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의학박사 학위논문
Genetic Polymorphisms in the
Folate Pathway Are Associated with
Efficacy and Toxicity of
Methotrexate in Pediatric
Osteosarcoma
소아 골육종 환자에서 Folate 대사
연관 유전자 다형성과
Methotrexate 치료 효과 및 독성의
연관성에 관한 연구
2013 년 2 월
서울대학교 대학원
의학과 분자종양의학 과정
박 정 아
ii
A thesis of the Degree of Doctor of Philosophy
소아 골육종 환자에서 Folate 대사
연관 유전자 다형성과
Methotrexate 치료 효과 및 독성의
연관성에 관한 연구
Genetic Polymorphisms in the
Folate Pathway Are Associated with
Efficacy and Toxicity of
Methotrexate in Pediatric
Osteosarcoma
February 2013
The Department of Molecular Oncology
Seoul National University
College of Medicine
Jeong A Park
i
ABSTRACT
Background & Purpose: Osteosarcoma is the most common childhood malignant
bone tumor. Efficacy of multi-agent chemotherapy for osteosarcoma including
methotrexate, adriamycin and cisplatin may be influenced by multiple cellular
pathways. Methotrexate (MTX), one of the main drugs used for osteosarcoma, is a
representative folic acid antagonist. Genetic polymorphisms in folate pathway genes
are expected to influence the response and toxicity of high-dose MTX therapy in
pediatric osteosarcoma, However, there are scarce data available regarding
associations between genetic polymorphisms and pediatric osteosarcoma. This
study evaluated the effect of common genetic polymorphisms in the folate
metabolic pathway on overall survival, event-free survival and histological response
to neoadjuvant chemotherapy including high-dose MTX. In addition, whether these
genetic polymorphisms affect the concentrations of MTX and toxicity after high-
dose MTX therapy for osteosarcoma was investigated.
Methods: Blood and tissue samples from 48 osteosarcoma patients (blood samples
from 19 patients, tissue samples from 22 patients, and both blood and tissue samples
from 7 patients) who had completed chemotherapy were obtained, and the
following polymorphisms were analyzed; RFC1 80G>A, DHFR 829C>T, MTHFR
677C>T, MTHFR 1298A>C, AMPD1 34C>T, ATIC 347C>G, and ITPA 94C>A.
Associations between candidate polymorphisms and survival, histological response
ii
(tumor necrosis rate) and MTX level and toxicity after high-dose MTX therapy
were analyzed.
Results: Event-free survival significantly decreased in DHFR 829 CC homozygote
(P=0.045). There were no statistically significant associations between genetic
polymorphisms and histological response, however, variant carriers of MTHFR
677C>T had tendency towards poor histological response (P=0.078). MTX
concentration was significantly associated with RFC1 80G>A polymorphism
(P=0.027). Liver toxicity after high-dose MTX was associated with ATIC 347C>G
(P=0.043) and tended to increase in carriers of MTHFR 677C>T (P=0.069). Severe
stomatitis was associated with RFC1 80G>A (P=0.012).
Conclusions: This study has demonstrated that several genetic polymorphisms in
folate pathway can significantly influence therapeutic response, clinical outcome
and MTX level and toxicity after high-dose MTX therapy in osteosarcoma patients.
If these associations are independently validated, these variants could be used as
genetic predictors of clinical outcome in the treatment of patients with osteosarcoma,
aiding the development of tailored therapeutic approaches.
----------------------------------------------------------------------------------------------------
Key words:
Osteosarcoma, methotrexate, folate, genetic, polymorphism
Student Number: 2006-30540
iii
CONTENTS
Abstract………………………………………………………………………i
Contents……………………………………………………………… …….iii
List of tables and figures……………………………………………….........iv
List of abbreviations…………………………………………………………v
Introduction …………………………………………………………..……..1
Patients and Methods………………………………………………...……. . 5
Results…………………………..…………………………..………………16
Discussion…....……..………………………………………………………31
Conclusion…………….……………………………………………………39
References………………………………………………………..…………40
Abstract in Korean………….………………………………………………49
iv
LIST OF TABLES AND FIGURES
List of Tables
Table 1. Clinical and pathological characteristics of patients with
osteosarcoma at diagnosis-----------------------------------------------------------7
Table 2. Sequencing by synthesis (SBS) primer sequences for multiple
amplification of methotrexate genes ----------------------------------------------11
Table 3. Allele-specific primer extension (ASPE) primer sequences ------------13
Table 4. Treatment and clinical outcome of patients with osteosarcoma-----17
Table 5. Genotype frequencies of patients with osteosarcoma ----------------22
Table 6. Associations between various polymorphisms and histological response to
neoadjuvant chemotherapy including high-dose methotrexate.----------------------26
Table 7. Genotypic associations with methotrexate concentration at 48 h after high-
dose methotrexate therapy.-------------------------------------------------------------- 28
Table 8. Genotypic associations with grade 3-4 toxicities after high-dose
methotrexate therapy ------------------------------------------------------------------30
List of Figures
Figure 1. Effect of methotrexate on folate metabolic pathway-----------------------2
Figure 2. Event-free survival (A) and overall survival curves (B) of patients with
osteosarcoma according to histological response ----------------------------------- 20
Figure 3. Event-free survival of patients with osteosarcoma according to
dihydrofolate reductase polymorphism-------------------------------------------------- 24
v
LIST OF ABBREVIATIONS
MTX; methotrexate
RFC; reduced folate carrier
DHFR; dihydrofolate reductase
MTHFR; 5, 10-methylenetetrahydrofolate reductase
TS; thymidylate synthase
GGH; γ-glutamyl hydrolase
AICAR; 5-aminoimidazole-4-carbosamide ribonucleotide
ATIC; 5-aminoimidazole-4-carbosamide ribonucleotide (ATIC)
formyltransferase
AMPD; adenosine monophosphate deaminase
ITPA; inosine triphosphate pyrophosphatase
SNP; single nucleotide polymorphism
1
INTRODUCTION
Folate metabolism is essential for cellular functioning, because it provides one-
carbon donors for the synthesis of de novo purines and pyrimidines necessary for
RNA and DNA synthesis, remethylation of homocysteine and DNA methylation.
Folate metabolism may become deranged by inadequate nutrition, altered cellular
transport and polymorphisms in folate-related genes. These polymorphisms may
influence cellular folate metabolism by lowering folate status, shifting cellular
folate distribution, hypomethylation of DNA and uracil misincorporation. There has
been a growing body of evidence that polymorphisms in these folate pathway genes
are important factors in carcinogenesis and influence the risk of various
malignancies (1-3). Also, they are associated with an altered response to folic acid
antagonists in patients with malignancy and autoimmune disease.
