PACLseq: A Standalone Diagnostic Method for Ph-Like Acute Lymphoblastic Leukemia Using Nanopore Sequencing

Abstract

Timely and accurate detection of Philadelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL)-related fusion gene is essential for treatment decisions. However, due to the complexity of possible gene fusion combinations of Ph-like ALL, current diagnostic workflows face critical limitations: prolonged turnaround (7–14 days), high costs, and deficiency in degraded specimens. In this study, we introduce Partial Anchored Capture and Long-Read Sequencing (PACLseq), a nanopore-sequencing-technology-based approach. We designed a detection panel associated with Ph-like ALL, specifically ABL2, CSF1R, PDGFRB, JAK2, ABL1, EPOR, and CRLF2 as target genes. Validated on 47 clinical samples, PACLseq achieved 93.3% sensitivity and 100% specificity in 26 degraded RNA samples (RIN > 3). Crucially, PACLseq maintained detection accuracy in nine low-RIN samples (RIN ≤ 3) with fragmented transcripts. The method requires only 10 ng of RNA input, delivers results in 3 days (vs. 7–14 days for conventional methods), and reduces costs by 50%. By offering rapid and accurate fusion detection, PACLseq has the potential to significantly improve diagnostic efficiency, facilitate timely treatment decisions, and enhance patient outcomes in the management of Ph-like ALL.

Keywords

gene fusion / long-read sequencing / Philadelphia chromosome–like acute lymphoblastic leukemia / Target transcriptome sequencing

References

[1] C. G. Mullighan, X. Su, J. Zhang, et al., “Deletion of IKZF1 and Prognosis in Acute Lymphoblastic Leukemia,” New England Journal of Medicine 360, no. 5 (2009): 470-480.

[2] M. L. Den Boer, M. van Slegtenhorst, R. X. De Menezes, “A Subtype of Childhood Acute Lymphoblastic Leukaemia With Poor Treatment Outcome: A Genome-Wide Classification Study,” Lancet Oncology 10, no. 2 (2009): 125-134.

[3] D. A. Arber, A. Orazi, R. P. Hasserjian, et al., “International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: Integrating Morphologic, Clinical, and Genomic Data,” Blood 140, no. 11 (2022): 1200-1228.

[4] R. Alaggio, C. Amador, I. Anagnostopoulos, et al., “The 5th Edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms,” Leukemia 36, no. 7 (2022): 1720-1748.

[5] S. K. Tasian, M. L. Loh, and S. Hunger, “Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia,” Blood 130, no. 19 (2017): 2064-2072.

[6] K. G. Roberts, Y. Li, D. Payne-Turner, et al., “Targetable Kinase-Activating Lesions in Ph-Like Acute Lymphoblastic Leukemia,” New England Journal of Medicine 371, no. 11 (2014): 1005-1015.

[7] Y. Y. Ding, J. W. Stern, T. F. Jubelirer, et al., “Clinical Efficacy of Ruxolitinib and Chemotherapy in a Child With Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia With GOLGA5-JAK2 Fusion and Induction Failure,” Haematologica 103, no. 9 (2018): e427-e431.

[8] L. M. Niswander, J. P. Loftus, É. Lainey, et al., “Therapeutic Potential of Ruxolitinib and Ponatinib in Patients With EPOR-Rearranged Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia,” Haematologica 106, no. 10 (2021): 2763-2767.

[9] X. Chen, L. Yuan, J. Zhou, et al. “Sustained Remission After Ruxolitinib and Chimeric Antigen Receptor T-Cell Therapy Bridged to a Second Allogeneic Hematopoietic Stem Cell Transplantation for Relapsed Philadelphia Chromosome-Like B-Cell Precursor Acute Lymphoblastic Leukemia With Novel NPHP3-JAK2 Fusion,” Genes, Chromosomes & Cancer 61, no. 1 (2022): 55-58.

