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Department of Internal Medicine II, Friedrich-Schiller-Universität Jena, D-07740 Jena, Germany [M. R., K. H.]; Oncoscreen GmbH, D-07745 Jena, Germany [W. S., P. H., D. B.]; and Academic Medical Center, Laboratory of Genetic Metabolic Diseases, 1100 DE Amsterdam, the Netherlands [A. B. P. V. K., A. H. V. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 min (5)
. The rate-limiting initial step of 5-FU catabolism is catalyzed by DPD (for a recent review, see Ref. 3
), and deficiency in DPD enzyme activity is correlated with a delay in the clearance of 5-FU from the plasma (6
, 7)
. Accordingly, toxic side effects of 5-FU have been linked to low levels of DPD enzyme activity in human blood cells (6
, 8, 9, 10, 11, 12, 13)
. Human DPD cDNA has been cloned and sequenced, and the genomic structure of the DPD gene has been reported (14, 15, 16) . Subsequently, a number of mutations and polymorphisms have been identified, some of which result in a decreased activity of the DPD enzyme (13 , 17, 18, 19, 20) . The most frequent of these mutations is a G to A point mutation within the 5'-splicing donor site of intron 14 (exon 14-skipping mutation; DPD splice site mutation; Refs. 13 and 19 ). Processing of pre-mRNA bearing this mutation results in a loss of exon 14. The resulting protein product is truncated by 55 amino acids; its catalytic activity is virtually absent (17) . A first report of a heterozygous carrier of the exon 14-skipping mutation who had experienced severe toxicity after 5-FU treatment was published in 1996 (11) . Subsequently, additional cases have been reported (13 , 21 , 22) . On the basis of these findings, it has been suggested to screen cancer patients routinely for the exon 14-skipping mutation before 5-FU treatment (11 , 13 , 21) . However, no statistically sound data on the prevalence of this mutation that warranted such a suggestion have been reported to date.
A very recent report provided first data indicating a high frequency (25%) of heterozygotes for the exon 14-skipping mutation among 37 Caucasian cancer patients who had experienced severe 5-FU-related toxicity (13) . In all cases, the mutation was linked to low DPD enzymatic activity in peripheral blood mononuclear cells. However, there have been conflicting data based on a group of 37 (presumably French) patients (20) . Within this group none of the 23 individuals with reduced DPD activity carried the exon 14-skipping mutation.
Here we report, for the first time, prevalence data showing that the exon 14-skipping mutation is present in 1% of the Caucasian population. In addition, we confirm, on the basis of a sample of 25 randomly selected cancer patients who had experienced WHO grade 4 toxicity upon 5-FU treatment, that the frequency of the exon 14-skipping mutation within this group is high (24%).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Control Population of Randomly Selected Hospital Patients.
The frequency of the heterozygous DPD exon 14-skipping mutation was evaluated in a cohort of randomly selected hospital patients by analyzing leukocytes (n = 572), frozen tissue (n = 95), or paraffin-embedded tissue (n = 133) from 800 Caucasian individuals (see Table 2
). The majority (533) of leukocyte samples were from a cell bank of the Department of Internal Medicine II, Friedrich-Schiller-University, Jena, Germany, and had been routinely collected from patients with various hematooncological diseases upon their entry into the clinics. A smaller number of leukocyte samples (39) was from randomly selected colorectal cancer patients (all tumor stages) before chemotherapy. Frozen primary tumor tissue or paraffin-embedded primary tumor tissue was from patients with colorectal lesions (all tumor stages) collected in a tissue bank of Oncoscreen GmbH, Jena, Germany.
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RNA Preparation from EDTA Blood Samples.
EDTA-blood was collected, and the leukocytes were isolated by hypotonic lysis using Erythrocyte Lysis Buffer (Qiagen) and subsequent centrifugation. RNA from leukocytes was isolated using TRIzol (Life Technologies, Inc., GmbH, Karlsruhe, Germany) according to the manufacturer’s recommendations. Average RNA yields were 6 µg/ml blood, which were in accordance with the value indicated by the manufacturer (6–7 µg/7 x 106 WBCs).
RNA Preparation from Fresh-frozen Tissue.
Approximately 5 mg of fresh-frozen tissue were pulverized, and the RNA was extracted using TRIzol (Life Technologies, Inc., GmbH). On average we obtained 15 mg/preparation, which is within the expected range.
RNA Preparation from Paraffin-embedded Tissue.
