About the project
In order to improve the treatment of osteosarcoma patients, we aim to identify genetic variants that predict or explain treatment outcome or toxicities observed in osteosarcoma patients.
Current work
The treatment of osteosarcoma did not improve significantly during the last 30 years. Whereas several studies have attempted to develop new treatment strategies, no large progress has been made in osteosarcoma survival. The GO-consortium uses genetic variation to predict or explain interindividual differences in treatment outcome and the development of toxicities. The new knowledge emerging from our project will pave the way for personalized interventions that improve osteosarcoma treatment.
Osteosarcoma is the most common bone tumor in children and adolescents. Chemotherapy treatment is based on cisplatin, doxorubicin and methotrexate, combined with surgical resection of the tumor. Despite this, 30-40% of the patients will not survive. Moreover, survivors experience chemotherapy-induced toxicities and a high disease burden even after treatment. Having a pharmacogenetic profile predicting outcome at treatment start, would facilitate optimization of treatment schedules and new agent development.
To address this, we have formed the GO-consortium. Within the GO-consortium, we aim to identify, validate and functionally describe genetic variants associated with osteosarcoma treatment outcome. The approaches we use are explained below.
Drug Metabolizing Enzymes and Transporters (DMET)
By doing an association analysis of the genetic variants on the DMET Plus array with clinical outcomes, we will identify genetic variants in drug metabolism and transporter genes that influence treatment outcome.
The GWAS will provide us with a genome wide view on genetic variation involved in osteosarcoma treatment outcome. Whereas osteosarcoma is relatively rare, collaboration between several groups within the GO-consortium will result in the largest GWAS in pharmacogenetics of osteosarcoma worldwide.
Replication in independent patient cohorts
Variants we find to be associated with treatment efficacy or toxicity, will be replicated in an independent patient cohort.
PhD Theses
The role of pharmacogenetics in Osteosarcoma
Melanie Hagleitner
This thesis focusses on both clinical and genetic risk factors to predict osteosarcoma treatment efficacy. For example, age was identified as a prognostic factor in osteosarcoma. Using a pathway-based candidate-gene approach, Melanie constructed a prediction model which combined 5 genetic variants to predict disease-free survival in osteosarcoma patients.
The publications that resulted from this thesis can be found below. Melanie successfully defended her thesis on March 17, 2015 in Nijmegen.
Pharmacogenetics of osteosarcoma treatment
Hanneke Vos
In the introduction of her thesis, Hanneke included a systematic review on pharamcogenetics of osteoasarcoma. She also established that DNA samples isolated from paraffin tissue can be used to genotype with the Drug Metabolizing Enzymes and Transporters array. This allows us to include deceased patients in our pharmacogenetic analyses, which is especially important in clinical outcomes that describe treatment efficacy. In addition, the association of the ACYP2 variant with cisplatin induced ototoxicity was replicated in Dutch osteosarcoma patients.
The publications that resulted from this thesis can be found below. Hanneke successfully defended her thesis on December 23, 2017 in Nijmegen.
PhD Theses
The role of pharmacogenetics in Osteosarcoma
Melanie Hagleitner
This thesis focusses on both clinical and genetic risk factors to predict osteosarcoma treatment efficacy. For example, age was identified as a prognostic factor in osteosarcoma. Using a pathway-based candidate-gene approach, Melanie constructed a prediction model which combined 5 genetic variants to predict disease-free survival in osteosarcoma patients.
The publications that resulted from this thesis can be found below. Melanie successfully defended her thesis on March 17, 2015 in Nijmegen.
Pharmacogenetics of osteosarcoma treatment
Hanneke Vos
In the introduction of her thesis, Hanneke included a systematic review on pharamcogenetics of osteoasarcoma. She also established that DNA samples isolated from paraffin tissue can be used to genotype with the Drug Metabolizing Enzymes and Transporters array. This allows us to include deceased patients in our pharmacogenetic analyses, which is especially important in clinical outcomes that describe treatment efficacy. In addition, the association of the ACYP2 variant with cisplatin induced ototoxicity was replicated in Dutch osteosarcoma patients.
The publications that resulted from this thesis can be found below. Hanneke successfully defended her thesis on December 23, 2017 in Nijmegen.
Publications
The role of pharmacogenetics in the treatment of osteosarcoma.
