Toxicity with chemotherapy Pharmacogenomics and Predictors of toxicity - Essay Example

?Toxi with chemotherapy – Pharmacogenomics and Predictors of toxi Background Fanconi anemia (FA) is a genetic disease characterized by physical abnormalities, such as short stature, abnormal skin pigmentation, internal organ malformation, thumb deformation, hypogonadism, and development delay, as well as bone marrow failure and increased risk of malignancy. In fact, acute myelogenous leukemia (AML) may be the first manifestation of FA (Alter and Kupfer, 2013). AML is the cancer of blood and bone marrow. Like the other cancer types, damage to the DNA, including but not limited to FA genes, is identified as the cause of this malignancy. Because of its rapid progression, treatment must be started immediately (Mayo Clinic, 2012). Chemotherapy with cytarabine and anthracyclines is the cornerstone of remission induction therapy, which aims to kill the cancerous bone marrow cells. With the vast heterogeneity of AML, benefits and risks vary from patient to patient (Emadi and Karp, 2012). However, cancer medications have poor therapeutic profile. The mutagenic capability of these substances may have been the etiology of the treatment’s side effects (Paugh, Stocco and Evans, 2010; Walko and Ikediobi, 2012). Thus, aside from the responsiveness to treatment being partially dependent on genes (Kim, et al., 2010), the susceptibility to side effects are also genetically determined. Because multiple genes are implicated in FA, mutations in one or more of these manifest as a spectrum of symptoms, depending on what and how many genes are affected. It is possible that multiple mutations may present more complications than a single mutation. Conversely, those with a single mutation may be healthy individuals. Aplastic anemia is one of the possible bad outcomes of AML chemotherapy. It is likely that the genes mutated during treatment, which may have caused this complication, is those implicated in FA, especially since the disease caused by these genes is similar with aplastic anemia. Thus, especially susceptible to this must be those that have single to few FA gene mutations, since just one or a few more chemically-induced mutations on these genes can push the individual to one who manifests FA-like symptoms like anemia. Aim Because secondary aplastic anemia is a fatal side effect of AML treatment, it is helpful to determine susceptibility to this complication before chemotherapy is ensued. One of the ways that this can be done is by looking for mutations in FA genes that predispose to the development of aplastic anemia. However, for this information to be clinically useful, testing for these genes must be quick, since AML treatment should be commenced immediately. This research thus asks, “In AML patients who have undergone chemotherapy, what FA genes are predictive of developing secondary aplastic anemia? To answer this, the experiment will be conducted, with the general objective of suggesting genes that can be tested in order to predict the occurrence of aplastic anemia secondary to chemotherapy. Specific objectives include, 1) comparing FA gene mutations in post-treatment AML patients with or without secondary aplastic anemia, 2) determining predictive values of the various genetic mutations for the occurrence of secondary aplastic anemia, and 3) planning a quick screening test for AML patients before initiation of chemotherapy. Research plan This retrospective study will employ a retrospective design to identify FA genes predictive of secondary aplastic anemia in AML patients. Sampling At least 200 adult individuals without FA that have a present or past medical history of AML and subsequent chemotherapy will be the subject of this study. They will be classified into those that had or have acquired secondary anaplastic anemia and to those that have not. Exclusion criteria will include previous bone marrow transplant and unstable health status that prevents bone marrow sampling. Methods 1. Baseline characteristics For each subjects, age, sex, and age diagnosed with AML will be tabulated. 2. Bone marrow extraction The procedure is adopted from that used in UCSF Medical Center (2008). Briefly, after applying lidocaine cream on the skin nearest to the right or left iliac crest, site will be punctured with bone marrow needle, and the needle will be advanced through the subcutaneous tissue, periosteum and marrow cavity. The stylet will then be replaced with a plain syringe to aspirate 0.5 ml marrow. 