Methotrexate (MTX), as a representative folic acid antagonist, is a potent inhibitor
of dihydrofolate reductase (DHFR), a key enzyme for intracellular folate
metabolism, which converts dihydrofolate to the active tetrahydrofolate. Inhibition
of DHFR leads to impairment of purine and pyrimidine nucleotide biosynthesis,
resulting in blocked DNA synthesis and cell death (4). Metabolized polyglutamated
forms of MTX inhibit many other enzymes involved in the folate cycle, such as
5,10-methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), 5-
aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (ATIC),
and inhibition of these enzymes contributes to the effects of MTX (Figure 1) (5).
2
Cell membraneTarget cell
Adenosine
Rc
RFC1
MTX
MTX-PG
DHF THF
MTHFR
TS ATIC
Pyrimidine
synthesis
Purine
biosynthesis
Adenosine
accumulation
MTX
DHFR
FPGS GGH
Efflux transporter
Figure 1. Effect of methotrexate on the folate metabolic pathway. Methotrexate
(MTX), carried by the reduced folate carrier-1 (RFC1) transporter, enters cells, is
converted to methotrexate polyglutamate (MTX-PG) by folypoly-γ-gluatamate
synthetase (FPGS) and is extruded after deconjugation by γ-glutamyl hydrolase
(GGH). MTX-PG inhibits dihydrofolate reductase (DHFR), thymidylate synthase
(TS) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)
formyltransferase (ATIC), which are involved in the folate metabolic pathway.
DHFR inhibition results in reduced conversion of dihydrofolate (DHF) to
tetrahydrofolate (THF) and consequently in reduced synthesis of 5,10-
methylenetetrahydrofolate to 5-methyltetrahydrofolate by 5,10-
methylenetetrahydrofolate reductase (MTHFR), which in turn results in increased
homocysteine levels. TS inhibition results in reduced pyrimidine synthesis. ATIC
inhibition decreases purine biosynthesis and increases extracellular concentrations
of adenosine.
3
MTX is commonly used to treat a variety of diseases including autoimmune
disease like rheumatoid arthritis and psoriasis, malignancies and for prevention of
graft-versus-host disease after transplantation. MTX efficacy and toxicity differ
widely depending on the disease and the MTX doses used (6). However, even when
given identical MTX doses, patients show significantly different responses and
patterns of toxicities, and this diversity can be linked with genetic polymorphisms
associated with drug absorption, metabolism, excretion, and effector targets.
Osteosarcoma is the most frequent malignant bone tumor in children and
adolescents. Treatment for osteosarcoma includes preoperative chemotherapy,
surgery and postoperative chemotherapy. MTX, adriamycin, cisplatin and
ifosfamide form the backbone of most standard treatment protocols. These multi-
agent chemotherapies result in improved survival from less than 20% after surgery
alone to 60 to 70% at 3 years (7, 8). For treatment of osteosarcoma, conventional
doses of MTX therapy have been proven ineffective, and several retrospective
studies have suggested that a threshold peak MTX level needs to be achieved to
obtain a good histological response to chemotherapy (9, 10). High-dose MTX with
leucovorin rescue has been a cornerstone of neoadjuvant chemotherapy for
osteosarcoma. However, resistance to MTX can occur through a variety of
mechanisms, including decreased accumulation due to impaired transport of the
drug via the reduced folate carrier-1 (RFC1), decreased intracellular retention as a
consequence of a lack of polyglutamation, amplification of DHFR, altered
(mutated) DHFR that binds MTX less avidly, and increased level of γ-glutamyl
4
hydrolase (GGH) that hydrolyses MTX polyglutamates (11, 12). Some studies have
reported that impairment of methotrexate influx and upregulation of DHFR occur in
osteosarcoma, and genetic alterations in these genes could influence drug resistance
and toxicity (13-16). Outside of that, many functional polymorphisms in the genes
involved in the folate pathway are expected to influence not only MTX toxicity, but
also responses to chemotherapy in osteosarcoma patients. However, there are scarce
data regarding associations between polymorphisms of folate pathway genes and
pediatric osteosarcoma, and these results are inconsistent among different
populations. Also, no study has examined associations of genetic polymorphisms of
the DHFR and adenosine pathways with treatment outcomes of pediatric
osteosarcoma. This study hypothesized that, first, common polymorphisms of
various genes in the folate metabolic pathway could be predictors of treatment
outcome for patients with osteosarcoma, and second, these polymorphisms might
affect the histological response to neoadjuvant chemotherapy including high-dose
MTX, and third, these genetic polymorphisms might affect plasma levels of MTX
and toxicities after high-dose MTX therapy. This study used multiplex-polymerase
chain reaction (PCR) for mass analysis of various genetic polymorphisms, and
pharmacogenomic profiling of osteosarcoma patients using multiplex-PCR could
facilitate the optimization of current chemotherapy with the aim of improved
outcome and decreased toxicity.
5
PATIENTS AND METHODS
Study population
This is a retrospective study comprising 40 patients at Seoul National University
Children’s Hospital and 8 patients at Asan medical Center given pathological
diagnosis of osteosarcoma and treated with high-dose MTX. Between 1986 and
2009, 124 children were diagnosed with osteosarcoma and were treated at Seoul
National University Children’s Hospital. Seventy patients who had not been treated
with high-dose MTX and 14 patients for whom informed consent was not obtained,
were excluded from this study. Eight patients at Asan Medical Center enrolled in
this study were diagnosed with osteosarcoma in 2009 and were treated with high-
dose MTX containing regimens. This study was approved by the Ethics Committee
of Institutional Review Board of the Seoul National University Hospital (IRB No.
H-0906-067-284). In all cases, informed consent was obtained from the patients,
parents or both.
Each patient received high-dose MTX according to the treatment protocols: CCG
7921 A, CCG 7921 B (7), and COG AOST 0331 (17). High-dose MTX 12 g/m2 was
administered separately by a period of 1 week, each of which was infused over 4
hours. Intravenous hydration and alkalization was achieved 12 hours before the start
of MTX therapy. High-dose MTX was started at urine pH >7.0 for protection
against MTX-induced renal dysfunction. Leucovorin (calcium folinate) rescue was
started 24 hours after start of MTX infusion at a dose rate of 15 mg/m2, and
6
leucovorin doses were repeated every 6 hours until the MTX plasma concentration
was below the cut-off value (0.1 μmol/L).
