Enrolled patients were more than 6 months, but less than 18 years of age, with radiologic evidence of recurrent, refractory, progressive or persistent primary brain tumors despite receiving standard therapy. Radiologic evidence of tumor location and size was obtained via magnetic resonance imaging (MRI) performed within 14 days of starting ANP.

Inclusion criteria included a histologically confirmed, primary malignant brain tumor considered to be incurable following standard therapy. Patients were at least 1) four weeks past any surgical therapy, having made a complete recovery; 2) eight weeks past the last dose of radiation therapy (RT); and 3) four weeks past the last dose of chemotherapy (six weeks for nitrosoureas) or immunotherapy. However, patients with clear evidence of disease progression during initial therapy could be enrolled earlier if the investigator had determined that it is safe to administer ANP to such patients. Exclusion criteria included active serious active infections, fever or other medical conditions that would interfere with the evaluation of ANP (i.e., severe heart or lung disease or hepatic failure) Patients with uncontrolled hypertension, a history of congestive heart failure or a history of renal disease were also excluded.

It is generally accepted that the diagnosis of BSG can be made by MRI without the necessity of a biopsy. In this way, 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 included). Tumors that involve less than 50% of the pons or are exophytic in nature were classified as DIPG if they had anaplastic, glioblastoma (GBM) or gliosarcoma (GS) histology [18] [19] [20] [21] . Other types of BSG include focal, exophytic, cervicomedullary and midbrain tumors [22] [23] .

All study patients and/or their legal guardians read, understood, and signed written Informed Consent Documents prior to enrollment. This study was conducted in accordance with the US 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 FDA’s Guidance for Industry. The study was sponsored by the BRI and conducted by the Burzynski Clinic (BC) in Houston, Texas. Patients received ANP free-of-charge.

The study was designed as a single-arm (i.e., no placebo group), two-stage, interventional Phase II trial of ANP as monotherapy in a high-risk, poor-prognosis study population [24] . The study was listed by the National Cancer Institute (NCI), performed under the supervision of an independent Institutional Review Board, the BRI-IRB, and conducted according to Protocol BT-10, which was submitted to the FDA under IND # 43,742. Study enrollment commenced on 9/16/1996 and continued until 7/19/2012. The protocol was amended on occasion by BRI, but none of the amendments altered the study objectives/outcomes or affected patient safety.

The primary endpoint was objective response to ANP, (i.e., complete response (CR) or partial response (PR)). Secondary endpoints included overall survival (OS) and progression-free survival (PFS). OS was measured from the first day of ANP administration until death from any cause. PFS was measured from the first day of ANP administration until the date of first observation of progressive disease (PD) or until death from any cause. The distributions of OS and PFS were estimated by a Kaplan-Meier analysis.

The sample size was calculated based upon the method described by Chang et al. [24] . In this two-stage study, an interim analysis was conducted after 20 patients had been enrolled. Since one or more patients had achieved an objective radiographic response, an additional twenty patients could be recruited. An objective response rate to ANP of ≥ 10% (i.e., four objective responses) was considered “of interest” and sufficient to warrant further study. Because six objective responses were observed after the enrollment of 34 patients, the study was terminated early.

Every four hours, ANP were administered through a dual channel infusion pump and subclavian vein catheter. The details of ANP therapy have previously been described [11] .

Medications that were considered necessary for the patients’ welfare and that did not interfere with the evaluation of ANP were given at the discretion of the investigator. The use of corticosteroids was carefully monitored. Patients received full supportive care when appropriate. Treatment with other antineoplastic or immunomodulatory agents was not permitted.

The initial three weeks of therapy were administered by BC staff on an outpatient basis, in Houston, Texas. Patients and/or their legal guardians were trained by clinic staff to self-administer ANP during this time. Beginning at week 4, ANP therapy was administered at home with 24-hour support from the clinic. At-home therapy and monitoring of the subject’s condition were carried out under the supervision of the subject’s local physician.

Within 14 days of the start of ANP, a gadolinium-enhanced MRI measured all contrast-enhancing lesions. The products of the two greatest perpendicular diameters of all lesions were calculated and totaled, providing a baseline evaluation (“sum”) for each study subject. While tumor measurements were based on the contrast enhanced lesions, tumor size was also measured utilizing T2 and FLAIR images [21] [25] . The details of the required laboratory tests and follow-up studies have previously been published [11] . Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events (CTCAE v.3.0). Pharmacokinetic studies have been carried out in earlier Phase I and other Phase II studies and were not included in the study. Based on these earlier studies there was no expectation of interference with ANP activity by ancillary medications, especially anti-seizure drugs.

Patient enrollment began 9/16/1996 and continued until 7/19/2012. As of 2/17/2015, all patients had been removed from ANP therapy due to a CR, PD, a worsening clinical condition, or subject/legal guardian request.

Thirty-four patients, with a median age of 10.4 years, were enrolled in the study. Thirty-two patients (94.1%), were Caucasians while 21 (61.8%) were female and 13 were male (38.2%).

Eleven patients (32.4%) suffered from BSG, of which nine (26.5%) had a DIPG. Thirteen patients (38.2%) suffered from high-grade tumors group, including five GBM and five anaplastic gliomas (AG), one gliosarcoma (GS), one primitive neuroectodermal tumor (PNET-medulloblastoma) and one supratentorial medulloepithelioma (sPNET).

