5 - Oncology 2

Question 1

Methotrexate SE

Answer 1

• Peak w/in 10-14 days post-dose
• Myelosuppression – increases risk of bleeding and infection
• SE subside w/in 2 weeks of d/c
• • Unless renal function is compromised by MTX metabolites

Question 2

What is the active form of methotrexate?

Answer 2

Methotrexate polyglutamate (converted by folylpolyglutamate synthase)

Question 3

What is the mechanism of methotrexate in cancer?

Answer 3

• Decreases DNA synthesis
• Prevents reduction of folic acid to DHF to THF
• Ultimately blocks cell growth and replication (G1 and S phase)

Question 4

What is the mechanism of methotrexate in RA?

Answer 4

• MTX-PG inhibits AICAR transformylase => increased AICAR which inhibits adenosine deaminase => increased adenosine
• Adenosine binds to adenosine receptors and has many effects:
• • Neutrophils -> decreases activation and superoxide
• • Macrophages -> decrease cytokine expression
• • T-cell -> inhibits T-cell signalling and activation

Question 5

Describe methotrexate metabolism

Answer 5

MTX -> 7-hydroxymethotrexate (7HO-MTX) by aldehyde oxidase

Question 6

Describe the toxicity of 7HO-MTX

Answer 6

• *Nephrotoxicity:
• MTX and 7HO-MTX precipitate in the renal tubules and cause renal dysfunction, further decreasing MTX excretion and increasing toxicity
• 7HO-MTX is excreted much slower than MTX and is therefore considered primarily responsible for renal toxicity
• Thrombocytopenia (less thrombocytes = less platelets to have aggregation)

Question 7

MTX polymorphisms

Answer 7

• Methylene-tetrahydrofolate reductase (MTHFR)
• Thymidylate synthase (TS)
• Dihydrofolate reductase (DHFR)
• RFC1 associated w/ cellular influx
• ABCG2 associated w/ drug resistance from efflux

Question 8

Dihydrofolate reductase (DHFR)

Answer 8

• Co-substrate w/ NADPH in conversion of folic acid -> DHF and DHF -> THF
• • MTX and MTX-PG inhibit both of these steps

Question 9

Describe DHFR expression w/ 1/1 genotype

Answer 9

Significantly higher DHFR expression

Question 10

What effect does DHFR expression have on MTX?

Answer 10

• Increasing expression of DHFR has the effect of decreasing the inhibitory potency of MTX
• • Increasing DHFR increases the IC50 for MTX
• Px who have 1/1 haplotype would require more MTX to achieve the same DHFR inhibition as those w/ other haplotypes
• • No dosing recommendations have been made yet

Question 11

Significance of DHFR haplotypes and EFS (event-free survival)

Answer 11

• Being homozygous w/ -317A and -1610C alleles is correlated w/ lower EFS
• • Those w/ 1/1 haplotype are more likely to relapse or otherwise die from complications
• • This is also correlated w/ higher DHFR expression levels w/ the 1/1 genotype
• Being homozygous for the -1610T allele was associated w/ better EFS (4/4)

Question 12

Describe methylene-THF-reductase (MTHFR)

Answer 12

• Co-substrate w/ FADH to reduce methylene-THF -> methyl-THF
• Reduction steps require FADH as a cofactor (ie: as a source of hydrogens or more correctly, a source of electrons)
• Methyl-THF is used to make methionine form homocysteine

Question 13

MTHFR SNPs impacting cancer

Answer 13

1. 677 C>T -> results in A222V substitution
2. 1298 A>C -> results in E249A glutamate to alanine substitution
   * *Both result in reduced MTHFR activity

Question 14

Describe MTHFR genotype/ phenotype

Answer 14

• 677 C/C and 1298 A/A = WT
• 677 SNPs = C/T or T/T
• 1298 SNPs = A/C or C/C
• Unknown if it is possible to have both 677 T/T and 1298 C/C

Question 15

MTHFR clinical significance regarding toxicity

Answer 15

• Those who are 1298 A/A and 677 C/C (ie: WT) have significantly less toxicity w/ MTX tx than those who are 1298 C/C and/or 677 T/T
• • May be due to the reduction in plasma folate that is observed w/ those who are 1298 C/C and/or 677 T/T
• Those who are 1298 C/C and/or 677 T/T have higher homocysteine levels (likely due to reduction in pool of methyl-THF)