Clinical data
All data were collected from the patients’ charts, including patients’ characteristics
and clinical variables at diagnosis: gender, age, weight, height, histology, primary
tumor site, tumor volume and presence of metastasis (Table 1). The median age of
the patients was 11.4 years. The primary site of osteosarcoma was most common in
femur (52%), the next in tibia and fibula (27%), and then in humerus and radius
(15%). Because the patients who did not receive MTX were not included, the
proportion of the patients with metastasis at diagnosis (67%) was much more than
those without. The majority of histological diagnoses were osteoblastic type (88%),
and median tumor volume was 125.6 cm3.
7
Table 1. Clinical and pathological characteristics of patients with osteosarcoma
at diagnosis
N %
Total No. of patients 48
Age at diagnosis (years)
Median 11.4
Range 5.0-15.7
Sex
Male 28 58.3
Female 20 41.7
Primary site
Femur 25 52.1
Tibia/fibula 13 27.1
Humerus/radius 7 14.6
Axial 3 6.3
Maxillofacial 0 0.0
Metastasis at diagnosis
Absent 16 33.3
Present 32 66.7
Histological subtype
Osteoblastic 42 87.5
Chondroblastic 3 6.3
Telangiectatic 3 6.3
Tumor volume (cm3)
Median 125.6
Range 2.7-2128
8
To evaluate clinical outcome, following data were collected; treatment protocol,
surgical intervention (reconstruction or amputation), histological response to
neoadjuvant chemotherapy (degree of tumor necrosis determined histologically in
the surgical resection specimen defined as good response [more than 90% necrosis]
or poor response [less than 90% necrosis]) (9), time to and site of relapse and length
of follow-up. Overall survival (OS) was considered as the time from diagnosis to
death or last follow-up, and event-free survival (EFS) was defined as the time from
diagnosis to the first of disease recurrence, development of lung or bone metastases,
and/or death.
MTX concentration and toxicity
Venous blood samples were collected from all patients through routine TDM
protocol at 24 hours, 48 hours and 72 hours from the beginning of the infusion of
high-dose MTX, and additional sampling continued until the MTX plasma
concentration was <0.1 μmol/L. Plasma MTX concentrations were measured by a
fluorescence polarization immunoassay on a TDx system (Abbot Laboratories,
Abbot Park, IL). To evaluate the MTX exposure-toxicity relationship, following
data for each course of high-dose MTX treatment: blood tests including complete
blood count (CBC), total bilirubin, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine and
grades of stomatitis, liver toxicity, kidney toxicity and neurotoxicity according to
the Common Terminology Criteria for Adverse Events version 3.0 (CTCAE)
(http://ctep.info.nih.gov/reporting/index.html) were collected.
9
DNA extraction and genotyping
The genomic DNA from peripheral blood and formalin-fixed, paraffin-embedded
tumor tissue of the 48 patients of this study were analyzed. Blood samples were
obtained from 19 patients, tissue samples from 22 patients, and both blood and
tissue samples from 7 patients. Genomic DNA was extracted from peripheral blood
lymphocytes and tumor tissues using the QIAamp DNA Mini Kit and QIAamp
DNA FFPE Tissue kit (QIAGEN, Hilden, Germany) according to the
manufacturer’s instructions. DNA yield, integrity and protein contamination were
determined by spectrophotometry. Genotyping was performed for RFC1 80G>A,
DHFR 829C>T, MTHFR 677C>T and 1298A>C, ATIC 347C>G, inosine
triphosphate pyrophosphatase (ITPA) 94C>A and adenosine monophosphate
deaminase-1 (AMPD1) 34C>T in the folate metabolic pathway. Multiplex-PCR was
used for mass analysis of polymorphisms of various genes, and the results of
multiplex PCR were compared with the results of standard real-time PCR in 2
patients using blood samples.
Gene amplification
Gene amplification was performed in a total volume of 20 μL mixture containing 5
μM forward and reverse SBS (sequencing by synthesis) primers (Table 1), 0.25 mM
dNTP mix, 112.5 mM Tris HCl (pH 9.0), 3 mM MgCl2, 75mM KCl, 30mM
(NH4)2SO4, and 1.5 U of Taq polymerase (Biotools, Madrid, Spain). The reactions
were preheated at 94℃ for 2 min, then 10 amplification cycles were carried out;
denaturation at 94℃ for 15 sec, annealing at 50℃ for 30 sec and extension at 72℃
10
for 1 min and then 40 amplification cycles using the following parameters:
denaturation at 94℃ for 15 sec, annealing at 60℃ for 230 sec and extension at
72℃ for 40 sec in a 96 well thermal cycler (Applied Biosystems, CA, USA).
Sixteen µL of PCR products was mixed with 4 μL of the activation solution (YeBT,
Seoul, Korea), and this was incubated at 37℃ for 60 min and 85℃ for 15 min.
11
Table 2. Sequencing by synthesis (SBS) primer sequences for multiple
amplification of methotrexate related genes
Gene position Sequence ( 5’ to 3’)
RFC1 c.80 Forward GTGGAACCTGGG AAAA TGACCCCGAGCTCC
Reverse TGATGAAGCTCTC AAAA CTGGCCGTATCTGC
MTHFR
c.677
Forward CTGTCATCCCTATT AAAA CAGGTTACCCCAAAG
Reverse GTGCATGCCTTC AAAA AAAGCGGAAGAATGTG
MTHFR
c.1298
Forward CCTTTGGGGA AAAA TGAAGGACTACTACC
Reverse GCAAGTCACTTTGT AAAA CCATTCCGGTTTGG
DHFR c.829 Forward TGCTATAACTAAGTGCAAAACTCCAAGACCCCAAC
Reverse GTGAAACTGCTCAGTAAAAAAGGGTATGTGGATCT
ATIC c.347 Forward TTTAATGACAGCCAGAAAAACGTCTTTGTTTTATAGA
Reverse AATCCCAAAACACAATAAAAAGAAGTAGCTATTTTCT
AMPD1 c.34 Forward TGATACTCTGACAAATAAAACAGCAAAAGTAATGCA
Reverse TGGAAGGCTGAGC AAAA AAATAACAGCAATC
ITPA c.94 Forward CTTTAGGAGATGGG AAAA GCAGAGTTATCGATGA
Reverse CACAGAAAGTCAGGT AAAA CAGGAAGACAGAGAAA
12
Labeling of activation products
Five μL of activated PCR product was added to 20 μL of an allele-specific primer
extension (ASPE) reaction mixture containing 75 mM Tris HCl (pH 9.0), 2 mM
MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, 25 nM ASPE primer mix (Table 2), 6 mM
biotin dCTP, 50 mM of dATP, dGTP, dTTP mix and 1.0 U of Taq polymerase
(Biotools, Spain). The reactions were denatured at 94℃ for 5 min, 35 amplification
cycles were then carried out, using the following parameters to label the amplicons:
94℃ for 30 s, 55℃ for 1 min, and 72℃ for 2 min and a final extension at 72℃
for 7 min.