Hanneke I. Vos, Marieke J.H. Coenen, Henk-Jan Guchelaar, and Dunja Maroeska W.M. te Loo
2016, Drug Discovery Today, 21: 1775-86.
In osteosarcoma, large variation is observed in the efficacy and toxicity of chemotherapeutic drugs among similarly treated patients. Treatment optimization using predictive factors or algorithms is of importance, because there has been a lack of improvement of treatment outcome and survival for decades. The outcome of cancer treatment is influenced by the genome, thus studying genetic variants involved in the efficacy and toxicity of the chemotherapeutic drugs used in the treatment of osteosarcoma could be an opportunity to optimize current treatments and improve our understanding of the individual’s drug response in osteosarcoma patients. This review discusses the current insights in the pharmacogenetics of the treatment response of osteosarcoma patients regarding efficacy and toxicity, and implications for future research and treatment.
Replication of a genetic variant in ACYP2 associated with cisplatin-induced hearing loss in patients with osteosarcoma
Hanneke I. Vos, Henk-Jan Guchelaar, Hans Gelderblom, Eveline S.J.M. de Bont, Leontien C.M. Kremer, Anne Marlies Naber, Marina H. Hakobjan, Winette T.A. van der Graaf, Marieke J.H. Coenen, and Dunja Maroeska W.M. te Loo
2016, Pharmacogenetics and Genomics, 26: 243-7.
OBJECTIVE: Irreversible hearing loss is a frequent side effect of the chemotherapeutic agent cisplatin and shows considerable interpatient variability. The variant rs1872328 in the ACYP2 gene was recently identified as a risk factor for the development of cisplatin-induced ototoxicity in children with brain tumors. We aimed to replicate this finding in patients with osteosarcoma.
METHODS: An independent cohort of 156 patients was genotyped for the rs1872328 variant and evaluated for the presence of cisplatin-induced ototoxicity.
RESULTS: A significant association was observed between carriership of the A allele and cisplatin-induced ototoxicity after the end of treatment (P=0.027).
CONCLUSION: This is the first study replicating the association of ACYP2 variant rs1872328 with cisplatin-induced ototoxicity in patients with osteosarcoma who did not receive potentially ototoxic cranial irradiation. Hence, the ACYP2 variant should be considered a predictive pharmacogenetic marker for hearing loss, which may be used to guide therapies for patients treated with cisplatin.
A First Step toward Personalized Medicine in Osteosarcoma: Pharmacogenetics as Predictive Marker of Outcome after Chemotherapy-Based Treatment
Melanie M. Hagleitner, Marieke J.H. Coenen, Hans Gelderblom, Remco R. Makkinje, Hanneke I. Vos, Eveline S.J.M. de Bont, Winette T.A. van der Graaf, H.W. Bart Schreuder, Uta Flucke, Frank N. van Leeuwen, Peter M. Hoogerbrugge, Henk-Jan Guchelaar, Dunja Maroeska W.M. te Loo 2015,
Clinical Cancer Research, 21: 3436-41.
PURPOSE: Overall survival in patients with osteosarcoma is only 60%. Poor response to chemotherapy is the dominant risk factor for poor survival. Pharmacogenetic research can offer possibilities to optimize treatment and improve outcome. We applied a pathway-based approach to evaluate the cumulative effect of genes involved in the metabolism of cisplatin and doxorubicin in relationship to clinical outcome.
EXPERIMENTAL DESIGN: We included 126 patients with osteosarcoma. To comprehensively assess common genetic variation in the 54 genes selected, linkage disequilibrium (LD; r(2) = 0.8)-based tag-single nucleotide polymorphisms (SNP) strategy was used. A final set of 384 SNPs was typed using Illumina Beadarray platform. SNPs significantly associated with 5-year progression-free survival (PFS) were replicated in another 64 patients with osteosarcoma.
RESULTS: We identified five variants in FasL, MSH2, ABCC5, CASP3, and CYP3A4 that were associated with 5-year PFS. Risk stratification based on the combined effects of the risk alleles showed a significant improvement of 5-year PFS. Patients that carried no or only one risk allele had a 5-year PFS of 100% compared with a 5-year PFS of 84.4% for carriers of two or three risk alleles, 66.7% PFS if a patient carried four to five alleles, and a 5-year PFS of 41.8% for patients with >5 risk alleles (P < 0.001).
