Comprehensive Genomic Profiling of Recurrent Classic Glioblastoma in a Patient Surviving Eleven Years Following Antineoplaston Therapy

Most patients with recurrent glioblastoma (RGBM) die within 6 months regardless of treatment. In phase II studies of Antineoplaston A10 and AS2-1 injections (ANP), our investigators have reported objective responses and long-term survival in RGBM. Using a next-generation sequencing (NGS) based assay of 343 cancer-related genes and introns, comprehensive genomic profiling of tumor tissue obtained from a RGBM patient (who remains alive and well) was performed 11 years after diagnosis and permitted assignment of the patient’s RGBM to the classical subgroup. The most important genomic alterations included amplification of epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A/B), loss of phosphatase and tensin homolog (PTEN) and telomerase reverse transcriptase (TERT) mutation. An analysis of the signaling networks and other targets of ANP therapy was recently performed and presented in this publication. Based on our findings, patients with classical RGBM have a reasonable possibility of responding to ANP therapy and experiencing long-term survival. It is proposed that this subgroup of RGBM patients be enrolled in a genomics-driven clinical trial of ANP therapy.


Patient and Methods
Patient 2 was admitted in September 2004 for ANP therapy, at Burzynski Clinic (BC), under Protocol BT-21 as a Special Exemption patient, which was permitted by the Food and Drug Administration (FDA) based on poor performance status.This phase II study was conducted under IND 43,742, sponsored by Burzynski Research Institute, Inc., and supervised by an independent Institutional Review Board (BRI-IRB).The details of the study were recently published (S.R. Burzynski, Janicki, & G. S. Burzynski, 2014;S. R. Burzynski, G. S. Burzynski, & Janicki, 2014).In December 2014, comprehensive genomic profiling of the patient's tumor tissue was performed by Foundation Medicine, Inc. of Cambridge, MA, using a next-generation sequencing (NGS) based assay of 343 cancer-related genes and introns.The list of the genes is located at www.foundationone.com.The assay development, validation and control procedure was described in detail in a prior publication (Frampton et al., 2013).The clinical genomic profiling assay does not report heterozygosity or homozygosity.The assessment of EGFR and other gene amplification via this assay is done by quantitative estimation of the degree of amplification.Flourescent in situ hybridization (FISH) for gene amplifications relies on a comparison between hybridization to the gene of interest and hybridization to the centrosome, ie the HER2/CEP17 ratio is used to gauge HER2 FISH.The amplification of EGFR is quantitatively estimated as 12x in this case.CDNK2A/B is homozygously deleted in this case (100% of the gene is lost).Frampton et al. (2013) contains a detailed description of the methodology used and its validation for clinical use.The methods for data analysis including pathway analysis were described before (Burzynski & Patil, 2014).The tumor tissue analyzed had been preserved in a paraffin block dated May 26, 2004, which correlates with the patient's second tumor resection.

Case Study
In March 2004, Patient 2, a 59-year-old Caucasian male and retired U.S. Marine Corps Lieutenant Colonel, developed severe headaches with diminished coordination and was found by magnetic resonance imaging (MRI) scan to have a contrast-enhancing tumor in the left frontal lobe.In April 2004, the patient was treated with a near-total tumor resection.Histologic examination of the submitted tumor specimen revealed a GBM.One month later, the patient underwent a second tumor resection and placement of Gliadel wafers.Subsequently, the patient received RT to a total dose of 6,000 cGy in 30 fractions and three cycles of temozolomide (TMZ) followed by isotretinoin.The tumor recurred despite this combination therapy.On the MRI scan, there was a doubling in tumor size compared to the baseline evaluation.With documented failure of the patient's initial therapy, Patient 2 was admitted to BC for ANP therapy under Protocol BT-21 as a Special Exemption patient.ANP therapy began in September 2004 and was interrupted after 7.5 months because of an infection of the patient's infusion catheter.At that time, a positron emission tomography (PET) scan showed no evidence of active tumor and ANP therapy was permanently discontinued.MRI performed one year post ANP therapy revealed an approximate 60% decrease in tumor size.MRI scans four years after discontinuation of ANP therapy confirmed resolution of the tumor.Repeated MRIs through February 2014 have shown no tumor recurrence.This case study was recently published (S.R. Burzynski, G. S. Burzynski, & Janicki, 2014).

Results of Comprehensive Genomic Profiling
In December 2014, comprehensive genomic profiling of the Patient 2's tumor tissue was performed by Foundation Medicine, Inc. of Cambridge, MA, using a NGS based assay of 343 cancer-related genes and introns.The tumor tissue analyzed had been preserved in a paraffin block dated May 26, 2004, which correlates with the patient's second tumor resection.This was a high quality specimen, scored at 78% by computational purity modeling (Figure 1).The most important genomic alterations included epidermal growth factor receptor (EGFR) amplification, phosphatase and tensin homolog (PTEN) mutation, R159fs21, cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A/B) loss, and mutation of telomerase reverse transcriptase (TERT) promoter-124C>T (Table 1).There were no alterations of platelet derived growth factor receptor, alpha (PDGFRA) and isocitrate dehydrogenase 1(IDH1).There were nine additional genetic mutations and one gene rearrangement of unknown significance (Table 1).The comprehensive genomic profile permitted classification of Patient 2's RGBM as the classical subtype (Van Meir et al., 2010;Sturm et al., 2012Sturm et al., , 2014)).Based on the genetic profile, targets for ANP therapy in the patient's RGBM have been proposed (Figures 2 and 3).