RNA from paraffin-embedded tissue was prepared as described by Coombs et al. (23)
, with modifications. Briefly, 200 µl of xylene (Roth, Karlsruhe, Germany) was added to each 10-µm section, heated for 10 min to 37°C, centrifuged for 3 min at 12,000 x g, and then xylene was removed by aspiration. The xylene washing was repeated three times. The pellet was washed in 100% ethanol and dried. The pellet was rehydrated in a 300-µl reaction buffer (10 mM TRIS; Life Technologies, Inc., GmbH), 5 mM EDTA (Life Technologies, Inc., GmbH), 0.5% N-lauroylsarcosine (pH 8.0; Sigma Chemical Co., Taufkirchen, Germany), and mechanically homogenized. Proteinase K (Sigma Chemical Co.) was added to a final concentration of 65 µg/ml. After incubation at 55°C for 3 h, the reaction mix was stopped by addition of sodium acetate (0.6 M final concentration; pH 4.0) and the RNA was extracted with phenol/chloroform/isoamylalcohol according to Chomczynski and Sacchi (Ref. 24
; reagents from Life Technologies, Inc., GmbH). For demodification, RNA was incubated for 60 min at 70°C before reverse transcription reaction according to Masuda et al. (25)
. From a 10-µm section (which roughly corresponds to 1 mg of tissue), on average, we obtained 2.4 µg, which was remarkably close to the RNA yield obtained from fresh-frozen tissue.
RT-PCR Analysis.
RNA was precipitated, washed twice in 70% ethanol (Roth), dried, resuspended in 40 µl of distilled water, and reverse transcribed with hexamers (Amersham Pharmacia, Freiburg, Germany) into cDNA using MMLV (Life Technologies, Inc., GmbH) according to the manufacturer’s instructions. Two µl of cDNA were used in a total 50-µl PCR reaction mix containing 47 µl of PCR Supermix (Life Technologies, Inc., GmbH) and 1 µl primer mix (12 µM each forward and reverse primer). The PCR was run for 35 cycles. The DPD mRNA sequence with the GenBank accession no. U09178 was used, with exon 14 ranging from nucleotide 1822 to 1986. For amplification of cDNA derived from blood and fresh-frozen tissue, primers were selected for amplification of a sequence ranging from nucleotide 1939 to nucleotide 2043, with forward primer F13 starting from nucleotide 1939 and reverse primer R15 starting from nucleotide 2043 (Fig. 1, A and B)
. For amplification of cDNA derived from paraffin-embedded tissue, a sequence from nucleotide 1929 to 2043 was amplified with forward primer R14, starting from nucleotide 1929, and reverse primer R15, described above (Fig. 1A)
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RT-PCR Analysis of cDNA Obtained from Paraffin-embedded Sections.
In addition to cDNA from leukocytes, we analyzed cDNA prepared from archival paraffin-embedded tissue to facilitate the analysis of patients who had died during their 5-FU chemotherapy. Preliminary experiments revealed that RNA extracted from paraffin-embedded tissue contained predominantly fragments smaller than 300 bp (data not shown). Although the mutant fragment of 140 bp was easily detectable in these samples, the 305-bp PCR product of the wild-type allele was unlikely to be amplified. For this reason we designed a second PCR reaction with the forward primer located on exon 14 and the reverse primer located on exon 15. PCR resulted in a band of predicted size (115 bp) when cDNA known to contain the wild-type allele was used as a template. RT-PCR-based screening of archival paraffin-embedded tissue thus consisted of two separate RT-PCRs.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As expected (see Fig. 1
), PCR products of the mutant allele, which resulted from the skipping of exon 14, were 165 bp shorter than those of the wild-type allele. Amplification of the wild-type allele from a homozygous control person resulted in a PCR product of 305 bp, whereas amplification of the homozygous mutant allele resulted in a band of 140 bp. PCR of cDNA from a person heterozygous for the exon 14-skipping mutation yielded three bands: a band at 140 bp (the PCR product of the mutant allele), a band at 305 bp (the PCR product of the wild-type allele), and a weaker band of 390 bp. The 390-bp band is caused by a heteroduplex formation of the mutant and wild-type amplicon during the PCR, which is a common phenomenon in competitive PCR (27)
. Only when a high amount of PCR product has accumulated is a very faint band of similar size visible in the wt/wt sample (Fig. 3)
. However, this band is clearly an artifact of PCR at high template/product concentration. In contrast with the other bands, it is no longer visible when PCR is limited to 30 cycles. The 390-bp band does not influence the exact analysis of the genotype. A typical result of the RT-PCR-based screening of cDNA obtained from leukocytes is shown in Fig. 3
. cDNA of leukocytes from a healthy donor known to be heterozygous for the exon 14-skipping mutation was used as a positive control. The identity of both the 140-bp band and the 305-bp band was confirmed by sequencing.
