1. ICNCT-16, June 2014, Helsinki Glioma heterogeneity and the L-Amino acid transporter-1 (LAT1): A first step to stratified BPA-based BNCT? D. Ngoga 1 ; C. L. Schütz 1 ; A. Detta 1 ; S. Green 2 ; G. Cruickshank 1 1 University of Birmingham, School of Cancer Sciences, Queen Elizabeth Hospital, Department of Neurosurgery 2 University of Birmingham, Queen Elizabeth Hospital, Department of Medical Physics

2. 2 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  To date, glioblastoma subject to the most extensive genomic profiling of any cancer (Dunn, et al Genes Dev. 2012 26: 756-784)  The Cancer Genome Atlas Research Network (Parsons et al. 2008) and other studies have enabled us to systematically and comprehensively define the genomic landscape of glioblastoma.  Malignant gliomas are characterized by genetic instability and complex alterations in:  Chromosome structure  Copy number.  Variation in post translational modification. Glioma Genetics

3. 3 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Patterns of gene expression have been collated to identify molecular subgroups with putative prognostic or predictive significance. Glioma Genetics Verhaak, et al. 2010

4. 4 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Perhaps the most important of the discoveries arising from the TCGA studies.  IDH-1 cytosolic component of the glycolytic pathway (Bleeker, et al. 2009)  A spontaneous mutation in the gene for IDH1 R132 identified in  12% of patients GBM.  12%–17% of AML (Mardis et al. 2009)  Majority of central and periosteal cartilaginous tumors (Amary et al. 2011)  23% of cholangiocarcinomas (Borger et al. 2012) Isocitrate Dehydrogenase – 1 mutation

5. 5 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Found in majority of LGG  Confers a significantly improved prognosis. Isocitrate Dehydrogenase – 1 mutation

6. 6 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  The DNA repair enzyme 0-6-methylguanine-DNA methyltransferase (MGMT) is responsible for reversing the damage caused by temozolomide.  Methylation of the promoter region encoding MGMT has a critical role in patients response to Temozolomide.  Only ~ 50% of patients with GBM have methylated MGMT. (Esteller, et al. 2000)  The remainder of patients with unmethylate, IDH WT tumours continue to have have poor prognosis. Methylation and treatment response

7. 7 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Understand how BNCT fit in this new framework of understanding of GBM biology?  Understand and explain differences in treatment response to BNCT.  Understand the impact of glioma heterogeneity on Boron uptake and therefore the effectiveness of BNCT. The challenge for BNCT

8. 8 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga 4-dihydroxyboryl-L-phenylalanine (BPA) no selectivity/specificity, but increased AA need (  LAT1) low toxicity, resistance in tumor cells, low background coupling to fructose / mannitol to increase solubility uptake mostly regulated via amino acid transporter 4F2hc/LAT1 BPA based BNCT

9. 9 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga 4F2hc/LAT1 - Structure heavy chain (also called CD98) light chain The monomers alone do not function as transporter! SLC3A2 has been shown to interact with SLC7A7 ( Pfeiffer, R;et al 1999)

10. 10 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  To understand role of LAT1 genetics in glioma  To determine the influence of LAT1 gene expression of patient survival.  Understand the interaction between LAT1 gene expression and know mutations relevant to GBM (IDH1 mutation) Study aims

11. 11 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Interrogate publically available gene expression databases (REMBRANDT)  Genes encoding the constituent proteins of the LAT1 transporter (SLC3A2 and SLC7A5) as well as related transporter SLC7A7 were analysed. Study Method 1

12. 12 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 1 – SLC3A2 (Heavy chain) Gene Expression Plot (SLC3A2) ALL GLIOMA (454) ASTROCYTOMA (148) GBM (228) MIXED: (11) NON_TUMOR: (28) OLIGODENDROGLIOMA: (67) UNKNOWN: (67) Kaplan-Meier Survival Plot for Samples with Differential SLC3A2 Gene Expression (All Glioma) Number of samples in group Up-Regulated:96 Intermediate:247 Log-rank p-value: Up-Regulated vs. Intermediate: 9.322837E-4

