1. This slide set is designed for educational presentations about TheraGuide 5-FUTM to audiences of healthcare professionals. It contains slides, supplemental (optional) slides and suggested speaker’s notes. These materials are provided as an educational service by Myriad Genetic Laboratories, Inc. of Salt Lake City, Utah.
These slides and text were updated in August of We welcome your comments on these slides and the accompanying notes. You can contact us by

2. Learning Objectives At the conclusion of this presentation participants should understand the following:
Use of pharmacogenetics in understanding patient susceptibility to 5-FU/capecitabine toxicity
Toxicity risk associated with variations in DPYD and TYMS
DPYD = DPD deficiency
TYMS= TS deficiency
Use of genetic test results in medical management
As you may be aware, the topic of toxicity risk following chemotherapy treatment has received a great deal of attention in professional and community publications alike. This presentation will address the use of pharmacogenetics to understand patient susceptibility to 5-FU/capecitabine toxicity and toxicity risk associated with variations in the DPYD and TYMS genes. DPYD gene variations are associated with DPD (dihydropyrimidine dehydrogenase) deficiency. TYMS gene variations are associated with TS (thymidylate synthase) deficiency.

3. Pharmacogenetics
The study of genetic variation that determines an individual’s response to drugs
Pharmacogenetic testing can be beneficial in oncology because it can help determine
How a patient will respond to chemotherapy
Example: cytochrome P450 2D6 (CYP2D6) genotype and ability to metabolize Tamoxifen
The likelihood that a patient will experience severe side effects
Example: TheraGuide 5-FU
It is known that individual variations in genetic makeup play a role in determining how patients will respond to drugs. The study of this process is called pharmacogenetics.
Pharmacogenetic testing can be beneficial in oncology because it can help determine:
-How a patient will respond to chemotherapy. Example: cytochrome P450 2D6 (“sip” 2D6 or CYP2D6) genotype and ability to metabolize Tamoxifen -OR- -The likelihood that a patient will experience severe side effects Example: TheraGuide 5-FU
This allows healthcare providers and their patients to make informed, individualized decisions about drug treatments for their patients.

4. 5-fluorouracil/capecitabine Mechanism of Action
The majority of 5-FU is rendered inactive by the DPD enzyme.
The remaining 5-FU is sufficient for cancer therapy and binds TS enzyme.
One of the most common chemotherapeutic drug therapies is 5-Fluorouracil/capecitabine. It is used to treat several types of cancers including colon, metastatic breast, head and neck and ovarian. It has different options for administration of treatment; individually or in combination with other drugs.
As with many chemotherapies, there can be significant side effects with 5-FU/capecitabine based chemotherapy including diarrhea, nausea, stomatitis and hand foot syndrome.
Normally when 5-FU enters the system, the majority of the drug is rendered inactive when it interacts with the dihydropyrimidine dehydrogenase (DPD) enzyme. The remaining active 5-FU is at a sufficient therapeutic dose. The 5-FU binds with thymidylate synthase (TS) to inhibit the production of thymidine thereby restricting DNA synthesis and cell division.

5. DPD Deficiency Mechanism of Action
Variations in DPYD can lead to DPD insufficiency.
This results in an inability to inactivate 5-FU leading to increased levels of active drug in the system that can result in toxicity.
In the presence of clinically significant variations in DPYD, the DPD enzyme is insufficient to inactivate the 5-FU. This results in higher levels of active drug in the system that can result in toxicity.

6. TS Deficiency Mechanism of Action
Variations in TYMS can lead to TS deficiency.
This results in lower amounts of the TS enzyme being available to bind with the active 5-FU.
This results in increased levels of active drug in the system that can result in toxicity.
In the case of TYMS variations, there is a decreased amount of working TS enzyme. When there is not enough TS enzyme available to bind with the active 5-FU, excess active drug remains in the system and toxicity results. Note that decreased levels and expression of TS may result in toxicity even if there are not increased levels of active 5-FU secondary to DPD function.

