1. Who benefits most of BNCT? –
A review on literature data on the prognostic value of protein expression of amino acid transporter 4F2hc/LAT1
C. L. Schütz1; D. Ngoga1; 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
ICNCT-16, June 2014, Helsinki 1

2. Patient selection for clinical trials
Location, dimension and stage of lesions: Historically in BNCT mostly glioma, H&N tumours and melanoma - Application of a neutron beam is comparably easy
Outcome of BNCT is unsystematic within many cohorts, the effectiveness of the applied protocols varies: While certain patients, even so-called lost cases, show complete response, other do not respond at all
Do we know the reason for the very different outcome?
Could we select patients accordingly before therapy?
To date the selection of patients for BNCT has been based on anatomical and technical constreints of delivering the therapy
Outcomes have in some cases been excellent but in others inexplicably variable.
Can we use tumour bology to explain these outcomes?
Can we begin to stratify patients and offer treatment to those that would benefit most?

3. Targeting and boron compounds

4. intercalation/binding
Targeting
Receptors/Integrins
hormones
vitamins
growth factors …
Oxygenation
nitromidazoles
Proliferation (DNA)
Transporters
nucleosides
intercalation/binding
carbohydrates
amino acids
vitamins
In the search for a targeted therapy, has focused in on a number of pathways
In the case of BNCT, both compounds currently in clinical use depend on transporter systems to target cancer cells.
Angiogenesis
Metabolism
enzyme substrates
carbohydrates
amino acids
cRGD-peptides

5. Clinically relevant boron compounds
no selectivity / specificity
lipophilic / crossing BBB
very high boron loading
enhanced permeability and retention effect (epr-effect)
protocols are well-established
ability for coupling chemistry
conjugates with carbohydrates
BSH is well known to many in the audience.
It has a number of advantages.
Na mercapto-undecahydro-
closo-dodecaborate (BSH) 5

6. Clinically relevant boron compounds
4-dihydroxyboryl-L-phenylalanine (BPA)
no selectivity/specificity, but increased AA need ( LAT1)
low loading  high blood levels of compound needed
low toxicity, resistance in tumor cells, low background
coupling to fructose / mannitol to increase solubility
uptake mostly regulated via amino acid transporter 4F2hc/LAT1
Likewise BPA is a well established compound in the context of BNCT
Crucially its uptake is mostly regulated via LAT1

7. Why is BPA-BNCT effective in tumour therapy?

8. Amino acid transporters
Amino acid transport is facilitated by a number of transport proteins, several of those being heterodimers – like 4F2hc/LAT1, an obligate exchanger, whose main role is to regulate amino acid transport in growing cells and across some endothelial / epithelial barriers
LAT 1 is a heterodimeric obligatory exchanger comprised of the LAT1 light chain and the 4F2hc heavy chain encoded by the genes: SLC7A5 and SLC3A2.

9. heavy chain (also called CD98)
4F2hc/LAT1 - structure
heavy chain (also called CD98)
light chain
The monomers alone do not function as transporter!

10. Mechanism and specificity of LAT1
Unidirectional amino acid transport in combination with the LAT exchanger
LAT1 shows high affinity for bulky, neutral amino acids like phenylalanine
It interacts in a complex system of neutral AA transporters

11. Substrates of LAT1 >> >
To act as substrate for LAT1, an amino acid must have a free -NH2 and –COOH group with a certain bonding angle and charge, without the C=O participating in hydrogen bonding (Uchino et al., 2002)
Size and branching of side chain is directly related to substrate affinity, most probably due to hydrophobic interaction with binding site
α-L-amino acids are preferred by both LAT1&2, though not obligatory
>>
>
Specific substrate conditions are necessary for binding to LAT1
Free Amine (1) and carboxyl groups
Charge, (1&4)
Bond angle and hydrophobicity
Substrate recognition increases with hydrophobicity