CONCLUSIONS: We identified several genes that showed association with PFS in patients with osteosarcoma. These pharmacogenetic risk factors might be useful to predict treatment outcome and to stratify patients immediately after diagnosis and offer the possibility to improve treatment and outcome.
High-quality genotyping data from formalin-fixed, paraffin-embedded tissue on the drug metabolizing enzymes and transporters plus array
Hanneke I. Vos, Tahar van der Straaten, Marieke J.H. Coenen, Uta Flucke, Dunja Maroeska W.M. te Loo, Henk-Jan Guchelaar
2015, The Journal of Molecular Diagnostics, 17: 4-9.
The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus array covers 1936 markers in 231 genes involved in drug metabolism and transport. Blood- and saliva-derived DNA works well on the DMET array, but the utility of DNA from FFPE tissue has not been reported for this array. As the ability to use DNA from FFPE tissue on the array could open the potential for large retrospective sample collections, we examined the performance and reliability of FFPE-derived DNA on the DMET Plus array. Germline DNA isolated from archived normal FFPE tissue blocks stored for 3 to 19 years and matched blood or saliva from 16 patients with osteosarcoma were genotyped on the DMET Plus array. Concordance was assessed by calculating agreement and the kappa-statistic. We observed high call rates for both the blood- or saliva-derived DNA samples (99.4%) and the FFPE-derived DNA samples (98.9%). Moreover, the concordance among the 16 blood- or saliva-derived DNA and FFPE DNA pairs was high (97.4%, kappa = 0.915). This is the first study showing that DNA from normal FFPE tissue provides accurate and reliable genotypes on the DMET Plus array compared with blood- or saliva-derived DNA. This finding provides an opportunity for pharmacogenetic studies in diseases with high mortality rates and prevents a bias in studies where otherwise only alive patients can be included.
Influence of genetic variants in TPMT and COMT associated with cisplatin induced hearing loss in patients with cancer: two new cohorts and a meta-analysis reveal significant heterogeneity between cohorts
Melanie M. Hagleitner, Marieke J.H. Coenen, Ana Patiño-García, Eveline S.J.M. de Bont, Anna González-Neira, Hanneke I. Vos, Frank N. van Leeuwen, Hans Gelderblom, Peter M. Hoogerbrugge, Henk-Jan Guchelaar, Dunja Maroeska W.M. te Loo
2014, PLoS One, 9: e115869.
Treatment with cisplatin-containing chemotherapy regimens causes hearing loss in 40-60% of cancer patients. It has been suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain interindividual variability in sensitivity to cisplatin-induced hearing loss. Two recently published studies however, sought to validate these findings and showed inconsistent results. The aim of this study was to evaluate the role of polymorphisms in the TPMT and COMT genes in cisplatin-induced ototoxicity. Therefore we investigated two independent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including all previously published studies resulting in a total population of 664 patients with cancer. With this largest meta-analysis performed to date, we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less important than previously suggested.
Taqman genotyping assays can be used on decalcified and paraffin-embedded tissue from patients with osteosarcoma
Melanie M. Hagleitner, Marieke J.H. Coenen, Judith W.M. Jeuken, Uta Flucke, Bart Schreuder, Dunja Maroeska W.M. te Loo, Peter M. Hoogerbrugge
2011, Pediatric Blood Cancer, 56: 35-8.
BACKGROUND: In cancers like osteosarcoma with a 5-year overall survival of 50-60%, archived histological specimens can be a useful source of biological material. However, this material generally has been decalcified and formalin-fixed for many years. In our study, we investigated whether DNA obtained from these tissues can be used for reliable single nucleotide polymorphism (SNP) genotyping.
PROCEDURE: We studied two SNPs in the drug transporter MDR1 using Taqman(R) SNP genotyping assays. Genotypes of the germ line DNA derived from freshly isolated DNA of 20 surviving patients with osteosarcoma were compared with genotypes obtained from archived material from decalcified formalin-fixed, paraffin-embedded (FFPE) blocks of the same patients.
RESULTS: Decalcified FFPE-derived DNA yielded smaller PCR fragments compared to DNA extracted from peripheral blood cells, with a reliable size of approximately 200 bp. However, we were able to evaluate each SNP in 19 of 20 cases included in this study. All successfully genotyped samples showed 100% concordance between genotypes obtained from DNA of FFPE tissue and the genotypes obtained from DNA of blood from the same patients.