7. Who benefits from TheraGuide 5-FU™?
Up to 1 in 3 patients will experience an adverse reaction to 5-FU/capecitabine based chemotherapy
Dependant on drug administration and regimen
Meta Analysis Group in Cancer study (JCO 1998)
6 randomized 5-FU clinical trials
Assessment in toxicity is key to determining treatment modality
31% of patients had grade 3 or 4 toxicity when given 5-FU bolus
Andre, et al JCO 2003
11% of patients had grade 3 or 4 toxicity with 5-FU and leucovorin semi-monthly
Who benefits from TheraGuide 5-FU?
Historically, as many as 1 in 3 patients have experienced dose limiting, sometimes life-threatening toxicity to 5-FU that is largely avoidable This is dependant on how the drug is administered and the regimen that is chosen.4
A meta analysis of 6 randomized 5-FU clinical trials found little difference in survival based on treatment modality but found significant difference in toxicity.
This group felt that assessment in toxicity is key to determining treatment modality out of 612 patients had grade 3 or 4 toxicity when given 5-FU bolus compared to continuous intravenous infusion (23 out of 607).4
In contrast,
Andre, et. al. showed that 11% of patients had grade 3 or 4 toxicity with 5-FU and leucovorin semi-monthly6. This data reinforces that there are differences in patient susceptibility to 5-FU, which can be dependant on how it is administered and the regimen that is chosen.
REFERENCES
1 van Kuilenburg, A.B. Screening for dihydropyrimidine dehydrogenase deficiency: to do or not to do, that’s the question. Cancer Invest Mar;24(2):215-7.
2 de Gramont, A., C. Tournigand., et.al. Adjuvant therapy for stage II and III colorectal cancer. Semin Oncol Apr;34(2 Suppl 1):S37-40.
3 Douillard, J.Y., J. Bennouna. Adjuvant chemotherapy for colon cancer: a confusing area! Ann Oncol Dec;16(12):
4 Meta Analysis Group in Cancer. Toxicity of Fluroruracil in Patients with Advanced Colorectal Cancer: Effects of Administration Schedule and Prognostic Factors. J. Clin. Onc :
5 Wagstaff, A.J.,K. Goa, T. Ibbotson. Capecitabine. A Review of its Pharmacology and Therapeutic Efficacy in the Management of Advanced Breast Cancer. Drugs (2):
6 Andre, et al. Semimonthly Versus Monthly Regimen of Fluorouracil and Leucovorin Administered for 24 or 36 Weeks as Adjuvant Therapy in Stage II and III Colon Cancer: Results of a Randomized Trial.J Clin Onc. 2003:21(15):
Cancer Invest Mar;24(2):215-7.
Semin Oncol Apr;34(2 Suppl 1):S37-40.
Ann Oncol Dec;16(12):
J. Clin. Onc :
Drugs (2):
J Clin Onc. 2003:21(15):

8. What are the risks?
Variations in DPYD and TYMS are associated with up to a 60% risk of severe to life-threatening toxicity to 5-FU/capecitabine.
Morel et al. study (Mol Cancer Ther 2006)
187 out of 487 patients had DPYD variations
44 had grade 3 or 4 toxicity
12 had grade 1 or 2 toxicity
3 specific variations caused level 3 or 4 toxicity in 60% of carriers
Approximately ½ of highly toxic reactions to 5-FU in patients are due to DPD deficiency.
What are the risks?
Studies indicate that there is a significant risk of toxicity in patients with variations in the DPYD and TYMS genes. The risk of grade 3 or 4 (severe or life-threatening) toxicity to 5-FU is approximately 60%. This is up to a 7-fold increased risk compared to the general population.1,2
In a study by Morel et. al., in an unselected population of 5-FU candidates, 187 out of 487 patients (38%) had DPYD variations. Of these patients, 44 had level 3 or 4 toxicity and 12 had level 1 or 2 toxicity. Three specific variations (IVS14 +1G>A, 2846A>T and 1679T>G) caused level 3 or 4 toxicity in 60% of carriers.1
Approximately ½ of highly toxic reactions to 5-FU in patients are due to DPD deficiency.2,3
REFERENCES
1 Morel, A.  Clinical relevance of different dihydropyrimidine dehhydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther (11):
2 Pullarkat, S. T., J. Stoehlmacher, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J (1):
3 van Kuilenburg, A.B. Screening for dihydropyrimidine dehydrogenase deficiency: to do or not to do, that’s the question. Cancer Invest Mar;24(2):215-7.
Mol Cancer Ther (11):
Pharmacogenomics J (1):
Cancer Invest Mar;24(2):215-7.