13. 13 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Number of samples in group: Up-Regulated:14 Intermediate:36 Log-rank p-value Up-Regulated vs. Intermediate: p=0.0062 Results 1 – SLC3A2 (Heavy chain) Kaplan-Meier Survival Plot for Samples with Differential SLC3A2 Gene Expression (Oligodendroglioma)

14. 14 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 1 – SLC7A5 (Light chain) Gene Expression Plot (SLC7A5) ALL GLIOMA (454) ASTROCYTOMA (148) GBM (228) MIXED: (11) NON_TUMOR: (28) OLIGODENDROGLIOMA: (67) UNKNOWN: (67) Kaplan-Meier Survival Plot for Samples with Differential SLC7A5 Gene Expression (All Glioma) Number of samples in group: Up-Regulated:8 Down-Regulated: 30 Intermediate:305 Log-rank p-value Up-Regulated vs. Down-Regulated: 0.073

15. 15 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 1 – SLC7A7 (y+LAT1) Gene Expression Plot (SLC7A7) ALL GLIOMA (454) ASTROCYTOMA (148) GBM (228) MIXED: (11) NON_TUMOR: (28) OLIGODENDROGLIOMA: (67) UNKNOWN: (67) Kaplan-Meier Survival Plot for Samples with Differential SLC7A7 Gene Expression (All Glioma) Number of samples in group: Up-Regulated:192 Intermediate:149 Down-Regulated:2 Log-rank p-value Up-Regulated vs. Intermediate:2.16x10 -8

16. 16 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Number of samples in group: Up-Regulated:51 Intermediate:54 Log-rank p-value(for significance of difference of survival between group of samples) Up-Regulated vs. Intermediate: 6.68x10 -4 Kaplan-Meier Survival Plot for Samples with Differential SLC7A7 Gene Expression (Astrocytoma) Results 1 – SLC7A7 (y+LAT1)

17. 17 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  SLC3A2  Increased expression in GBM compared with normal brain and Low grade glioma  Up-regulation is associated with a significantly worse prognosis when considering all glioma  Particular prognostic significance in oligodendroglioma.  SLC7A5  Expression not upregulated in GBM  increased expression possibly associated with better prognosis (Not significant)  SLC7A7  Increased expression in GBM compared with normal brain and Low grade glioma  Particular prognostic significance in astrocytoma. Summary – Results 1

18. 18 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Identified patients treated at the Queen Elizabeth Hospital, Birmingham for low grade glioma who had an overall survival of > 5 years (Long survival) and <5 years (short survival).  RNA extraction from paraffin embedded tumour samples  Performed whole exome sequencing (Oxford Gene Technology)  Correlated gene expression of SLC3A2, SLC7A5 and SLC7A7 to patient survival.  Identify if there was any association of LAT1 gene expression with IDH1 mutation. Study Method 2

19. 19 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 2 – SLC3A2 (Heavy chain)

20. 20 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 2 – SLC7A5 (Light chain)

21. 21 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 2 – SLC7A7

22. 22 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga Results 2 – IDH1 status

23. 23 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  SLC3A2  Increased expression in these grade 2 glioma samples did not appear to influence survival.  SLC7A5  Increased expression in was associated with a significantly worse prognosis. (p = 0.001)  SLC7A7  Increased expression in was associated with a significantly worse prognosis. (p = 0.016) Summary – Results 2

24. 24 Glioma heterogeneity and the LAT-1: A first step to stratified BPA-based BNCT? ICNCT-16, Helsinki, June 2014 / Desire Ngoga  Though numbers of samples were small, we were able to show the significant impact of the gene expression of the LAT1 transporter on the treatment and survival of glioma patients.  The fact that transporter up-regulation is associated with IDH1 wild type patients and a significantly worse prognosis offers the potential to stratify these patients for BNCT Discussion

25. Thank you for your attention Further questions? d.g.ngoga@bham.ac.ukd.g.ngoga@bham.ac.uk