13
Table 3. Allele-specific primer extension (ASPE) primer sequences
Gene Taq sequence Sequence (5’ to 3’)
RFC1 80G TCAAAATCTCAAATACTCAAATCA AAAGGTAGCACACGAGGC
RFC1 80A CAATTCAAATCACAATAATCAATC AAAGGTAGCACACGAGGT
MTHFR 677C
MTHFR 677T
CTAACTAACAATAATCTAACTAAC TGCGTGATGATGAAATCGG
AATCTTACTACAAATCCTTTCTTT TGCGTGATGATGAAATCGA
MTHFR 1298A
MTHFR 1298C
CTACAAACAAACAAACATTATCAA GAGCTGACCAGTGAAGA
CAATATACCAATATCATCATTTAC GAGCTGACCAGTGAAGC
DHFR 829C
DHFR 829T
TACAAATCATCAATCACTTTAATC TGGAATGGCAGCTCACTG
TACACTTTCTTTCTTTCTTTCTTT TGGAATGGCAGCTCACTA
ATIC 347C
ATIC 347G
TCAATTACCTTTTCAATACAATAC CACAGCCTCCTCAACAG
TTACTCAAAATCTACACTTTTTCA CACAGCCTCCTCAACAC
AMPD1 34C
AMPD1 34T
TCATTTACCAATCTTTCTTTATAC TCATACAGCTGAAGAGAAAC
TCATTTCAATCAATCATCAACAAT TCATACAGCTGAAGAGAAAT
ITPA 94C
ITPA 94A
CAATATCATCATCTTTATCATTAC TGCCACCAAAGTGCATGG
TAACATTACAACTATACTATCTAC TGCCACCAAAGTGCATGT
14
Hybridization of amplicons
Microspheres (FlexMAP beads, Tm Bioscience, Toronto, Canada) with anti-tags
for each allele specific primer were added to 22 μL of the ASPE reaction product
for a final volume of 42 μL in 2× Hybrisol (YeBT, Seoul, Korea). The mixtures
were denatured for 10 min at 95℃, and then incubated for 30 min at 37oC.
Microspheres were washed 3 times in 160 μL of Tm hybridization buffer (0.2 M
NaCl, 0.1 M Tris (pH 8.0), 0.08% Triton X-100). Streptavidin-R-phycoerythrin
(SAPE, MOSS, Pasadena, MD) was diluted 1:500 in Tm hybridization buffer, and
100 μL was added to the microsphere-hybridized ASPE reaction products. The
mixture was incubated for 15 min at room temperature.
Data analysis
Microsphere fluorescence was measured using a Luminex 200 cytometer
(Luminex, Austin, TX). Data were collected from a minimum of 50 microspheres of
each type. Masterplex GT software (Miraibio, San Francisco, CA) was used to
analyze single nucleotide polymorphism (SNP) genotypes. Homozygous alleles are
discriminated by differences of 25% in median fluorescence intensity (MFI). The
differences ratio was calculated by dividing the net MFI of one allele by the sum of
the net MFI of all alleles and multiplying by 100.
Statistical analysis
All statistical analyses were performed using SAS ver. 9.3. The x2 test was used
to analyze categorical variables, and the student’s t-test, ANOVA test, Wilcoxon
rank sum test, Kruskal-Wallis test were used for continuous variables. The
15
associations between SNPs and toxicity were analyzed by Fisher’s exact test. SNPs
were assessed in relation to OS and EFS using Kaplan-Meier method. Log-rank test
and Cox-regression analysis were used to evaluate the significance of survival
curves, and P-value of less than 0.05 was considered statistically significant. Due to
insufficient number of patients, multivariate analysis was not performed.
16
RESULTS
Treatment and clinical outcome of patients with osteosarcoma
Data of forty-eight patients with osteosarcoma were analyzed. Treatment and
clinical outcome of the patients are displayed in Table 4. The median follow-up
period was 51.5 months. Twenty-five patients were treated with high-dose MTX as
neoadjuvant chemotherapy (CCG 7921 A/B and COG AOST0331 protocol), and 23
patients were treated according to different regimens not included high-dose MTX
[IA CDDP+ IV ADR (18) and BCD regimen (19)]. Forty-six patients received high-
dose MTX as an adjuvant chemotherapy, but two patients were treated with other
adjuvant protocols not including high-dose MTX [one patient with BCD regimen
and the other patient with POG-ICE regimen (20)] due to significant renal toxicity
after high-dose MTX therapy. All patients, except 4, who received an amputation of
an affected limb, received wide excision and reconstruction. After neoadjuvant
chemotherapy (regardless of the protocol used), 19 of 48 patients showed good
histological response. Among 25 patients who received neoadjuvant chemotherapy
with high-dose MTX, 7 patients showed good response (more than 90% necrosis),
and 18 patients showed poor histological response (less than 90% necrosis). By the
time of analysis, 22 patients experienced relapse, and 32 patients were alive.
17
Table 4. Treatment and clinical outcome of patients with osteosarcoma
N %
Total No. of patients 48
Follow-up (months)
Median 51.5
Range 10-298
Neoadjuvant chemotherapy
CCG7921A (CDDP, ADR, HD-MTX) 17 35.4
CCG7921B (Ifos, ADR, HD-MTX) 2 4.2
IA CDDP + IV ADR(18) 20 41.7
COG AOST0331 (CDDP, ADR, HD-MTX) 6 12.5
Others (BCD; Bleo, CPM, ACTD) 3 6.3
Adjuvant chemotherapy
CCG7921A (CDDP, ADR, HD-MTX) 22 45.8
CCG7921B (CDDP, ADR, HD-MTX, Ifos) 19 39.6
COG AOST0331 (CDDP, ADR, HD-MTX /Ifos, VP16) 5 13.2
Others 2 4.2
Surgical intervention
Reconstruction 40 90
Amputation 4 10
Histological response
Good 19 39.6
Poor 29 60.4
Histological response to neoadjuvant chemotherapy
including HD-MTX
Good 7 28
Poor 18 72
Relapse
No 22 45.8
Yes 26 54.2
18
Last follow-up status
Alive 32 66.7
Dead 16 33.3
CDDP, cisplatin; ADR, adriamycin; HD-MTX, high-dose methotrexate; Ifos,
ifosfamide; Bleo, bleomycin; CPM, cyclophosphamide; ACTD, actinomycin-D;
VP16, etoposide.
