Detection of XPO1E571K Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR
Abstract
1. Introduction
2. Results
2.1. Network Analysis of XPO1 in Lymphoma
2.2. Assessment of Plasma and FFPE Tissue Samples for XPO1 Gene Amplification and Sequencing in Lymphoma Patients
2.3. Optimal Critical Temperature (Tc) of FAST-COLD-PCR Assay and Verification of FAST-COLD-PCR Assay for XPO1E571K Detection
2.4. Limit of Detection and of FAST-COLD-PCR Assay for XPO1E571K Mutation Detection
2.5. Detecting XPO1E571K Gene Mutation in Patient Samples and FFPE Tissue Samples Using the FAST-COLD-PCR Assay
3. Discussion
4. Materials and Methods
4.1. Bioinformatics Analysis
4.2. Patients and Sample Collections
4.3. Genomic DNA and cfDNA Preparations
4.4. Detection of XPO1E571K in Patient Samples and FFPE Tissue Samples Using Conventional PCR Assay
4.5. Development of FAST-COLD-PCR Assay
4.6. Assesment of the Limit of Detection of FAST-COLD-PCR Assay
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BCL | B-cell lymphoma |
BL | Burkitt’s lymphoma |
BP | Biological process |
c-Myc | Cellular myelocytomatosis oncogene |
CC | Cellular component |
cfDNA | Cell-free circulating tumor DNA |
cHL | Classical form of Hodgkin lymphoma |
CRM1 | Chromosome region maintenance 1 |
CMIR | Cell-mediated immune response |
DLBCL | Diffuse large B-cell lymphoma |
DNA | Deoxyribonucleic acid. |
E571K | A substitution of glutamate 571 to lysine |
FFPE | Formalin-fixed, paraffin-embedded |
GO | Gene Ontology |
HBB | Hemoglobin Subunit Beta |
HIR | Humoral immune response |
HL | Hodgkin lymphoma |
HRM | High-resolution melt |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
LOD | Limit of detection |
MF | Molecular function |
NHL | Non-Hodgkin lymphoma |
PCR | Polymerase chain reaction |
PMBL | Primary mediastinal B-cell lymphoma |
PPI | Protein–protein interaction |
Tc | Critical temperature |
Tm | Average melting temperature |
UD | Undiluted |
XPO1 | Exportin1 |
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Cycle Step | Temp | Time | Cycles |
---|---|---|---|
Initial denaturation | 94.0 °C | 2 min | 1 |
Denaturation | 94.0 °C | 30 s | 10 |
Annealing | 60.0 °C | 30 s | |
Extension | 68.0 °C | 30 s | |
Denaturation (Tc) | 73.3 °C | 30 s | 30 |
Annealing | 60.0 °C | 30 s | |
Extension | 68.0 °C | 30 s | |
Hold | 4.0 °C | ∞ |
Sample (Patient No.) | Sex | Age | Lymphoma Type | Patient Status | XPO1E571K Mutation |
---|---|---|---|---|---|
1 | Female | 67 | DLBCL | New | + |
2 | Female | 54 | DLBCL | Refractory | + |
3 | Female | 64 | DLBCL | Refractory | + |
4 | Male | 70 | BCL | Refractory | + |
5 | Female | 71 | NHL | New | + |
6 | Female | 71 | NHL | Refractory | + |
7 | Male | 61 | DLBCL | Refractory | + |
8 | Female | 69 | DLBCL | Refractory | + |
9 | Female | 20 | NHL | Refractory | − |
10 | Female | NA | N/A | N/A | − |
11 | Male | 70 | NHL | N/A | − |
12 | Female | N/A | N/A | N/A | − |
13 | Male | N/A | N/A | N/A | − |
14 | Male | N/A | N/A | N/A | − |
15 | Female | 20 | NHL | N/A | − |
16 | Male | 73 | N/A | N/A | − |
17 | Male | 29 | NHL | N/A | − |
18 | Male | 58 | N/A | N/A | − |
19 | Male | 28 | HL | Refractory | − |
20 | Female | N/A | BCL | N/A | − |
21 | Female | 51 | BCL | N/A | − |
22 | Male | 60 | HL | N/A | − |
23 | Female | 53 | NHL | N/A | − |
24 | Female | 32 | NHL | N/A | − |
25 | Male | 40 | N/A | N/A | − |
26 | Female | 21 | HL | N/A | − |
27 | Male | N/A | NHL | N/A | − |
28 | Female | 64 | DLBCL | New | − |
29 | Female | N/A | N/A | N/A | − |
30 | Female | 55 | N/A | N/A | − |
Sample (Patient No.) | Sex | Age | Lymphoma Type | Patient Status | XPO1E571K Mutation |
---|---|---|---|---|---|
1 | Male | 44 | DLBCL | Refractory | − |
2 | Female | 54 | DLBCL | Refractory | − |
3 | Male | 53 | DLBCL | New | − |
4 | Male | 56 | DLBCL | New | − |
5 | Male | 21 | DLBCL | New | − |
6 | Male | 59 | DLBCL | New | − |
7 | Female | 44 | DLBCL | New | − |
8 | Female | 55 | DLBCL | New | − |
9 | Female | 64 | DLBCL | New | − |
10 | Male | 73 | DLBCL | New | − |
11 | Male | 75 | DLBCL | Refractory | − |
12 | Female | 66 | DLBCL | Refractory | − |
13 | Male | 74 | DLBCL | New | − |
14 | Female | 68 | DLBCL | New | − |
15 | Male | 68 | DLBCL | New | − |
16 | Female | 64 | NHL | Relapsed | − |
17 | Male | 66 | DLBCL | Relapsed | − |
18 | Female | 68 | DLBCL | Relapsed | − |
19 | Female | 80 | DLBCL | New | − |
20 | Male | 75 | DLBCL | New | − |
21 | Female | 71 | DLBCL | New | − |
22 | Female | 68 | DLBCL | New | − |
23 | Male | 66 | DLBCL | New | − |
24 | Female | 64 | DLBCL | New | − |
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Duangmano, S.; Viriyaadhammaa, N.; Khamphikham, P.; Intasai, N.; Tantiworawit, A.; Daroontum, T.; Chiampanichayakul, S.; Anuchapreeda, S. Detection of XPO1E571K Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR. Int. J. Mol. Sci. 2025, 26, 7324. https://doi.org/10.3390/ijms26157324
Duangmano S, Viriyaadhammaa N, Khamphikham P, Intasai N, Tantiworawit A, Daroontum T, Chiampanichayakul S, Anuchapreeda S. Detection of XPO1E571K Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR. International Journal of Molecular Sciences. 2025; 26(15):7324. https://doi.org/10.3390/ijms26157324
Chicago/Turabian StyleDuangmano, Suwit, Natsima Viriyaadhammaa, Pinyaphat Khamphikham, Nutjeera Intasai, Adisak Tantiworawit, Teerada Daroontum, Sawitree Chiampanichayakul, and Songyot Anuchapreeda. 2025. "Detection of XPO1E571K Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR" International Journal of Molecular Sciences 26, no. 15: 7324. https://doi.org/10.3390/ijms26157324
APA StyleDuangmano, S., Viriyaadhammaa, N., Khamphikham, P., Intasai, N., Tantiworawit, A., Daroontum, T., Chiampanichayakul, S., & Anuchapreeda, S. (2025). Detection of XPO1E571K Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR. International Journal of Molecular Sciences, 26(15), 7324. https://doi.org/10.3390/ijms26157324