icnct-16, june 2014, helsinki glioma heterogeneity and the l-amino acid transporter-1 (lat1): a...

ICNCT-16, June 2014, Helsinki

Glioma heterogeneity and the L-Amino acid transporter-1 (LAT1):

A first step to stratified BPA-based BNCT?

D. Ngoga1; C. L. Schütz1; A. Detta1; S. Green2; G. Cruickshank1

1University of Birmingham, School of Cancer Sciences, Queen Elizabeth Hospital, Department of Neurosurgery

2University of Birmingham, Queen Elizabeth Hospital, Department of Medical Physics

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

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Patterns of gene expression have been collated to identify molecular subgroups with putative prognostic or predictive significance.

Glioma Genetics

Verhaak, et al. 2010

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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 IDH1R132 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

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Found in majority of LGG

Confers a significantly improved prognosis.

Isocitrate Dehydrogenase – 1 mutation

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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

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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

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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

B

OH

HO NH2

O

OH

BPA based BNCT

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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)

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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

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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

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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 groupUp-Regulated: 96Intermediate: 247Log-rank p-value:Up-Regulated vs. Intermediate: 9.322837E-4

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Number of samples in group:Up-Regulated: 14

Intermediate: 36

Log-rank p-value

Up-Regulated vs. Intermediate: p=0.0062


Kaplan-Meier Survival Plot for Samples with Differential SLC3A2 Gene Expression (Oligodendroglioma)

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Results 1 – SLC7A5 (Light chain)




Number of samples in group:Up-Regulated: 8Down-Regulated: 30 Intermediate: 305

Log-rank p-valueUp-Regulated vs. Down-Regulated: 0.073

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Results 1 – SLC7A7 (y+LAT1)




Number of samples in group:Up-Regulated: 192Intermediate: 149Down-Regulated: 2Log-rank p-valueUp-Regulated vs. Intermediate: 2.16x10-8

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Number of samples in group:Up-Regulated: 51Intermediate: 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)

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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

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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

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Results 2 – SLC7A5 (Light chain)

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Results 2 – SLC7A7

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Results 2 – IDH1 status

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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

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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

Thank you for your attention

Further questions? [email protected]

icnct-16, june 2014, helsinki glioma heterogeneity and the l-amino acid transporter-1 (lat1): a...

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