19
Histological response and survival of the patients
There were no statistically significant associations between histological response
and gender, age (younger than 10 years vs. older than 10 years), primary tumor site,
histological subtype, metastasis at diagnosis, tumor volume (less than 125.6 cm3 vs.
more than 125.6 cm3) or types of neoadjuvant chemotherapy (including high-dose
MTX or not). EFS and OS were significantly associated with histological response
(P=0.016 and P=0.034) (Figure 2). Cox regression analysis for EFS and OS also
showed significant associations with histological response (OR 0.383, 95% CI
0.161-0.915, P= 0.031, and OR 0.263, 95% CI 0.074-0.942, P= 0.040, respectively).
Age, gender, histological subtype, primary tumor site, presence of metastasis, tumor
volume and types of chemotherapy did not influence survival.
20
Figure 2. Event-free survival (A) and overall survival curves (B) of patients
with osteosarcoma according to histological response.
0 50 100 150 200 250 300
Months from diagnosis (OS)
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
lati
ve
surv
ival
P=0.034
Good response
Poor response
B
0 50 100 150 200 250 300
Months from diagnosis (EFS)
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
lati
ve
surv
iva
l
Good response
Poor response
P=0.016
A
21
Toxicity after high-dose MTX therapy
High-dose MTX induced toxicity was recorded and graded according to CACTE.
At least one episode of grade 3-4 liver toxicity was experienced by 27 patients
(56.3%), grade 3-4 kidney toxicity was observed in 3 patients (6.3%). Over grade 3
of neurotoxicity was recorded in only one patient (2.1%), and over grade 3 of
stomatitis was experienced by 6 patients (12.5%).
Genotypes of folate pathway genes
SNP genotype results of multiplex analysis were compared with genotype analyses
of standard sequencing method, and both results were entirely consistent. In seven
patients, peripheral blood and tissue samples were analyzed concurrently using
multiplex-PCR, and their SNP results coincided with each other. Average DNA
yields of paraffin embedded tumor tissues were low, and there was a difficulty in
obtaining adequate amounts of DNA for multiplex-PCR analysis for some patients.
Genotype frequencies of polymorphisms are listed in Table 5. In an analysis of
AMPD1 34C>T, most patients showed CC genotype (97.3%), and this SNP was
excluded from further analysis. Genetic distributions of RFC1 80G>A were 19% for
GG, 31% for GA, and 50% for AA genotype. For MTHFR, CT genotype was the
most common (51%) in 677C>T, and AA genotype (65%) was the most common in
1298A>C polymorphism. For DHFR 829C>T, the frequency of CT genotype (89%)
was much more than that of CC genotype (11%). For ATIC 347C>G and ITPA
94C>A, homozygous alleles of CC predominated. Genotypes showed no association
with age, gender, primary tumor site, or metastasis at diagnosis.
22
Table 5. Genotype frequencies of patients with osteosarcoma
Polymorphism Genotype No. of Patients (%)
RFC1 80G>A GG 9 (18.8)
GA 15 (31.3)
AA 24 (50)
DHFR 829C>T CC 5 (11.1)
CT 40 (88.9)
TT 0 (0)
MTHFR 677C>T CC 12 (26.7)
CT 23 (51.1)
TT 10 (22.2)
MTHFR 1298A>C AA 31 (64.6)
AC 16 (33.3)
CC 1 (2.1)
ATIC 347C>G CC 21 (67.7)
CG 8 (25.8)
GG 2 (6.5)
AMPD1 34C>T CC 36 (97.3)
CT 1 (2.7)
TT 0
ITPA 94C>A CC 24 (85.7)
CA 4 (14.3)
AA 0 (0)
23
Correlation between genotyping and event-free and overall survival
In univariate analysis using log-rank test and Cox-regression analysis, there were
no significant associations between these SNPs and overall survival. However,
decreased EFS was observed in the carriers of DHFR 829CC homozygous allele
(P=0.045 in log-rank test and OR 2.88, 95% CI 0.971-8.55, P=0.057 in Cox-
regression analysis) (Figure 3), and this statistical significance was increased in an
analysis on 25 patients who received neoadjuvant chemotherapy including high-
dose MTX (P=0.0018 in log-rank test and OR 21.49, 95% CI 1.34-343.70, P= 0.030
in Cox regression analysis).
24
Figure 3. Event-free survival of patients with osteosarcoma according to
dihydrofolate reductase polymorphism. Event-free survival significantly differed
by polymorphisms of DHFR 829C>T. Patients with the CC genotype showed lower
EFS than patients with the CT genotype.
0 50 100 150 200 250 300
Months from diagnosis (EFS)
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
lati
ve
surv
ival
DHFR 829 CC genotype
DHFR 829 CT genotype
P=0.045
25
Correlation between genotyping and histological response after neoadjuvant
chemotherapy
This analysis was conducted on 25 patients who received neoadjuvant
chemotherapies including high-dose MTX. Among these patients, 7 patients showed
good response (more than 90% necrosis), and 18 patients showed poor histological
response (less than 90% necrosis). In the analyses of the relationship between
histological response and various genotypes in these 25 patients, there was no
significantly associated polymorphism referring to histological response. While,
poor histological response showed a trend of increasing in the patients with variants
of MTHFR 677C>T (P=0.078). The associations between each polymorphism and
histological response are presented in Table 6.
26
Table 6. Associations between various polymorphisms and histological response
to neoadjuvant chemotherapy including high-dose methotrexate
Polymorphism Genotype No. of
Patients (%)
Histological
response, P-value
Poor histological
response, OR [95% CI]
RFC1 80G>A GG 6 (24)
1.000
GA 9 (36)
AA 10 (40)
GA/AA 19 (76) 0.74 1.4 [0.19-10.14]
DHFR 829C>T CC 3 (12)
CT 22 (88)
TT 0
CT/TT 22 (88)) 0.636 0.67 [0.22-2.01]
MTHFR 677C>T CC 5 (22)
0.222
CT 11 (48)
TT 7 (30)
CT/TT 18 (78) 0.078 3.5 [0.84-14.56]
MTHFR 1298A>C AA 16 (67)
0.389
AC 7 (29)
CC 1 (4)
AC/CC 8 (33) 0.937 0.95 [0.28-3.19]
ATIC 347C>G CC 17 (68)
0.130
CG 6 (24)
GG 2 (8)
CG/GG 8 (32) 0.154 0.17 [0.007-3.89]
ITPA 94C>A CC 21 (84)
1.000
CA 4 (16)
AA 0
CA/AA 4 (16) 0.826 1.31 [0.115-14.93]
27
Correlation between MTX concentration and genotyping
Plasma MTX concentrations were measured at 24, 48 and 72 hours from the start
of infusion of high-dose MTX. Because there were quite a few missing data, the
statistical analyses of plasma levels of MTX did not involve all 48 patients. The
median MTX concentration at 24 hours (24hr MTX) was 4.66 µmol/L (range, 0.19-
378.6 µmol/L) in 24 patients, and median MTX concentrations at 48 hours (48hr
MTX) and 72 hours (72hr MTX) were 0.65 µmol/L (range, 0.09-97.3 µmol/L) in 36
patients and 0.21 µmol/L (range, 0.02-9.75 µmol/L) in 32 patients, respectively. An
analysis of association between genotypes and MTX concentration showed that
48hr MTX significantly increased in variants of RFC1 80G>A (P=0.0275). While
other polymorphisms of ATIC 347C>G, DHFR 829C>T, ITPA 94C>A, MTHFR
677C>T and MTHFR 1298A>C did not show any association with serial plasma
MTX levels (Table 7).