12. Substrates of LAT1: Phe derivatives
Phenylalanine
p-[10B]borono-phenylalanine
L-p-hydroxiphenylalanine / L-tyrosine
2-[18F]fluoro-L-tyrosine (FTyr)
L-[3-18F]‑α‑methyltyrosine (L-[18F]FAMT)
L-3,4-dihydroxiphenylalanine / L-DOPA
3-O-methyl-6-[18F]fluoro-L-DOPA (18F-OMFD)
Phenylalanine and its derivatives therefore make ideal substrates for LAT1
2-amino-bicyclo[2.2.1]-2-carboxylic acid (BCH)
Gabapentin
L-Thyroxine

13. The role and potential of 4F2hc/LAT in tumour therapy

14. What do we need for clinical success?
A suitable neutron source providing the right beam or field
A non toxic, highly selective boron compound
The biological effectiveness of the applied boron compound in combination with neutron irradiation must be proven and detailed knowledge about the pharmacokinetics and uptake behaviour must be given
Online dose monitoring and monitoring of blood or extracellular boron levels during treatment
Selection of the right patients

15. Protein expression of LAT1 in esophageal carc.
Normal esophageal mucosa, esophageal carcinoma (all paraff. human tissue), Kobayashi et al., Journal of Surgical Oncology 2005;90:233–238
LAT1 expression according to histopathological grade. LAT1 ratio in G1 (well differentiated), G2 (moderately differentiated), and G3 (poorly differentiated) tumors

16. Confirmed protein expression of LAT1 in H&N
Normal esophageal mucosa, esophageal carcinoma (all paraff. human tissue), Kobayashi et al., Journal of Surgical Oncology 2005;90:233–238
LAT1 expression according to depth of invasion
LAT1 expression according to stage

17. Prognostic value of LAT1: Non small cell lung ca
Non adeno
Stage I
Stage IA
Stage IB
At all stages, survival is significantly worse with increased expression of LAT1

18. Prognostic value of LAT1 alone – Pancreatic Ca
Though Proliferative index (Ki67) and tumour grade were also predicitve of survival,
LAT1 expressionwas an independent predictor.
LAT1 prognostic in pancreatic ductal adenocarcinoma (PDAC)
In this study: no correlation to Ki-67

19. Prognostic value of 4F2hc/LAT1 together
...in transitional cell carcinoma
Urothelial ca
Worse survival with increased expression of LAT1 in combination with 4F2hc (Heavy chain)

20. Prognostic value of 4F2hc/LAT1 – Adenoid cystic Ca.
Adenoid cystic carcinoma (Head and neck)
Overall survival
Progression free survival

21. Prognostic value of 4F2hc/LAT1: Tripple negative breast Ca.
(part 3)
4F2hc/LAT1 together have more prognostic significance than LAT1 alone!

22. LAT1 vs 4F2hc(CD98): prognostic value of LAT1
LAT1 is a prognostic factor for a considerable number of tumours (including glioma, NSCLC, SCLC, SCC and neuro-endocrine tumours of the lung, esophageal cancer, prostate cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, triple negative breast cancer, and transitional cell carcinoma), but not for all that have been tested!
Statistical correlation of outcome and progression with the whole transporter (4F2hc/LAT1) is usually higher than with LAT1 alone
The correlation to ki-67, VEGF and CD31/34 is random and was never proved through double staining, only statistical evidence
Most samples were obtained from patients before/without radiotherapy or chemotherapy and usually before surgery

23. The potential of BPA-BNCT – if offered to the right patient

24. The potential of BPA-BNCT
Uptake of BPA is mostly regulated by 4F2hc/LAT1, an obligate exchanger regulating the transport of large neutral amino acids
4F2hc/LAT1 protein expression can be used for the prognosis of the disease for several kinds of tumours (including glioma, NSCLC, SCLC, SCC and neuro-endocrine tumours of the lung, esophageal cancer, prostate cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, triple negative breast cancer, and transitional cell carcinoma).
BPA-BNCT has a demonstrated potential for therapeutic efficacy and benefit even for progressed diseases, possibly because of 4F2hc/LAT1 expression
4F2hc/LAT1 expression levels offer an opportunity for stratified BNCT and a means of trgeting patients for whom conventional therapy is inneffective.

25. Implications of high LAT1 expression
Could we conclude…?
The more aggressive the tumour
> LAT1 expression
> BPA-uptake?
Selection of patients?
> Efficacy of BNCT?

26. Thank you for your attention
Further questions? // 26