Question 16

Effects of MTHFR polymorphisms

Answer 16

Lower plasma [folate] means less folate to compete w/ MTX for DHFR, thereby increasing MTX toxicity

Question 17

Clinical significance of MTHFR polymorphisms in dosing of MTX for cancer

Answer 17

High dose MTX may not be tolerated w/ px who are 1298 C/C and/or 677 T/T

Question 18

Clinical significance of MTHFR polymorphisms in dosing of MTX for RA

Answer 18

677C>T and 1298A>C MTHFR SNPs are not correlated w/ response to MTX tx in RA

Question 19

Thymidylate synthase (TS)

Answer 19

• Inhibition of TS by 5FU or MTX-PG => decreased dTMP synthesis and eventually decreased DNA synthesis
• Affects mostly rapidly growing cells such as cancer cells
• TS also a target for fluorouracil, so almost everything we talk about will also apply to fluorouracil

Question 20

Describe the TS E-box enhancer

Answer 20

• For TS, the relevant enhancer is a 28bp sequence w/ a sequence (E-box) which binds to a transcription factor called the upstream stimulatory factor (USF) which increases transcription of TS gene
• Enhancer sequence comes in tandem repeats of the 28bp sequence, repeats of 1 to 5 are known

Question 21

TS polymorphisms

Answer 21

• Polymorphisms consist of from 1 to 5 tandem repeats of 28bp of the enhancer sequence located in the TS enhancer (ex: 2R has 2 28bp repeats, 3R has 3, etc.)
• Some of the repeats have an E-box w/ a SNP in the last position
• • The SNP disrupts that E-box preventing binding of the transcription factor USF at that repeat

Question 22

TS alleles

Answer 22

• G considered functioning
• C considered non-functioning
• WT = 2RGC and 3RGGC

Question 23

Activity of TS alleles

Answer 23

• As # of functional E-box repeats increases, the expression of TS increases
• WT 2RGC and 3RGGC have highest TS expression (3RGGC more than 2RGC)

Question 24

TS genotype/ phenotype

Answer 24

• Those w/ genotypes containing 2 or less functional E-boxes (G in the genotype) are in the low expression phenotype
• Those w/ genotypes containing > 2 functional E-boxes are in the high expression phenotype

Question 25

TS clinical significance

Answer 25

• Those w/ the high expression TS genotypes (that contain more E-boxes) have lower sensitivity to MTX and 5-FU
• These individuals are at greater risk for tx failure (low expression individuals are fine)
• No dosing recommendations yet

Question 26

RFC-1

Answer 26

• Reduced folate carrier 1 (RFC-1) is a transmembrane protein which transfers folic acid across the membrane
• Folic acid is hydrophilic and can’t diffuse easily across the membrane
• RFC-1 recognizes the negatively changed glutamate residue of folic acid

Question 27

When is RFC-1 important?

Answer 27

• More important as a method to transfer MTX across the membrane for the low doses used to treat RA
• For the high doses used to treat cancer, diffusion of MTX into the cell is a more important mechanism for getting into the cell

Question 28

Effect of RFC-1 80 G>A on methotrexate

Answer 28

• MTX concentrations higher when taking relatively low doses used to treat RA
• Not a concern w/ doses used to treat cancer

Question 29

RFC-1 clinical significance in RA

Answer 29

• Probability of remission of RA sx was higher in carriers of 80 A/A genotype compared w/ px who have 80G/G genotype
• Higher plasma folate and MTX in RFC-1 80 A/A individuals suggest that less folate and MTX is being taken up into cells
• May be that there is less intracellular folate to compete w/ the MTX and this offsets the lower MTX uptake

Question 30

Problem w/ MTX

Answer 30

• Main problem w/ developing dosing guidelines for MTX based on genotypes
• • Many enzymes involved in metabolism and activity of MTX and many have polymorphisms
• • Dose may have to be based on genotype of several polymorphisms for several genes