9. What are the risks?
TYMS gene variations are associated with a 2.5-fold increased risk of severe toxicity
Meta analysis (Lecomte, Pullarkat, Ichikawa)
200 unselected patients treated with 5-FU
43 patients (22%) had grade 3 to 4 toxicity
52% of patients with TYMS high risk genotype had grade 3 to 4 toxicity
What are the risks?
Studies indicate that there is an increased risk of toxicity in patients with variations in the TYMS gene. The risk of grade 3 or 4 (severe or life-threatening) toxicity to 5-FU is increased up to a 2.5-fold risk compared to the general population. Three papers have examined the relationship of alleles (or variants) as predictors of severe or life-threatening (grades 3 and 4, respectively) 5-FU toxicity.1-3 In the aggregate, these studies examined a total of 200 unselected patients treated with 5-FU, of which 44 had grade 3 or 4 toxicity.
43 patients (22%) had grade 3 to 4 toxicity. 13 of 25 patients with TYMS high risk genotype (52%) had grade 3 to 4 toxicity. 52% divided by 22% (which was the unselected population average) equates to a 2.5-fold increased risk of grade 3 to 4 toxicity.
Meta analysis
13 2R/2R
18 2R/3R
12 3R/3R
Total
REFERENCES:
1 Pullarkat, S. T., J. Stoehlmacher, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J (1):
2 Lecomte T, Ferraz JM, Zinzindohoue F, et al. Thymidylate synthase gene polymorphism predicts toxicity in colorectal cancer patients receiving 5-fluorouracil- based chemotherapy. Clin Cancer Res Sep 1;10(17):
3 Ichikawa W, Takahashi T, Suto K, et al. Orotate phosphoribosyltransferase gene polymorphism predicts toxicity in patients treated with bolus 5-fluorouracil regimen. Clin Cancer Res Jul 1;12(13):
Pharmacogenomics J (1):
Clin Cancer Res Sep 1;10(17):
Clin Cancer Res Jul 1;12(13):

10. What is included in TheraGuide 5-FU™ analysis?
The only clinical test that performs:
Full sequencing of the DPYD gene and
Analysis of the TYMS gene promoter region
What is included in TheraGuide 5-FU™ analysis?
TheraGuide 5-FU™ is the only clinical test that allows for full sequencing of the DPYD gene. This test also allows for the analysis of the TYMS gene promoter region.

11. TheraGuide 5-FUTM includes full sequencing of DPYD
DPYD (DPD deficiency)
Three common variations account for the majority of known 5-FU toxicity to date
IVS14+1 G>A, D949V, and I560S
More than 40 different variations in DPYD have been identified as causing DPD deficiency
Full sequencing is the “gold standard” for identifying mutations
TheraGuide 5-FUTM includes full sequencing of DPYD
We know that there are three common variations that account for the majority of DPD deficiency and resulting 5-FU toxicity. However, more than 40 different variations in DPYD have been identified as causes of DPD deficiency.1
Because full sequencing is the most sensitive method for detecting variations, additional variations in DPYD (as well as the common variations) will be detected with TheraGuide 5-FU™.
The DPYD gene is located on chromosome 1.
REFERENCES
1 Morel, A.  Clinical relevance of different dihydropyrimidine dehhydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther (11):
Mol Cancer Ther (11):

12. TheraGuide 5-FUTM includes analysis of TYMS
TYMS variations
2R/2R
2R/3R
3R/3R
4R variations have also been described
The 2R/2R variation is considered high-risk
TheraGuide 5-FUTM includes analysis of TYMS
The variations that will be analyzed in the TYMS gene are located in the promoter (or beginning) region of the gene. There is a section of DNA that is normally repeated in this area. Sometimes it is repeated 2 times (2R), sometimes it is repeated 3 times (3R), and occasionally it can be repeated more times (4-9R). Everyone has some version of this repeated segment, and everyone has two versions of the repeat – one on the gene inherited from mom, and the other on the gene inherited from dad. The results will reflect each copy we inherited, for example, a patient who inherits a 2R copy from mom and a 3R copy from dad would be designated 2R/3R.
In general, individuals with the 2R/2R combination are considered “high risk” for toxicity, while 2R/3R, 3R/3R, and other combinations are considered low risk.
TYMS is located on chromosome 18.