The low-grade tumor group included ten patients. Three patients suffered from a ganglioglioma (GG) while seven patients suffered from other low-grade tumors, including astrocytoma, pilocytic astrocytoma, oligodendroglioma, optic pathway glioma (OPG), neurocytoma and craniopharyngioma.

There were three cases with leptomeningeal involvement and/or disseminated, multicentric tumors (astrocytoma, pilocytic astrocytoma and ganglioglioma) and one case of a multicentric OPG. Table 1 describes the patient characteristics.

The median dosage of A10 was 11.64 g/kg/d (range, 2.89 - 19.26 g/kg/d) while for AS2-1 it was 0.36 g/kg/d (range, 0.16 - 0.57 g/kg/d). The duration of ANP therapy ranged from 0.2 to 46.6 months with a median of 3.1 months.

The median maximum effective daily dose of A10 in patients with an OR (calculated at the first MRI showing an OR) was 13.92 g/kg/d (range, 6.23 to 21.02 g/kg/d) while the median time to an OR (six patients) was 4.7 months of ANP (range, 1.3 - 7.8 months).

Responses to treatment and survival are summarized in Table 2, which describes the data for all patients as well as for particular subgroups. Six patients obtained an OR (17.6%). There were two CRs (5.9%) and four PRs (11.8%). An additional six patients (17.6%) had stable disease (SD) while 18 patients developed PD (52.9%). Four patients (11.8%) were not evaluable because they elected to discontinue ANP prematurely without having a follow-up MRI to evaluate their response to ANP.

In the DIPG group (n = 9) one patient achieved a CR (11.1%), one achieved a PR (11.1%), one patient had SD (11.1%), and four patients developed PD (44.4%). In the other two patients with a BSG, midbrain one patient achieved a CR (50.0%) and one patient developed PD (50.0%).

In the LG group, two GG patients obtained a PR and one pilocytic astrocytoma patient obtained a PR.

In summary, all ORs occurred in the BSG and LG patient groups. In the BSG group, two of the nine patients with DIPG developed an OR; there was one CR and one PR. Both patients subsequently passed away. The patient who achieved a PR did not show signs of tumor progression and most likely died from an intratumoral hemorrhage. Of the two patients with a BSG, midbrain, one achieved

Note. B―African-American, BSG―brainstem glioma, CH―chemotherapy, DIPG―diffuse intrinsic pontine glioma, GBM―glioblastoma multiforme, HG―high-grade, LG―low-grade, M―Latin American, PNET―primitive neuroectodermal tumor, sPNET―supratentorial primitive neuroectodermal tumor, SU―surgery, RT―radiation therapy, W―Caucasian.

Note. BSG―brainstem glioma, CH―chemotherapy, CR―complete response, D―disseminated, DIPG―diffuse intrinsic pontine glioma, L―leptomeningeal, MC―multicentric, NA―not applicable, OS―overall survival from start, PFS―progression-free survival, PR―partial response, SU―surgery, RT―radiation therapy. Tumor measurements were performed on T1 contrast images.

a CR and is currently alive, having survived 8 and 8 months at last contact. This patient did not receive any treatment before ANP (Figure 1). The other BSG patient failed chemotherapy, received ANP and developed PD. However, after discontinuation of ANP, the patient is alive, having survived over seven years.

In the group of patients with LG, one patient relapsed after two surgical resections, but then achieved a PR following treatment with ANP. This patient is alive, tumor-free, off all treatment, and surviving for more than 15 years at last contact. Two additional patients who relapsed after surgical resection (one patient after two resections) achieved a PR.

Objective response, PFS and OS data for all patients, for BSG, for high-grade tumors, and for low-grade tumors are shown in Table 3. Kaplan-Meier survival curves are shown in Figures 2-5.

The seven LG patients who did not obtain an OR are also alive. Among these surviving patients, there are cases of sPNET and neurocytoma which developed PD while on ANP treatment. After discontinuation of ANP, only one patient with a neurocytoma received chemotherapy. The patient diagnosed with oligodendroglioma is alive and surviving over 16 years at last contact despite maintaining only SD while receiving ANP. Table 4 presents the diagnosis, response to treatment, and OS survival data for the seven LG patients who did not achieve an OR while on ANP.

Safety assessments were based upon the total number of enrolled patients in the study (n = 34). Intense, systematic monitoring of patient safety was conducted during the first two months of therapy and involved daily direct questioning

Note. BSG―brainstem glioma, CR―complete response, N―number, PR―partial response, SD―stable disease.

concerning adverse events, first at the clinic and then followed by phone calls during the home administration. After two months, telephone contact was conducted on a weekly basis. Adverse events were coded and graded according to

Version 3.0 of the Common Terminology Criteria for Adverse Events (CTCAE v 3.0).

Adverse Drug Events (ADEs) included grade 4 hypernatremia (x2); grade 3 hypokalemia (x3); grade 1 headache (x1), somnolence (x1), anemia (x1) and fatigue (x1). No long-term ADE to ANP has been reported.

Brain tumor patients frequently receive corticosteroids to reduce cerebral edema around tumors. The use of corticosteroids and the infusion of large volumes of sodium-containing solutions during ANP therapy predispose patients

to serum sodium concentration abnormalities. Grade 4 reversible hypernatremia, possibly related to ANP was reported in 2 cases (5.8%).