28
Table 7. Genotypic associations with methotrexate concentration at 48 hours
after high-dose methotrexate therapy
Polymorphism Genotype No. of patients
Median 48 hr
MTX (µmol/L) SD P-value
ATIC 347C>G CC 18 1.71 2.33
0.8984 CG 7 0.62 0.39
GG 2 0.94 1.03
DHFR 829C>T CC 4 1.16 1.24 0.5746
CT 30 1.25 1.86
ITPA 94C>A CC 22 1.21 2 0.1886
CA 4 1.83 2.11
MTHFR 677C>T CC 8 0.72 0.36
0.9118 CT 17 7.06 23.36
TT 9 1.5 1.61
MTHFR 1298A>C AA 21 5.68 21.02
0.3906 AC 14 1.4 2.45
AC/CC 1 1.36 .
RFC1 80G>A GG 8 0.55 0.51
0.0275 GA 15 2.22 2.36
AA 13 8.79 26.67
29
Correlation between genotyping and toxicity after high-dose MTX therapy
Significant genotypic associations with grade 3-4 toxicity after high-dose MTX
therapy are displayed in Table 8. RFC1 80 G>A was associated with over grade 3
stomatitis (P=0.012), and carriers of MTHFR 677 C>T polymorphisms had a
tendency towards grade 3-4 liver toxicity (P=0.067). MTHFR 1298 A>C were not
associated with liver, kidney, CNS or GI toxicities. DHFR 829 C>T were not
associated with any severe toxicities, either. Although ATIC 347C>G was associated
with grade 3-4 liver toxicity (P=0.043), another polymorphisms of adenosine
pathway genes did not correlate with significant toxicities. Grade 3-4 kidney
toxicity and neurological toxicity developed in only 3 and 1 patients, respectively,
and no significant association with these SNPs was found in this study.
30
Table 8. Genotypic associations with grade 3-4 toxicities after high-dose
methotrexate therapy
Toxicity Polymorphism Genotype Toxicity,
P-value
Toxicity,
OR [95% CI]
Stomatitis RFC1 80G>A GG
0.008
GA
AA
GA/AA 0.012 7.5 [1.7-33.8]
Liver toxicity MTHFR 677C>T CC
0.093
CT
TT
CT/TT 0.069 1.68 [0.8-3.6]
ATIC 347C>G CC
0.043
CG
GG
CG/GG 0.083 3.5 [0.66-18.5]
31
DISCUSSION
Methotrexate is an effective anti-folate agent which is successfully used for the
treatement of various diseases, but the wide array of applications of the treatment
has hampered the establishment of definite conclusions with respect to clinical
impact of genetic polymorphisms. Due to the diversity of usage, greatly different
doses and treatment protocols have been tested, even for the same disease [e.g.,
high-dose MTX vs. maintenance dose of MTX in acute lymphoblastic leukemia
(ALL)], which makes direct comparisons across a range of studies difficult.
Folate metabolism plays an essential role in both DNA synthesis and methylation.
Several functional polymorphisms in the genes encoding enzymes and transporters
in the folate pathway affect the risk of various malignancies (2, 21-23). However,
for osteosarcoma, the subject hitherto has received but scant attention, and not much
is known about the influence of genetic polymorphisms on susceptibility to
osteosarcoma, or indeed toxicity and response to chemotherapy. Understanding and
overcoming the inter-individual differences in drug response and toxicity remains a
major challenge in osteosarcoma chemotherapy.
This study searched diverse kinds of genetic polymorphisms in the folate-MTX
metabolic pathway and investigated possible associations between these
polymorphisms and clinical data. The selection of candidate polymorphisms was
based on established pharmacological pathways of methotrexate and associations
which had previously been reported in literature. Although this study is limited by a
small sample size, due largely to the retrospective nature of this study and the rarity
32
of this tumor, all patients were treated with the same dose of MTX and standardized
leucovorin rescue, as well as having the same ethnicity. Herein, this study observed
several novel polymorphic associations, as well as confirming several previously
reported findings. In addition, this study observed that frequencies of some
polymorphisms differed from the reported genotype frequencies. In most SNPs,
their frequencies were consistent with previously reported studies. However,
analysis of AMPD1 34C>T showed the dominance of the CC genotype (97.3%)
unlike other studies described that the genetic distributions of AMPD1 34C>T were
74% for CC, 25% for CT and 1% for TT genotype (24, 25). Also, unlike a previous
report of CC dominant distribution of DHFR 829C>T in the general population (26),
the CT genotype was much more prevalent than CC genotype. Indeed, there exist
ethnic-related differences in the frequency distribution of genetic polymorphisms in
the folate pathway genes (27-29). Furthermore, because genetic variations not only
affect treatment response, but also the risk of developing malignancies (2, 21, 23, 28,
30, 31), further studies comparing these polymorphisms with a normal control
population in Korean are warranted.
Genetic polymorphisms of folate pathway and treatment outcome
For osteosarcoma, increased DHFR expression was reported to be associated with
metastatic or recurrent disease (13, 15). One of the important mechanisms of MTX
resistance is an increase in DHFR activity due to amplification of the DHFR gene or
decreased binding of MTX to DHFR due to mutations in the DHFR gene (12, 15).