13. How are TheraGuide 5-FUTM results reported?
As many as 1 in 4 individuals have a variation in DPYD or TYMS that increases the risk for 5-FU/capecitabine-related toxicity
TheraGuide 5-FU™ is used to determine a patient’s likelihood of 5-FU toxicity
High Risk (up to 60% risk for Grade 3 or Grade 4 toxicity)
Low Risk
Indeterminate
FDA 2003 warning had been issued stating capecitabine and 5-FU are contraindicated in patients with a known DPD deficiency
How are TheraGuide 5-FUTM results reported?
As many as 1 in 4 individuals (25%) will be at increased risk of 5-FU toxicity because of a variation in DPYD or TYMS.1
TheraGuide 5-FU™ test results indicate a likelihood of risk for a patient to experience toxicity to 5-FU/capecitabine. Depending on the combination of genetic test results for both genes, patients will be placed into the category of either High Risk, Low Risk, or Indeterminate Risk.
By predicting 5-FU or capecitabine toxicity, oncologists can provide enhanced therapeutic choices for improved patient outcomes (either by dose reduction or choice of alternative therapy).
In March 2003, the safety label on Xeloda® (capecitabine) was changed, following FDA indications. The FDA and package insert read that “XELODA® is contraindicated in patients with known dihydropyrimidine dehydrogenase (DPD) deficiency.” Additionally, the manufacturer of 5-Fluorouracil warns physicians on the drug’s insert that 5-FU is contraindicated in patients with DPD enzyme deficiency.
REFERENCES
1 Morel, A.  Clinical relevance of different dihydropyrimidine dehhydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther (11):
2 FDA package warning – Xeloda®:
3 Package warning – fluorouracil:
Mol Cancer Ther (11):
Pharmacogenomics J (1):
FDA package warnings –

14. How are TheraGuide 5-FUTM results used?
Identifies high risk patients before beginning their chemotherapy
Allows for personalized treatment options for cancer therapy
enhanced patient monitoring
dose reduction considerations
alternate chemotherapies
How are TheraGuide 5-FUTM results used?
TheraGuide 5-FU™ Identifies high risk patients before beginning their chemotherapy
-AND-
Allows for personalized treatment options for cancer therapy1-7
- enhanced patient monitoring
- dose reduction considerations
- alternate chemotherapies
REFERENCES
1 Morel, A.  Clinical relevance of different dihydropyrimidine dehhydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther (11):
2 Pullarkat, S. T., J. Stoehlmacher, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J (1):
3 van Kuilenburg, A.B. Screening for dihydropyrimidine dehydrogenase deficiency: to do or not to do, that’s the question. Cancer Invest Mar;24(2):215-7.
4 de Gramont, A., C. Tournigand., et.al. Adjuvant therapy for stage II and III colorectal cancer. Semin Oncol Apr;34(2 Suppl 1):S37-40.
5 Douillard, J.Y., J. Bennouna. Adjuvant chemotherapy for colon cancer: a confusing area! Ann Oncol Dec;16(12):
6 Meta Analysis Group in Cancer. Toxicity of Fluroruracil in Patients with Advanced Colorectal Cancer: Effects of Administration Schedule and Prognostic Factors. J. Clin. Onc :
7 Wagstaff, A.J.,K. Goa, T. Ibbotson. Capecitabine. A Review of its Pharmacology and Therapeutic Efficacy in the Management of Advanced Breast Cancer. Drugs (2):
Mol Cancer Ther (11): Pharmacogenomics J (1): Cancer Invest Mar;24(2):215-7 Semin Oncol Apr;34(2 Suppl 1):S37-40 Ann Oncol Dec;16(12): J. Clin. Onc : Drugs (2):

15. In Summary
TheraGuide 5-FU™ can help predict a patient’s risk of toxicity to 5-FU.
Patient management can be personalized based on results.
Avoiding adverse events can help physicians save time, money, and patient quality of life.
In Summary
TheraGuide 5-FU™ is the most comprehensive test that can help to predict a patient’s risk of toxicity to 5-FU allowing for patient management to be personalized based on results so to avoid adverse events which can help physicians save time, money, and patient quality of life.
Disclaimer: This information is provided to help answer questions with respect to pharmacogenetic testing and chemotherapy toxicity risks. It is general in nature and is not intended to provide a comprehensive, definitive analysis of specific risk factors for chemotherapy treatments. The information provided herein should not be relied upon; but rather, should be taken into consideration with other medical and research information regarding chemotherapy risks and pharmacogenetic testing.
Myriad, the Myriad logo and TheraGuide 5-FU are either trademarks or registered trademarks of Myriad Genetics, Inc. in the United States and other jurisdictions.