Prediction of severe toxicity in adult patients under treatment with 5-Fluorouracil: a prospective cohort study
Supplemental Material
Carolina Vázquez a, b, María Orlova b, Federico Angriman b, c, José Nicolás Minatta d, Paula Scibona a, María Alicia Verzura d, Esteban Gabriel Jáuregui a, Heidy Díaz de Arce e, María Guadalupe Pallotta d, Waldo Horacio Belloso a.
a. Clinical Pharmacology Section. Internal Medicine Department. Hospital Italiano de Buenos Aires.
Buenos Aires, Argentina.
b. Internal Medicine Department. Hospital Italiano de Buenos Aires. Buenos Aires, Argentina.
c. Pharmacology Department. University of Buenos Aires, School of Medicine. Buenos Aires, Argentina.
d. Clinical Oncology Section. Internal Medicine Department. Hospital Italiano de Buenos Aires. Buenos Aires, Argentina.
e. Molecular Biology Section, Central Laboratory. Hospital Italiano de Buenos Aires. Buenos Aires, Argentina
Corresponding Author: Dr. Carolina Vázquez. [email protected]
J.D. Perón 4190. Farmacología Clínica. Hospital Italiano de Buenos Aires. ZIP: (C1181ACH). Ciudad Autónoma de Buenos Aires. Argentina. Tel: +54-11-4959-0866
DNA processing and Genotyping
Regarding sample collection and DNA extraction, following the informed consent process, 10 mL of peripheral blood were collected from each subject in 5-mL EDTA tubes. Whole blood samples were stored at 4ºC until the time of processing. Genomic DNA was extracted and purified using the QIAmp DNA Blood Mini kit (QIAGEN ® Germany). Quality and quantity of DNA was checked by
spectrophotometry (NanoDrop ® Thermo Fisher-Scientific, USA).
The genotyping assays were based on PCR and Sanger sequencing that is considered to be the gold standard in determining gene variations providing a high degree of accuracy and high sensitivity, especially in detecting genetic variants in genomic DNA. For the genotyping of MTHFR exon 4 and 7 SNPs, we performed PCR and Sanger sequencing. In each PCR reaction, genomic DNA was amplified using the same sequence-specific primers originally designed. For MTHFR exon 4 SNP (677C>T,
rs1801133) region: 5-AAGATCAGAGCCCCCAAAGC-3 (forward) and 5-CGGGATCTTTCCCATCCAGG-3 (reverse) (IDT® USA) generating an amplicon of 309 bp. For MTHFR exon 7 SNP (1298A>C, rs1801131) region: 5-GCATGCTTGTGGTTGACCTG-3 (forward) and 5-CGGGAGAACCAAACCGGAAT-3 (reverse) (IDT®) generating an amplicon of 294 bp. The PCR was performed in a 50-ul mixture containing final volume: 1X PCR buffer (Invitrogen® USA), 1.5 mM Magnesium Chloride (Invitrogen®), 200 uM dNTPs (Sigma® USA), 500 nM of each primer, 1.5 U Taq polymerase (Invitrogen®) and 100 ng genomic DNA. Reactions were performed in an Applied Biosystems® Thermocycler (USA). Cycling parameters for regions of MTHFR exon 4 and 7 were as follows: initial denaturation at 94ºC for 2 min and 35 cycles of 94ºC for 30 sec, 50ºC for 30 sec, and 72ºC for 30 sec, and a final extension step at 72ºC for 7 min followed by 4°C ∞. In order to characterize the amplicon identity, DNA fragments of all expected sizes were submitted for
purification and sequencing to an external laboratory (Macrogen®, South Korea). The quality of each determined sequence was analyzed manually using Chromas 1.56 program,
(http://www.technelysium.com.au/chromas.html) and each one was compared for sequence similarity with sequences deposited in the GenBank database using a BLAST search at NCBI site
(http://www.ncbi.nlm.nih.gov). For the purpose of study samples, amplification products were checked by electrophoresis on a 2% agarose gel, stained with ethidium bromide (0.5 g/mL) and cleaned directly by QIAquick PCR Purification Kit (Qiagen®). DNA sequencing reactions for the four amplified strands were prepared with GenomeLab® Dye Terminator Cycle Sequencing Quick Start Kit, following the manufacturer’s directions (Beckman Coulter®-USA). Sequencing products were read on a Genome Lab GeXP Genetic Analysis System (Beckman Coulter®).
For genotyping the TYMS 5-UTR polymorphism a PCR and polyacrylamide gel electrophoresis were performed as previously described with minor modifications [23]. In each PCR reaction, genomic DNA was amplified using the same sequence-specific primers for TYMS 5-UTR region (rs45445694): 5- AGGCGCGCGGAAGGGGTCCT-3 (forward) and 5-TCCGAGCCGGCCACAGGCAT-3 (reverse) (IDT®) generating
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a 113 bp amplicon in 2R/2R genotype, and a 141 bp amplicon in 3R/3R genotype. The PCR was
performed in a 50-ul mixture containing final volume: 1X PCR buffer (Invitrogen®), 1.5 mM Magnesium Chloride (Invitrogen®), 200 uM dNTPs (Sigma®), 500 nM of each primer, 1.5 U Taq polymerase
(Invitrogen®), 1 X Betaine (Qiagen®), and 100 ng genomic DNA. Reactions were performed in an Applied Biosystems ™ Thermocycler. Cycling parameters for TYMS were as follows: initial denaturation at 95ºC for 15 min and 30 cycles of 91ºC for 1 min, 60ºC for 1 min, and 72ºC for 1 min, and a final extension step at 72ºC for 10 min followed by 4°C ∞. In order to characterize the amplicon identity, DNA fragments of all expected sizes were submitted for purification and sequencing to an external laboratory (Macrogen®).