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show that the wild-type 305-bp band is detected only in the heterozygous control derived from leukocytes (Lane 6) but not in any of the four cDNA specimens derived from paraffin-embedded tissue (Fig. 4A
, Lanes 1–4). Primer set F13/R15 did, however, give rise to the 140-bp mutant band, even in paraffin-derived cDNA samples (Fig. 4A
, Lane 4). Thus, an additional primer set F14/R15 (see Fig. 1A
) was designed to amplify a 115-bp band specific for wild-type molecules only. Fig. 4B
shows that this 115-bp wild-type sequence was amplified in all four cDNAs derived from paraffin-embedded tissue as well as in the positive control cDNA. Thus, the specimen in Lane 4 was heterozygous, and the other three paraffin-derived specimens were homozygous for wild-type.
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, patient 8) was proved to be a homozygous mutant using the approach described above. Quantitative TaqMan RT-PCR on the ABI 7700 Prism machine was applied to confirm this result. The TaqMan signal derived from a common sequence (26)
was compared with the TaqMan signal derived from the wild-type-specific PCR product obtained with the primer set F14/R15 (Fig. 1A)
. A TaqMan RT-PCR signal was detected around cycle 27 for the common DPD sequence of all specimens. There was a sound wild-type-specific TaqMan RT-PCR signal around cycle 30 from paraffin-derived cDNA previously tested to be wt/wt (Table 1
, patient 13) or heterozygous (Table 1
, patient 19). However, no wild-type-specific TaqMan signal was detectable from patient 8 after 40 PCR cycles.
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. The remaining 19 patients (76%) did not carry this mutation.
Clinical Presentation of the Patient Homozygous for the DPD Exon 14-skipping Mutation.
Patient 8, a woman 48 years of age with postmenopausal breast cancer (American Joint Committee on Cancer stage IIA), was treated with cyclophosphamide (400 mg/m2), methotrexate (40 mg/m2), and 5-fluorouracil (600 mg/m2) on days 1 and 8 in an adjuvant setting. Three days after completion of the first cycle, she experienced acute bone marrow aplasia. Broad-spectrum antibiotics and granulocyte colony-stimulating factor were administered because of febrile neutropenia. Despite these treatments at the intensive care unit, she suffered from a rapid, progressive, therapy-resistant pneumonia and developed cardiopulmonary failure. The patient died in a multiorgan failure 12 days after the end of the first chemotherapy cycle.
Clinical Presentation of Patients Heterozygous for the DPD Exon 14-skipping Mutation.
Patient 14, a man 63 years of age with UICC stage III colon cancer, received chemotherapy with 5-FU (425 mg/m2) and folinic acid (20 mg/m2) for 5 days as an adjuvant treatment. Ten days after completion of the first cycle, the patient was readmitted to the hospital with leukopenia WHO grade IV, sepsis, and disseminated intravascular coagulation. The patient died 4 days after admission from multiorgan failure.
Patient 19, a man 78 years of age with pulmonary metastasis of a colon cancer, had palliative treatment with 5-FU (425 mg/m2) and folinic acid (20 mg/m2) for 5 days. Five days after the end of the first chemotherapy cycle, the patient was readmitted to the hospital with a photodermatosis. During the subsequent week, the patient developed severe neutropenia and thrombopenia. Despite intensive care treatment, he died from Staphylococcus aureus sepsis in a myocardial failure 8 days after admission.
Patient 7, a man 73 years of age with UICC stage III rectal cancer, was treated with 5-FU (500 mg/m2) for 5 days in an adjuvant setting. After completion of the first cycle, the patient developed WHO stage IV neutropenia and sepsis. After treatment with broad-spectrum antibiotics and cytokines the patient recovered.
Patient 18, a woman 50 years of age with a UICC stage III rectal cancer, was treated with adjuvant 5-FU (500 mg/m2) for 5 days. Therapy had to be terminated after the first cycle because of WHO grade IV mucositis and neutropenia. In-patient treatment was needed for 2 weeks, with administration of i.v. antibiotics and cytokines until the patient recovered.
Patient 24, a woman 72 years of age with a double carcinoma of the sigma (UICC stage III) and coecum (UICC stage III), was treated with adjuvant 5-FU (425 mg/m2) and folinic acid (20 mg/m2) for 5 days. Therapy had to be terminated after the first cycle because of WHO grade IV mucositis and neutropenia. After a period of 8 days, the patient gradually improved, returning to her previous state of health.
Frequency of the DPD Exon 14-skipping Mutation in the Caucasian Control Population.
In an attempt to obtain reliable information on the frequency of the exon 14-skipping mutation within the Caucasian population, cDNA from a total of 851 individuals was tested for the presence of the mutation. Eight of 851 (0.94%) specimens were heterozygous for the exon 14-skipping mutation (Figs. 3
and 4
; see Table 2
for details). The remaining 843 individuals (99.06%) were homozygous for the wild-type allele. None of the individuals tested was homozygous for the DPD exon 14-skipping mutation.
One of two heterozygote mutants initially detected on the basis of paraffin-embedded tumor tissue was confirmed by a blood specimen. From the second patient, no blood could be obtained.
To rule out that heterozygote PCR signals from malignant blood specimens were caused by cells of a homozygote wild-type contaminated by somatically mutated tumor cells, a titration experiment was performed. cDNA of a known heterozygote was added to wild-type cDNA in various proportions. A PCR was performed from this titration series and compared with PCR products of five heterozygotes detected within the cohort of 572 blood specimens. The results presented in Fig. 5
indicate that the ratio wild-type:mutant sequence clearly is 1:1 in all five patients; thus, they are real heterozygotes.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We now present data indicating that the mutation occurs with a frequency of 0.94% within the Caucasian population. These findings are based on 851 donors, the largest cohort screened thus far. In the light of our data, it is obvious why the results of previous studies based on sample numbers between 30 and 131 have been conflicting. We detected the mutation in a heterozygous allelic state in 1 of 51 leukocyte specimens from healthy donors (1.96%), in 5 of 572 leukocyte specimens from patients with various hematooncological disorders (0.87%), and 2 of 228 colorectal tumor specimens (0.88%). No homozygous mutant was detected in our screening study. One may suspect a bias among the donors with malignant disorders, because somatic exon 14-skipping mutations might arise within the tumor tissue, thus resulting in false-positives. However, we were able to rule out this possibility. Thus, the value of nearly 1% heterozygote mutant individuals is representative of the prevalence within the Caucasian population.
To test the hypothesis of heterozygote patients being at significantly increased risk of severe 5-FU-related toxicity, we screened for the occurrence of the exon 14-skipping mutation a cohort of cancer patients who had experienced WHO grade 3–4 toxicity upon 5-FU treatment. The specimens were recruited randomly from German physicians who had reported cases of 5-FU-related WHO grade 3–4 toxicity. Five of 25 patients were heterozygous and one was homozygous for the exon 14-skipping mutation. The homozygote and two of the heterozygotes experienced therapy-resistant sepsis with lethal outcome. WHO grade 4 leukopenia was observed in all of the patients carrying the DPD exon 14-skipping mutation, independent of their allelic status.
A previous report based on a cohort of 37 cancer patients has posed doubts about a high prevalence of the exon 14-skipping mutation among DPD-deficient patients (20) . Our data, however, correspond to a second study published very recently (13) , which also presents evidence that 25% of patients with WHO grade 3–4 deficiency are heterozygous for the exon-skipping mutation. These data are remarkably close to the results we present independently here.
We have to emphasize, however, that the exon 14-skipping mutation clearly is not the only cause for severe or lethal toxic 5-FU side effects (Table 1)
. These findings are in line with recent reports: 43% of 5-FU-related toxicity probably are not attributable to DPD deficiency (13)
, and, in addition to the exon 14-skipping, other loss-of-function mutations of the DPD gene have been detected (13
, 20
, 30
, 31)
. However, exon 14-skipping accounts for approximately one-half of DPD deficiency, rendering this mutation attractive for routine screening (13
, 19)
. Our report adds additional evidence that the exon 14-skipping mutation is very common among patients who suffer from WHO grade 4 leukopenia.
We conclude that approximately one-fourth of patients who experience severe 5-FU-related toxicity carry the exon 14-skipping mutation, with the majority of them being heterozygous. The data presented here and by others (13) strongly indicate that these patients are at high risk of experiencing severe to life-threatening toxicity upon 5-FU treatment. On the basis of a prevalence of nearly 1% within the Caucasian population, it therefore appears that routine genetic screening for this common splice-site mutation should be carried out before 5-FU chemotherapy.
However, it should be pointed out that not everybody who is heterozygous for the mutation may actually suffer severe 5-FU side effects. The individual risk of heterozygous patients suffering from severe or life-threatening 5-FU-related toxicity can only be addressed by pharmacokinetic measurements of the individual 5-FU turnover in heterozygous patients using very low doses of 5-FU. Thus, routine screening for the exon 14-skipping mutation and subsequent individual determination of the 5-FU pharmacokinetics of heterozygous patients provides a concept of individualized therapy and allows the avoidance of undesired treatment toxicity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Thüringer Ministerium für Wissenschaft, Forschung und Kultur, Grant B 309-99008. 
2 To whom requests for reprints should be addressed, at Imperial Cancer Research Fund, Growth Factor Group, Institute of Molecular Medicine, John Radcliffe Hospital Oxford, United Kingdom, OX3 9DS. Phone: 01865-222420; Fax: 01865-222431; E-mail: m.raida{at}icrf.icnet.uk
3 The abbreviations used are: 5-FU, 5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; RT-PCR, reverse transcription-PCR; UICC, Union International Contre Cancer; WT, wild type.
Received 10/ 6/01; revised 5/29/01; accepted 6/ 5/01.
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REFERENCES |
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