This suggests that genetic polymorphisms which affect the expression of DHFR
33
may modulate the pharmacological response to MTX. Although, in comparison with
other genes involved in the folate pathway, the polymorphisms of DHFR gene have
received relatively little attention. DHFR 829C>T is known to increase the
expression of DHFR, but the clinical role of this SNP in osteosarcoma has not yet
been evaluated (26, 32). This study could not find any significant difference in the
frequencies of DHFR 829C>T SNPs between the patients with and without
metastasis at diagnosis. However, interestingly enough, this study found that
patients with the CT genotype had better EFS than patients with the CC genotype. T
allele, which has been regarded to increase the expression of DHFR, showed better
EFS in this analysis. A similar conclusion about this correlation between
unfavorable EFS and low DHFR expression has been made by a CCG report of
Levy et al. (33). In his study of 40 children with newly diagnosed ALL, he found
that low DHFR was associated with poor outcome. DHFR expression was inversely
related to proliferative cell nuclear antigens necessary for G1-S transit, and it
signified that DHFR was increased in the S phase. He suggested that DHFR
expression might be a marker for cell proliferation in patient’s blasts other than drug
resistance. The results of this study also reflect a hypothesis that childhood high-
grade osteosarcoma composed of fast dividing cells in the S phase, which have high
DHFR activity, might be more susceptible to multi-agent chemotherapy including
high-dose MTX. This study is the first to report on the DHFR 829C>T
polymorphism as a pharmacogenetic determinant of treatment outcome for
osteosarcoma. This study suggests that this genetic polymorphism of DHFR might
34
be an important prognostic factor in osteosarcoma. Additional studies are necessary
in order to clarify the mechanisms for the correlation of DHFR 829 CC homozygote
and poor outcome.
The effects of MTX on adenosine metabolism have recently received a lot of
attention. Although polymorphisms of ITPA 94C>A and ATIC 347C>G were
reported to affect the treatment outcome in rheumatoid diseases (24, 34, 35), this
study failed to find any significant association between these SNPs in the adenosine
pathway and osteosarcoma disease outcome. The anti-inflammatory effect of MTX
via this adenosine pathway might not be enough to affect the treatment outcome in
osteosarcoma patients
Genetic polymorphisms of folate pathway and histological response
The relationship between poor histological response and inferior survival has been
well established in osteosarcoma (36-38). Many researchers have tried to find a
means of identifying patients who are at risk of poor response at diagnosis, and
some factors, including genetic variations, have been suggested to be associated
with poor responses to chemotherapy (9, 38, 39). In this study, MTHFR 677T allele
showed a tendency towards poor histological response. Although this finding did
not reach statistical significance, considering that the MTHFR 677T allele is related
to poor pharmacological response and a negative prognostic value in hematological
malignancies (40-43), it might suggest a correlation between MTHFR 677T allele
and poor histological response in osteosarcoma.
35
Decreased RFC1 expression has been suggested as another mechanism of
methotrexate resistance in osteosarcoma (13, 15, 44). In some studies of childhood
ALL, the 80A variant of RFC1 was found to result in the impaired intracellular
transport of MTX, and this was related to poor prognosis (33, 45). However, a
search for their association did not return any significant results in this study. RFC1
polymorphisms did not affect the tumor necrosis rate after neoadjuvant
chemotherapy. Although this might be attributable, in part, to the limited numbers of
patients in this analysis, histological response to high-dose MTX can occur as a
result of many mechanisms involved in the MTX-folate pathway, moreover,
histological response is not due to MTX alone but rather to the combined effect of
many chemotherapeutic agents, including MTX, adriamycin, and cisplatin (39, 46).
Additionally, the MTX transport capacity of RFC1 might significantly influence the
MTX effect in low-dose MTX settings, whereas the trivial difference of this
capacity might not be so important in this high-dose MTX setting for osteosarcoma,
as with the use of high-dose MTX, as the difference in propensity for MTX-
polyglutamation has less influence on cure rates (47).
Genetic polymorphisms of folate pathway and MTX concentration
Plasma levels of MTX at 48 hours after MTX administration were significantly
higher in patients with the RFC1 80A allele, providing additional support for
differential carrier activity among those with the variant allele. RFC1 is a major
route of entry for MTX into the cell. A polymorphism in the RFC1 gene (80G>A,
Arg27His) was found to be associated with decreased carrier function or affinity for
36
folate (4, 48). The A allele was associated with higher plasma folate levels, and the
G allele correlated with lower plasma folate and higher homocysteine levels (49,
50). Our result indicates a decrease in the MTX uptake capacity of RFC1 in carriers
of the 80A allele as compared to 80G allele, and it is consistent with the previous
report in ALL which showed that patients with a homozygous AA genotype had
significantly higher concentrations of MTX than the other genotype groups (45).
Genetic polymorphisms of folate pathway and toxicity after high-dose MTX
therapy
High-dose MTX therapy causes significant side effects in some patients. The early
identification of patients at risk of severe toxicity may facilitate improved
monitoring and early intervention to minimize complications. This study identified
that several polymorphisms influenced MTX toxicities in osteosarcoma. RFC1 80
G>A was associated with severe stomatitis, ATIC 347 C>G was associated with
liver toxicity, and MTHFR 677 C>T showed a tendency towards increased liver
toxicity.
Some studies pointed to 80A allele of RFC1 as a marker of increased occurrence
of adverse reactions (51-53), but this finding regarding an association with RFC1
80G>A and stomatitis in osteosarcoma has not been previously reported. The risk of
stomatitis is related to a long duration of MTX exposure and high plasma levels of
MTX (54-56), and it might be caused by the vulnerability of the mucosal membrane
to MTX (57).
37
The majority of studies about the relationship between polymorphisms in the
adenosine pathway and outcome of MTX treatment have been based on rheumatoid
arthritis and psoriasis. Although many studies still hold widely different views on
the impact of the ATIC 347G allele on MTX efficacy, with regard to toxicity, ATIC
347G allele has been associated with a greater likelihood of developing significant
toxicities (24, 48, 58). This study also showed that the G allele of ATIC 347C>G
has a higher risk of developing liver toxicity after high-dose MTX in osteosarcoma
patients, and this finding has not been previously reported.
MTHFR has two well-described, non-synonymous SNPs, 677C>T and 1298A>C.
The variant alleles for both of these polymorphisms have been shown to result in
decreased enzyme activity, and therefore, to affect the toxicity of MTX (43, 59, 60).
MTHFR 677C>T has been associated with a 40% decrease in enzyme activity in
heterozygous carriers, and up to a 70% decrease in homozygous carriers. There is a
growing body of evidence of association between the 677T allele and a higher risk
of MTX toxicity in various diseases, including acute leukemia, non-Hodgkin
lymphoma, solid tumors and rheumatoid arthritis (40, 61-63). Our results also show
that the carriers of this 677T allele had an increased risk of liver toxicity compared
to the individuals with the 677CC homozygous allele. On the other hand, the
functional impact of the MTHFR 1298A>C polymorphism has been relatively less
well characterized. This 1298C allele also seems to decrease enzyme activity but to
a lesser degree than 677T, and the results of studies on the impact of this SNP on
MTX related toxicity and efficacy have been inconsistent (64-66). With regard to
38
toxicity, under conditions of extreme folate depletion, such as by high-dose MTX
therapy, the effect of this SNP might be clinically relevant. Though, this study did
not reveal any significant association between this SNP and toxicity after high-dose
MTX.