[10] Y. Wouters, L. Nevejan, A. Louwagie, et al., “Efficacy of Ruxolitinib in B-Lymphoblastic Leukaemia With the PCM1-JAK2 Fusion Gene,” British Journal of Haematology 192, no. 4 (2021): e112-e115.

[11] K. Inokuchi, S. Wakita, T. Hirakawa, et al., “RCSD1-ABL1-Positive B Lymphoblastic Leukemia Is Sensitive to Dexamethasone and Tyrosine Kinase Inhibitors and Rapidly Evolves Clonally by Chromosomal Translocations,” International Journal of Hematology 94, no. 3 (2011): 255-260.

[12] E. Lengline, K. Beldjord, H. Dombret, J. Soulier, N. Boissel, and E. J. H. Clappier, “Successful Tyrosine Kinase Inhibitor Therapy in a Refractory B-Cell Precursor Acute Lymphoblastic Leukemia With EBF1-PDGFRB Fusion,” Haematologica 98, no. 11 (2013): e146.

[13] B. W. Weston, M. A. Hayden, K. G. Roberts, et al., “Tyrosine Kinase Inhibitor Therapy Induces Remission in a Patient With Refractory EBF1-PDGFRB-Positive Acute Lymphoblastic Leukemia,” Journal of Clinical Oncology 31, no. 25 (2013): e413-e416.

[14] C. Schwab, S. L. Ryan, L. Chilton, et al., “EBF1-PDGFRB Fusion in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Genetic Profile and Clinical Implications,” Blood 127, no. 18 (2016): 2214-2218.

[15] K. G. Roberts, “Why and How to Treat Ph-Like ALL?,” Best Practice & Research Clinical Haematology 31, no. 4 (2018): 351-356.

[16] R. C. Harvey, H. Kang, K. G. Roberts, et al., “Development and Validation of a Highly Sensitive and Specific Gene Expression Classifier To Prospectively Screen and Identify B-Precursor Acute Lymphoblastic Leukemia (ALL) Patients with a Philadelphia Chromosome-Like (“Ph-Like” or “BCR-ABL1-Like”) Signature for Therapeutic Targeting and Clinical Intervention,” Blood 122, no. 21 (2013): 826-826.

[17] H. Inaba, E. M. Azzato, and C. G. Mullighan, “Integration of Next-Generation Sequencing to Treat Acute Lymphoblastic Leukemia With Targetable Lesions: The St. Jude Children's Research Hospital Approach,” Frontiers in Pediatrics 5 (2017): 258.

[18] E. E. Heyer, I. W. Deveson, D. Wooi, et al., “Diagnosis of Fusion Genes Using Targeted RNA Sequencing,” Nature Communications 10, no. 1 (2019): 1388.

[19] G. M. Sheynkman, K. S. Tuttle, F. Laval, et al., “ORF Capture-Seq as a Versatile Method for Targeted Identification of Full-Length Isoforms,” Nature Communications 11, no. 1 (2020): 2326.

[20] Q. Liu, Y. Hu, A. Stucky, L. Fang, J. F. Zhong, and K. Wang, “LongGF: Computational Algorithm and Software Tool for Fast and Accurate Detection of Gene Fusions by Long-Read Transcriptome Sequencing,” BMC Genomics 21, no. 11 (2020): 793.

[21] N. M. Davidson, Y. Chen, T. Sadras, et al., “JAFFAL: Detecting Fusion Genes With Long-Read Transcriptome Sequencing,” Genome Biology 23, no. 1 (2022): 10.

[22] F. Karaoglanoglu, C. Chauve, and F. Hach, “Genion, an Accurate Tool to Detect Gene Fusion From Long Transcriptomics Reads,” BMC Genomics 23, no. 1 (2022): 129.

[23] Y. Chen, Y. Wang, W. Chen, et al., “Gene Fusion Detection and Characterization in Long-Read Cancer Transcriptome Sequencing Data With FusionSeeker,” Cancer Research 83, no. 1 (2023): 28-33.

[24] I. Iacobucci, Y. Li, K. G. