Mind the Mix

Pharmacogenetics in allogeneic stem cell Transplant Patients

Mind the Mix

M.H. ten Brink ¹ , H. Bouwsma ² , R. Baak-Pablo ¹ , H-J Guchelaar ¹ , T. van der Straaten ¹ , J.J. Swen ¹

1

Department of Clinical Pharmacy and Toxicology and

Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands

m.h.ten_brink@lumc.nl

Background

• Pharmacogenetic information is accumulating rapidly and is beginning to show consistent reproducible results for an increasing number of genetic markers for drug response.

• Several consortia have published guidelines to aid physicians and pharmacists with the interpretation and clinical translation of pharmacogenetic test results.

• An increasing number of medical centers have acquired clinical genotyping facilities.

• Among the first medical centers to implement pharmacogenetics there are many highly specialized care centers with complex patient populations.

• These patients may present some unexpected challenges as is exemplified by the following case description.

Table 2. The occurrence of acute and delayed chemotherapy induced nausea and vomiting

Case

• Female patient, 20 years old.

• Admitted to the LUMC for living related kidney transplantation.

• Adequate tacrolimus exposure early after transplantation is essential.

• Prior to kidney transplantation, patient is

preemptively genotyped for CYP3A5*3 (rs776746) and CYP3A5*6 (rs10264272).

• Genotyping: in duplicate by two independent techniques, to mimimize the risk of potential errors.

• Genotyping results from Pyrosequencing and Taqman were conflicting.

• Pyrosequencing  CYP3A5*1/*3

• TaqMan  CYP3A5*3/*3

• Further research: second blood sample and consulting the nephrologist

Tacrolimus and kidney transplantation

• Patients receive standard quadruple

immunosuppressive regime: basiliximab, tacrolimus, mycophenolate, prednisolone.

• Adequate tacrolimus exposure early after transplantation is essential.

• Tacrolimus is metabolized into active and inactive metabolites by CYP3A4 and CYP3A5.

• Patients carrying at least one copy of the CYP3A5*1 allele require a significantly increased tacrolimus dose to attain therapeutic blood concentrations.

 All patients undergoing a kidney transplantation in the LUMC from 2009 onward are

preemptively genotyped for the CYP3A5*3 (rs776746) and CYP3A5*6 (rs10264272) polymorphisms

Plasmid control

First patient blood sample

Patient saliva sample

Donor saliva sample

CYP3A5*3 (G)

CYP3A5*1 (A)

CYP3A5*1 (A)

CYP3A5*3 (G)

First blood sample

Second blood sample

Saliva samples

CYP3A5*1 (A)

CYP3A5*3 (G)

A

B

C

D

F

G

H

Results Case

• 1992: 1^st allogeneic stem cell transplantation to treat beta-thalassemia major. Transplant rejected.

• 2009: 2^nd allo-SCT from a second donor. Result is a mixed hematopoietic chimerism (28% autologous, 72% donor).

• Saliva samples from patient and donor were collected and genotyped for the CYP3A5*3 and CYP3A5*6.

• The donor was autocalled CYP3A5*3/*3

• The patient was autocalled CYP3A5*1/*3.

• This genotype is in line with the relatively low tacrolimus trough level (5.5 ug/L).

• AUC levels of 110 µg*hours/L were achieved with a tacrolimus dose of 8 mg twice daily.

References

1, Swen, J. J. et al. Pharmacogenetics: From Bench to Byte— An Update of Guidelines. 662–673

2, Relling, M. V. & Klein, T. E. CPIC: Clinical Pharmacogenetics Implementation Consortium of the Pharmacogenomics Research Network. 464–467.

3. Press, R. R. et al. Explaining Variability in Tacrolimus Pharmacokinetics to Optimize Early Exposure in Adult Kidney Transplant Recipients. 187–197.

4. Straaten, T. van der, Swen, J., Baak-Pablo, R. & Guchelaar, H.-J. Use of plasmid-derived external quality control samples in pharmacogenetic testing. 1261- 1266.

5. Thiede, C., Prange-Krex, G., Freiberg-Richter, J., Bornhäuser, M. & Ehninger, G.

Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplants 575–577

Figure 1. Genotyping results of different samples based on pyrosequencing (A-D) and the TaqMan assay (E-G).

1A-D Pyrosequencing results.

Results for patient blood sample (A), for plasmid CYP3A5*1/*3 control (B), saliva sample of patient (C) and saliva sample of donor (D). A- peak indicating presence of the CYP3A5*1 allele, G-peak indicating presence of the CYP3A5*3 allele. Pyrosequencing results from patients’

blood sample (A) showed inconsistencies in peak proportion between the A and the G peak compared to the results obtained with plasmid control (B). Saliva sample from the patient is auto-called CYP3A5*1/*3 (C) and the sample of the donor is auto-called CYP3A5*3/*3 (D).

1E-G TaqMan results.

Blue dots indicate samples called as CYP3A5*3/*3; Green dots indicate samples called as CYP3A5*1/*3; Red dots indicate samples called as CYP3A5*1/*1. Encircled dots are results obtained with DNA from the patient (triplos). Squared dots are results obtained with DNA from the donor. Conflicting results obtained with the first (E) and second (F)blood sample. Saliva sample from the patient is called

CYP3A5*1/*3 and the sample of the donor is auto-called CYP3A5*3/*3 (G).

Discussion

• This case illustrates

 The challenging aspects of pharmacogenetic testing

 The importance of proper quality control mechanisms

 Selection of the proper source of DNA to determine the genotype in Tx patients

• To determine germline DNA in SCT patients:

buccal swabs should be used.

• Pharmacogenetic testing in solid organ transplantation recipients should be handled with great care.

• Taking the transplant type, the metabolic pathway, mechanism of action and toxicity of the applied drugs into consideration.