3. Sequence analysis The following procedure is based on the study by Meetei, et al. (2004), which sequenced and analyzed FANCB gene and protein. a. DNA extraction DNA will be extracted from the samples as well as the controls using the QIAGEN blood mini kit (Qiagen, USA). b. Amplification of sequences For amplification, controls to be used will be the HeLa cell line and lymphoblastoid cell line from a normal individual (WT). 200 ng of Extracted DNA will then undergo polymerase chain reaction (PCR), with the forward- and backward-sequencing primer pairs designed based on the FA complementation gene sequences posted on Online Mendelian Inheritance in Man (OMIM) website of Johns Hopkins University. 15 primer pairs will be designed, each of which will correspond to a particular FA complementation gene. Long-range PCR is preferred to cover the mutations in different sites, and for this, Platinum Taq polymerase (Invitrogen), together with Elongase system (Invitrogen), will be used. Thus, for a sample, 15 PCRs will be done. c. DNA sequencing After purification of PCR products by SAP/EXO treatment (Amersham), sequencing reactions will be prepared using 10 pmol of primer and a Big Dye terminator cycle sequencing kit (Applied Biosystems). GeneAmp PCR system 9700 (Applied Biosystems) and ABI 3730 DNA Analyzer (Applied Biosystems) will be used as per manufacturer instructions. Data analysis a. Recording of results Once sequences have been obtained, data on the nature of mutations, if present, will be tabulated. Example table is shown below. Fig. x Mutations on FA complementation genes FA complementation gene Mutations seen Number of anemic Subjects with the mutation (a) Number of anemic subjects without the mutation (c) Number of non-anemic subjects with mutation (b) Number of non-anemic subjects without the mutation (d) FANCA none N/A N/A N/A N/A FANCB 1-BP insertion, 1838T 1 na - 1 2 nna – 2 3314-BP, deleted 10 na – 10 1 nna – 1 na: total number of subjects with aplastic anemia nna: total number of subjects without aplastic anemia b. Statistical analysis After tabulation of the results, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each mutation will be calculated. Sensitivity is the proportion of anemic patients with the mutation (a/(a+c)), while specificity is the proportion of non-anemic patients without the mutation (d/(b+d)). On the other hand, PPV will be calculated by obtaining the proportion of persons with the mutation who correctly turn out to have anemia (a/(a+b)), while NPV is the proportion of persons without mutation who correctly turn out to have no anemia (d/(c+d)) (Dans, Dans and Silvestre, 2008). References Alter B. P., and Kupfer, G., 2013. Fanconi Anemia. In: Pagon R. A., Bird, T. D., Dolan, C. R., Stephens, K., and Adam, M. P., eds. GeneReviews™ [online]. Seattle (WA): University of Washington, Seattle; 1993-. Available at: http://www.ncbi.nlm.nih.gov/books/NBK1401/ Dans, A. L., Dans, L. F., and Silvestre, M. A., 2008. Painless Evidence-Based Medicine. England: Wiley, p. 50 Emadi, A., and Karp, J. E., 2012. The clinically relevant pharmacogenomics changes in acute myelogenous leukemia. Pharmacogenomics. 13(11), pp. 1257-1269. Kim, D. H., Han, B., Lee, K., Ahn, T., Son, D. S., Sohn. I., Jung, S. H., Park, K., Jang, J. H., Kim, K., Kim, H. J., Sohn, S. K., Moon, J. H., Kim, J., Huh, N., and Jung, C. W., 2010. Pharmacogenomics-based drug response prediction model for acute myeloid leukemia with normal karyotype. Florida, USA. 5 December 2010. Washington, USA: American Society of Hematology Mayo Clinic, 2012. Acute Myelogenous Leukemia. [online]. Available at: < http://www.mayoclinic.com/health/acute-myelogenous-leukemia/DS00548> Meetei, A. R., Levitus, M., Xue, Y., Medhurst, A. L., Zwaan, M., Ling, C., Rooimans, M. A., Bier, P., Hoatlin, M., Pals, G., de Winter, J. P., Wang, W., and Joenje, H., 2004. X-linked inheritance of Fanconi anemia complementation group B. Nature Genetics, 36, pp. 1219-1224. Online Mendelian Inheritance in Man, 2012. FANCB GENE; FANCB. [online]. Available at: http://omim.org/allelicVariant/300515 Paugh, S. W., Stocco, G., and Evans, W. E., 2010. Pharmacogenomics in Pediatric Leukemia. Current Opinion in Pediatric, 22(6), pp. 703-710 UCSF Medical Center, 2008. Standardized Procedure: Bone Marrow Aspiration. [online]. Available at: Walko, C. M. and Ikediobi, O., 2012. Pharmacogenomic Applications in Oncology, J Pharm Pract, 25(4), pp. 439-446. Read More