Many of the studies about genetic polymorphisms undertaken to date, including
this study, have been limited by small sample size. While the appropriate research
object number determined for this study was more than 150 patients for each
analysis, this study was limited due to retrospective recruitment and the rarity of
osteosarcoma. Also, this study was limited by inadequate DNA yields of paraffin
embedded tumor tissues, and thus, for some of patients, there was difficulty
obtaining an adequate amount of DNA for multiplex-PCR analysis, and all
genotypic information of these 48 patients could not be obtained. In addition, due to
the characteristics of the retrospective study, there was a considerable loss of
clinical data. However, despite the small number of patients and retrospective
nature of the analysis, strengths of this study include the extensive genotype data
and clinical and toxicity data from diagnosis to disease outcome, and that some
results may have important implications for patient management and deserve
validation in a larger, prospective cohort.
In order to identify combinations of genotypes that may influence the clinical
response to MTX, future research needs to consider various genetic polymorphisms
in the folate pathway, which are known to alter enzyme function, alone and in
combination, in a much larger cohort of patients. Additionally, in the near term,
39
analyses of various polymorphisms in the genes for the enzymes involved in the
metabolic pathways of chemotherapeutic agents may directly provide gene-based
information that predicts drug response to treatment of cancer patients, and such
information which may ultimately allow for tailored chemotherapy regimens.
40
CONCLUSIONS
In this study of 48 children with osteosarcoma who were treated with high-dose
MTX, it was identified that common genetic polymorphisms of the folate metabolic
pathway could affect the treatment outcome and toxicity after high-dose MTX
therapy. Patients with DHFR 829CC genotype had significant lower EFS than
patients with the 829CT genotype. MTHFR 677C>T showed a tendency towards
poor histological response to neoadjuvant chemotherapy including high-dose MTX.
Children with the RFC1 80A allele had higher plasma levels of MTX and a higher
risk of severe stomatitis. Patients with lower MTHFR activity (MTHFR 677 CT and
TT genotypes) were subject to greater hepatic toxicity after high-dose MTX therapy.
With regard to adenosine pathway genes, there were no significant associations
between SNPs and the outcome of MTX therapy, except ATIC 347C>G. and liver
toxicity. This study provides considerable information which will further increase
the knowledge of how to use host genetic variations to tailor chemotherapy for
individuals with osteosarcoma.
41
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50
국문 초록
배경 및 목적: 골육종은 소아청소년기에 가장 많이 발생하는 악성
골종양이다. Methotrexate, adriamycin, cisplatin을 포함하는 다 약제
병합요법은 괄목할 만한 치료성적의 향상을 가져왔으나, 이러한 약물의
효과는 약물대사에 관여하는 여러 대사과정에 의해 영향을 받는다. 특히,
methotrexate는 대표적인 folic acid 길항체로서 골육종 치료에
사용되는 중요한 약제이다. Folate 대사과정에 관여하는 유전자의
다양성은 소아골육종 환자에서 고용량 methotrexate 치료 후 반응 및
독성에 중대한 영향을 미칠 것으로 예상된다.
대상 및 방법: 48명의 소아 골육종 환자를 대상으로 혈액과 조직 검체를
이용하여 DNA를 추출하여 분석하였다. 19명의 환자에서 혈액 검체를
이용하였고, 22명의 환자에서 paraffin 보관 처리된 종양 조직 검체를
이용하였으며, 7명의 환자에서는 종양 조직과 혈액 검체 모두에서
DNA를 추출하여 양 검체 간의 유전자 다형성의 차이가 있는지
확인하였다. 환자들은 골육종 진단을 받고 고용량 methotrexate가
포함된 항암치료를 받았고, 현재 치료를 모두 종료한 환자들을 대상으로
하였다. Methotrexate 및 folate의 대사에 관여하는 6개의 유전자를
대상으로 유전자 다형성을 검사하였다. RFC1 유전자의 80G>A, DHFR
51
유전자의 829C>T, MTHFR 유전자의 677C>T와 1298A>C, AMPD1
유전자의 34C>T, ATIC 유전자의 347C>G, ITPA 유전자의 94C>A를
각각 분석하였고, 각 유전자 다형성이 골육종 환자들의 생존율, 고용량
methotrexate를 포함하는 선행항암치료 후 조직학적 괴사 정도,
methotrexate 치료 이후의 부작용 등에 미치는 영향을 비교 분석하였다.
결과: 전체 생존율에 대한 분석에서, DHFR유전자의 발현을
증가시킨다고 알려져 있는 DHFR 829C>T 변이형을 가진 환자의
무병생존율이 CC 유전형을 가지고 있는 환자보다 유의하게 높은 것을
관찰하였다(P=0.045). 선행항암치료 후 조직학적 반응(종양의 괴사
정도)과 통계적인 유의성을 보이는 유전자 다형성은 관찰되지 않았다.
그러나, MTHFR 677C>T 변이형에서 조직학적 반응이 떨어지는 것을
관찰하였다(P=0.078). 고용량 methotrexate 치료 48시간 후 혈중
methotrexate의 농도는 RFC1 80G>A 변이형에서 유의하게
높았고(P=0.027), RFC1 80G>A 변이형에서 grade 3 이상의 구내염이
발생할 위험 역시 유의하게 높았다(P=0.012). 통계적인 유의성은
떨어지나 MTHFR 677C>T 변이형에서 고용량 methotrexate 치료 후
grade 3 이상의 간독성이 발생할 위험이 높았고(P=0.069), 그 외
ATIC 347C>G 변이형에서 간독성과의 연관성이 관찰되었다(P=0.043).
결론: 본 연구는 folate 대사과정에 관여하는 여러 가지 유전자의
다형성이 골육종 환자들의 치료반응, 임상적 결과, 혈중
52
methotrexate 의 농도 및 독성 부작용의 발생 등에 유의한 영향을 미칠
수 있음을 보여주었다. 더 많은 환자를 대상으로 추가적인 연구가
필요하며, 이러한 유전적 다형성과 임상적 결과와의 관련성이 기반이
되어, 향후 골육종 환자의 치료 시, 환자 개개인의 유전적 다형성은
임상적 치료 결과에 대한 유전적 예측인자로 사용될 수 있을 것이며,
개별화된 항암치료를 가능하게 할 것 이다.
주요어: methotrexate, folate, 유전적, 다형성
학번: 2006-30540