CONCLUSIONS: In conclusion, we have demonstrated that decalcified FFPE tissue can be used for genetic polymorphism analysis using Taqman(R) allelic discrimination assays. This forms a unique opportunity to combine new insights in genetic research with historical patient cohorts.
Survival trends and long-term toxicity in pediatric patients with osteosarcoma.
Melanie M. Hagleitner, Eveline S.J.M. de Bont, Dunja Maroeska W.M. te Loo
2012, Sarcoma
BACKGROUND: This study was conducted to investigate the clinical characteristics and treatment results of osteosarcoma in pediatric patients during the past 30 years. Trends in survival rates and long-term toxicity were analyzed.
PROCEDURE: 130 pediatric patients under the age of 20 years with primary localized or metastatic high-grade osteosarcoma were analyzed regarding demographic, treatment-related variables, long-term toxicity, and survival data.
RESULTS: Comparison of the different time periods of treatment showed that the 5-year OS improved from 58.6% for children diagnosed during 1979-1983 to 78.6% for those diagnosed during 2003-2008 (P = 0.13). Interestingly, the basic treatment agents including cisplatin, doxorubicin, and methotrexate remained the same. Treatment reduction due to acute toxicity was less frequent in patients treated in the last era (7.1% versus 24.1% in patients treated in 1979-1983; P = 0.04). Furthermore, late cardiac effects and secondary malignancies can become evident many years after treatment.
CONCLUSION: We elucidate the prevalence of toxicity to therapy of patients with osteosarcoma over the past 30 years. The overall improvement in survival may in part be attributed to improved supportive care allowing regimens to be administered to best advantage with higher tolerance of chemotherapy and therefore less chemotherapy-related toxicity.
Is there a role for the MTHFR 677C>T and 1298A>C polymorphisms in methotrexate-induced liver toxicity?
Dunja Maroeska W.M. te Loo, Melanie M. Hagleitner, Marieke J.H. Coenen 2014,
Pharmacogenomics, 15:1401-3
KEYWORDS: MTX toxicity; meta-analysis; pharmacogenetics; pharmacogenomics; replication study
The role of the MTHFR 677C>T polymorphism in methotrexate-induced liver toxicity: a meta-analysis in patients with cancer.
Melanie M. Hagleitner, Marieke J.H. Coenen, R. Aplenc, Ana Patiño-Garcia, P Chiusolo, D Gemmati, M De Mattei, A Ongaro, M Krajinovic, PM Hoogerbrugge, Sita H.H.M. Vermeulen, Dunja Maroeska W.M. te Loo
2014, Pharmacogenomics Journal, 14:115-9
Methotrexate (MTX), one of the important pillars in the treatment of different forms of cancer, is associated with the development of hepatotoxicity. The 677C>T variant (rs1801133) in the methylenetetrahydrofolate reductase (MTHFR) gene might affect the development of hepatotoxicity. Results in literature are, however, contradictive. The aim of this study was to evaluate the role of the MTHFR 677C>T polymorphism in MTX-induced hepatotoxicity by analyzing a Dutch cohort of pediatric patients treated with high doses of MTX and subsequently performing a meta-analysis. Ninety-eight patients receiving 542 courses of high-dose MTX were genotyped for the MTHFR 677C>T variant. Hepatotoxicity was evaluated retrospectively according to common terminology criteria for adverse events-National Cancer Institute criteria. The influence of MTHFR 677C>T on hepatotoxicity was examined using a generalized estimating equation (GEE) analysis. A fixed-effect meta-analysis based on this and previous studies investigating the association between the MTHFR 677C>T polymorphism and uniformly coded hepatotoxicity was performed. The GEE analysis showed an increased risk of developing hepatotoxicity for T versus C allele (odds ratio (OR) 1.8; 95% confidence interval (CI) 1.0-3.2, P=0.04). This finding was not supported by the meta-analysis including seven studies and 1044 patients; the OR for the 677T versus C allele was 1.1 (95% CI 0.84-1.5, P=0.25). Heterogeneity between studies was observed, possibly related to differences in MTX dose and leucovorin rescue. In conclusion, in patients with cancer, the MTHFR 677T allele has only a minor role in the development of MTX-induced hepatotoxicity. Observed heterogeneity between studies warrants further study into (tailored) leucovorin rescue.
