Cancer chemotherapy

Certain SNP's correlate with the toxicity conferred by chemotherapeutic agents. The Geneblitz® genotyping service is pleased to offer genetic testing for the following genes, with their cognate chemotherapeutic.

Gene	Concentration needed:
Dihydro pyrimidine dehydrogenase (DPD)	5-Flurouracil (5-FU)
Thymidylate synthase (TS)	5-Flurouracil (5-FU)
Methylene tetrahydrofolate reductase (MTHFR)	Methotrexate (MTX)
Thiopurine methyl transferase (TPMT)	6-Mercaptopurine (6-MP)

DPD and 5-FU

Upon administration, 5-FU is normally metabolised by DPD. Polymorphisms in DPD, most notably the splice site SNP IVS14+1G>A can reduce the activity of this enzyme.

Between 80 and 90% of the drug is degraded by DPD and if present in reduced amounts, e.g. due to polymorphism then lowered levels of DPD can have two effects:

a) a decrease in 5-FU clearance and a consequential increase in exposure to the drug results in toxicity
b) an increase in the efficiency of 5-FU in the tumour.

We offer a genotyping assay for the splice site SNP in DPD as indicated above.

TS and 5-FU

If not metabolised by DPD, 5-FU is converted to 5-flurodeoxuridine, an inhibitor of TS. Certain repeat sequences in the promoter region of the TS gene correlate with clinical outcome, with fewer repeats resulting in decreased expression of TS. As before, this will increase the toxicity due to 5-FU and will increase the efficacy of the drug in the tumour issue.

We offer a genotyping assay for the number of repeats in the TS promoter.

MTHFR and MTX

MTX toxicity occurs in patients with a homozygous polymorphism (C677T) in MTHFR gene. This polymorphism occurs in around 10 to 12% of the population and reduces the activity of the MTHFR enzyme which in turn increases the potency of MTX. Genotype data may allow the use of reduced doses of antifolates and hence reduce toxicity.

We offer a genotyping assay for the C677T polymorphism in MTX.

TPMT and 6-MP

The anti leukaemia 6-MP is activated in vivo to 6 thioguanosine in part using a pathway involving TPMT. Polymorphisms in TPMT correlate with reduced TPMT activity, increased 6-MP levels and 6-MP induced toxicity. When genotype data is applied to dosing regimens, reduced dosing allows TPMT deficient patients to tolerate 6-MP therapy without toxicity.

We offer genotyping assays for the following spectrum of TPMT SNP's:

TPMT designation	Nucleotide	Protein
*2	238 G to C	Ala 80 Pro
*3A	460 G to A	Ala 154 Thr
	719 A to G	Tyr 240 Cys
*3B	460 G to A	Ala 154 Thr
*3C	710 A to G	Tyr 240 Cys
*4	IVS9AS G to A -1	No effect
*8	644 G to A	Arg 15 His

Transcriptional profiling of tumour issue

The intratumour levels of DPD, TS and Thymidine phosphorylase (TP) can be used to predict responsiveness to treatment with 5-FU/leukovorin.

Using our Taqman® System for analysis of gene expression, we profile a service to determine the levels of these particular mRNA's. Freshly excised and frozen (liquid nitrogen) samples will be required.

Low intratumoral mRNA levels of:	% Response rate
TS	57
TS and TP	79
TS and DPD	92
TS, TP and DPD	100

Other cancer associated genes

For tumour analysis we offer a range of SNP genotyping assays (including for example p53, Ha-, Ki-, N-ras), assays for other polymorphisms (e.g. LOH, if we can use a microsatellite based PCR reaction to distinguish the maternal and paternal alleles) and gene amplifications (including for example c-myc and N-myc). For example, in the case of p53 we offer the following series of genotyping assays:

Allelic Variants of p53

Nucleotide	Protein	Association (where known)	Nucleotide	Protein	Association (where known)
C to T	Arg 248 Trp		A to T	Lys 120 Ter	Osteosarcoma
G to A	Glu 258 Lys		C to T	Arg 282 Trp	Osteosarcoma
G to T	Gly 245 Lys		G to A	Gly 245 Ser	Osteosarcoma
T to C	Leu 252 Pro		G to A	Arg 273 His	Thyroid Carcinoma
C to G	Pro 72 Arg		G to T	Gly 325 Val
G to T	Arg 249 Ser	Hepatocellular Carcinoma	C to T	Arg 282 Trp	Osteosarcoma
G to T	Val 157 Phe	Hepatocellular Carcinoma	G to A	Arg 181 His	Breast cancer
G to A	Cys 242 Tyr		G to C	Arg 280 Thr	Nasopharyngeal Carcinoma
G to A	Gly 245 Asp		C to A	Pro 151 Thr	Breast cancer
G to A	Arg 248 Gln	Osteosarcoma	C to T	Pro 151 Ser	Breast cancer
T to C	Met 133 Thr		G to T	Leu 35 Phe	Pancreatic cancer
G to T	Val 272 Teu		T to A	Leu 257 Gln	Multiple malignancy
	Ser 241 Phe	Osteosarcoma	G to A	Arg 175 His
Insert C	1BPIns,151C		T to C	Leu 344 Pro
Del 2 bases	2 BP Del, 209-210	Histiocytoma	G to C
	1 BP Ins	Osteosarcoma
	Codon 71-72

We undertake projects which require investigation of associated oncogenes, polymorphisms and SNP's and analyse these in any number of your samples, clinical or otherwise. Together with our transcription profiling service, this represents a comprehensive set of tests whereby you can rapidly obtain tumour genotyping and expression data.