Discussion
The most important molecular features of classical GBM are EGFR mutation, amplification or overexpression, PTEN loss or mutation, CDKN2A loss, and Notch homolog protein (Notch) or Hedgehog signaling (Shh) pathway activation (Van Meir et al., 2010;Sturm et al., 2012;Sturm et al., 2014).All of these alterations were seen in Patient 2's RGBM.The significance of these alterations is discussed below, as is the significance of other alterations found in the patient's tumor.
The EGFR gene encodes the epidermal growth factor receptor, a cell surface receptor.Ligands bind to EGFR, producing a chemical message which exerts control on the growth rate of cells.EGFR amplification is identified in 44% to 66% of cases of GBM (Huang et al., 2007;Brennan et al., 2013;Furnari et al., 2015).In GBM, EGFR variant III (EGFRvIII) occurs in 40% of cases.This mutation affects the extracellular domain of the protein and produces a constitutively active receptor (Huang et al., 2007).In a clinical trial with erlotinib, which is an EGFR kinase inhibitor and has sensitivity to EGFRvIII, there was no evidence of efficacy in GBM (Mellinghoff et al., 2005;Bastien et al., 2015).
According to The Cancer Genome Atlas (TCGA) dataset, homozygous deletion of CDKN2A/B occurs in over 50% of GBMs and is associated with a poor outcome (Brennan et al., 2013;Feng et al., 2012).The Cdk4/6 inhibitor palbociclib shows preclinical activity in GBM cell culture (Cen et al., 2012).Theoretically, MDM2 inhibitors would be candidates for clinical trials.
TERT represents a catalytic subunit of the telomerase complex which is necessary for chromosome elongation (Shay & Wright, 2011).TERT promoter mutations, as seen in Patient 2's GBM, are found in over 80% of GBMs (Liu et al., 2013;Nonoguchi et al., 2013).EGFR amplification is frequently associated with TERT promoter mutation and is a poor prognostic factor in GBM (Nonoguchi et al., 2013).Validated therapeutic options for targeting mutated TERT in GBM are not currently available.
The STAT3 pathway is among the most promising targets for cancer therapy (Hua et al., 2014).GBM with increased signaling along the STAT3 pathway has a more aggressive behavior (Sturm et al., 2014).Inhibition of this pathway resulted in tumor progression in preclinical studies (Carro et al., 2010).STAT3 induces methylation of the CDKN2A promoter which silences this important tumor suppressor (Lee et al., 2012).Activating tyrosine-protein phosphatase non-receptor type 11 (PTPN11) mutants inhibit STAT3 (Zhang et al., 2009).Rearrangement of PTPN11 was seen in Patient 2's GBM (Table 1).
The comprehensive genomic profile of Patient 2's GBM suggests a central role of signaling pathways, PI3K/AKT and RAS, originating from an amplified EGFR and enforced by HGF/mesenchymal epithelial transition factor (c-MET) and G-protein-coupled receptors (GPCR)/GNAS.The tumor suppressor system may be compromised by the mutation of PTEN and MAG12.The STAT3 pathway contribution to disease progression may be due to lack of down-regulation by rearranged PTPN11.Another important pathway that appears compromised is CDKN4/6/cyclin/Rb, through the loss of CDKN2A/B and possibly through mutations of BLM, ARID1B and MLL3.
Based on analyses of the effect of ANP therapy on the GBM genome, we propose that the main inhibitory effects of ANP therapy are directed against the PI3K/AKT and RAS pathways through the inhibition of AKT and RAS, the up-regulation of PTEN, and restoration of the G1-S checkpoint (Figure 2) (Burzynski SR, Burzynski GS, Janicki TJ, 2014;Burzynski & Patil, 2014).Another important mechanism is promotion of apoptosis mediated through p53.Inhibition of GCS can be explained by the effect of ANP therapy on AKT and PTEN.
While comprehensive genomic profiling constitutes an exciting development in the diagnosis and treatment of GBM, incomplete data on the extremely complex GBM genome are obtained.This article has discussed approximately 2% of the genetic variants in the GBM genome.Additional genetic variants and important pathways in GBM that are affected by ANP therapy have been previously described, including promotion of neoplastic growth and invasion, cell cycle control, cellular metabolism, autophagy, GCS and improved tumor penetration.(Burzynski SR, Burzynski GS, Janicki TJ, 2014;Burzynski & Patil, 2014).It is important to notice that there are many variants of unknown significance listed in Table 1 and the explanation of their effects remains hypothetical.The alterations have been previously observed in peer-reviewed literature or the database Catalogue of Somatic Mutations in Cancer (COSMIC).

Conclusion
Large scale genomic and epigenomic analyses have permitted classification of GBM into four major subgroups, each with a different genomic profile.Each of these subgroups may require a different therapeutic approach.Under IND 43,742, Phase II studies of ANP therapy for GBM started in 1995 when comprehensive genomic profiling was not practiced.Among the GBM patients treated are long-term survivors (up to 20 years survival).
Through the initiative of Patient 2's local neuro-oncologist, comprehensive genomic analysis was performed to determine the genetic profile that had responded favorably to ANP therapy.From that analysis, we propose that patients with classical RGBM have a reasonable possibility of responding to ANP therapy and experiencing long-term survival.It is proposed that this subgroup of RGBM be enrolled in a genomics-driven clinical trial of ANP therapy.

Figure 1 .
Figure 1.HE staining representing the area used for comprehensive genomic profiling