluable; PA?pilocytic astrocytoma; PD?progressive disease; PET?positron emission tomography; RT?radiation therapy; S?surgery; SD?stable disease.

The median daily dose of ANP A10 for NDIPG (11 patients) ranged from 2.79 to 13.23 g/kg/d with a median of 8.58 g/kg/d. For AS2-1, the median daily dose was 0.30 g/kg/d, with a range of 0.23 to 0.49 g/kg/d.

The median daily dose of A10 in subjects until OR was observed was 9.23 g/kg/d; (range 7.18 to 10.72 g/kg/d) and the median daily dose of AS2-1 was 0.30 g/kg/d; (range 0.23 to 0.34 g/kg/d). The median time to first OR was 20.14 weeks (range 15 to 36 weeks).

In the group of subjects with a SD response, the median daily dose of A10 was 14.31 g/kg/d (range 4.50 to 21.01 g/kg/d) and the median daily dose of AS2-1 was 0.39 g/kg/d (range 0.30 to 0.50 g/kg/d). The duration of IV ANP therapy ranged from 19 to 102 weeks with a median of 44 weeks.

All CR patients from the NDIPG group were eligible for a maintenance oral treatment of A10 and AS2-1 capsules following the ANP intravenous (IV) therapy.

Responses to treatment are summarized in Table 3. The table shows the data for all eligible RPDIPG and NDIPG populations.

In the RPDIPG group (N = 17) there were five cases of OR (29.5%); one CR (6%) and four PR (23.5%), three patients (17.5%) had stable disease (SD), seven patients (41%) developed PD and two cases (12%) were non-evaluable (NE) (no follow-up MRI).

In the NDIPG group (N = 11), there were four cases of CR (36%), three cases of SD (27.5%), one case of PD (9%) and three NE cases (27.5%) (Figure 1).

The Kaplan-Meier overall survival curve for the NDIPG group is shown in Figure 2.

Estimated overall survival for the NDIPG group at 1, 5, 10 and 15 years is 82%, 73%, 62% and 50% correspondingly to March 3, 2015. The data on 6 patients who survived from over 8 to 18 years are shown in Table 4. One of these patients died, but there were 6 patients currently surviving in very good condition from the commencement of treatment from 8 to over 18 years.

Three patients diagnosed with focal pontine and midbrain tumors are surviving tumor-free and in excellent condition from over 14 to 18 years from the treatment start. One of these patients delivered two healthy children. An additional patient diagnosed with a cervicomedullary tumor underwent tumor resection in 2000 and lives a normal tumor-free life at present. Another patient with focal pontine glioma, whose response was classified as SD, still has the tumor, but is asymptomatic and surviving in excellent condition. Patient ID 20 was initially diagnosed with von Hippel-Lindau (VHL) disease and multifocal pontine glioma. Molecular genetic studies (MGS) revealed the complete deletion of one allele of the VHL gene. He obtained OR after five months of ANP treatment. A repeat MGS, after three months of treatment, did not show a detectable lesion or point mutation in the VHL gene. Five years later, his local oncologist was of the opinion that the patient developed central nervous system (CNS) lymphoma, for which there was no pathology confirmation. Subsequently, he was treated with chemotherapy and passed away 10 years after the treatment start with ANP.

Note: CR?complete response; N?number; NDIPG?non-DIPG; NE?non-evaluable; PD? progressive disease; PR?partial response; RPDIPG?recurrent pediatric DIPG; SD?stable disease.

Safety assessments were analyzed based upon the total number of NDIPG patients in the study (N = 11) and are presented in Table 5 [13] .

A profile of grades 3 and 4 adverse drug events (ADEs) identified and reported by the patients in this study, in comparison to the study with temozolomide, is shown in Table 6 [26] . Brain tumor patients frequently receive corticosteroids as part of their therapeutic regimen to reduce cerebral edema around tumors. The use of corticosteroids, the infusion of large volumes of sodium-containing solutions during ANP therapy, and the brain tumor itself predispose a patient to an increased incidence of serum sodium and potassium concentration abnormalities. As a result, Grade 4 hypokalemia (2.4 mmol/L) was reported in 1 patient (9%), but there was no Grade 3. No long-term ADEs to ANP were reported.