The quality of each determined sequence was analyzed manually using Chromas 1.56 program
(http://www.technelysium.com.au/chromas.html) and each one was compared for sequence similarity with sequences deposited in the GenBank database using a BLAST search at NCBI site
(http://www.ncbi.nlm.nih.gov). Aliquots of PCR products were run in an 8% polyacrylamide gel and stained with ethidium bromide, and bands were visualized with a UV transilluminator and genotyping was completed (Figure 1s: 5’-UTR-TYMS variants).
Figure 1s. 5’-UTR-TYMS variants genotyping with PCR on 8% polyacrylamide gel, stained with ethidium bromide. A 141 bp band corresponding with 3R/3R genotype, two bands of 141/113 bp corresponding to 3R/2R heterozygous genotype and a 113 bp band corresponding to 2R/2R genotype. On the right and left side of the figure, 50 kb ladder for reference.
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Figure 2s. Sigmoid-like relationship of 5-FU dose per cycle in g/m2 with the log hazard of severe toxicity
Table 1s: Univariate cox regression analysis between toxicity and mortality/treatment completion
Endpoint considered Crude HR (95% CI) p1
Mortality upon end of follow up 1.05 (0.50-2.18) 0.89
Completion of schedule chemotherapy 0.68 (0.44-1.06) 0.09
1. P value based on a univariate Cox regression model
Table 2s: Baseline inflammatory parameters of patients with and without severe toxicity during follow up Baseline Covariates Severe Toxicity
(n:79)
Without Severe Toxicity (n: 118)
p1
Albumin (mg/dl) (mean, SD) 3.7 ± 0.8 3.6 ± 0.9 0.57
White blood cells (by mm3) (mean, SD) 7948.1 ± 2401.8 8408.74 ± 2652.4 0.21 Platelets to lymphocytes ratio (PLR) (median,
IQR 75)
Neutrophils to lymphocyte ratio (NLR) (median, IQR 75)
136.5 [94.9]
2.6 [1.4]
146.2 [85.4]
2.6 [ 1.9]
0.54 0.31
Non-normal distributed variables are expressed in median and interquartile range (IQR, 25th - 75th percentile).
1. Two sided p value. Means are compared with t tests, medians with Mann-Whitney U tests.
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Table 3s: Accelerated failure time model for first severe toxicity event
Candidate covariate Time ratio (95% CI) p1 MTHFR exon 4 (677 C>T) TT 0.80 (0.44-1.44) 0.45 MTHFR exon 7 (1298 A>C) CC 1.32 (0.63-2.76) 0.46
5´-UTR TYMS 2R/2R 0.98 (0.59-1.61) 0.93
EORTC FS 1.02 (1.00-1.03) 0.02
Tumor type*
Anus 0.38 (0.15-0.97) 0.04
Head and neck, esophagus or
stomach 0.44 (0.24-0.78) < 0.01
Others (pancreas, small bowel) 0.62 (0.21-1.87) 0.40 5-FU Dose per cycle (g/m2)
0.3-3.0 0.90 (0.30-2.74) 0.85
3.1-5.0 1.00 (0.56-1.81) 0.99
5-FU infusion regimens# Continuous infusion over 24-36 hs
Continuous infusion over 4-5 days
1.04 (0.37-2.94) 0.19 (0.06-0.59)
0.94
< 0.01
Concomitant radiotherapy 0.57 (0.26-1.24) 0.15
1. Accelerated failure time model with gamma distribution.
* Reference level: colorectal cancer # Reference level: bolus infusion
Abbreviations: EORTC: European Organization for Research and Treatment of Cancer. FS:
functional scale, MTHFR: methylenetetrahydrofolate reductase gene, TYMS: thymidylate synthase gene
Table 4s. Inverse probability weighting analysis of a cox proportional hazards model in order to account for censoring
#Candidate covariate Hazard Ratio 95% CI p
MTHFR exon 4 (677 C>T) TT 1.02 0.46-2.27 0.97 MTHFR exon 7 (1298 A>C) CC 0.51 0.18-1.43 0.20
5´-UTR TYMS 2R/2R 0.96 0.51-1.78 0.89
EORTC FS 0.98 0.97-1.00 0.01
Tumor type*
Head and neck, esophagus or stomach 3.07 1.68-5.60 < 0.01
Others (pancreas, small bowel) 2.00 0.76-5.27 0.16
5-FU Dose per cycle (g/m2)
< 3 g/m2
> 3 g/m2
1.39 1.04
0.31-6.16 0.61 - 1.79
0.66 0.89
Concomitant radiotherapy 1.41 0.65-3.03 0.38
Infusion1 24 - 36 hs 4 - 5 days
1.22 4.18
0.36 - 3.49 1.06 - 16.42
0.84 0.04
Abbreviations: EORTC: European Organization for Research and Treatment of Cancer. FS: functional scale, MTHFR: methylenetetrahydrofolate reductase gene, TYMS: thymidylate synthase gene
# the analysis was performed including 25 censored patients (no recorded toxicity) from the categories: death and lost to follow-up
*Reference level: colorectal cancer and anus 1. Reference level: bolus infusion.
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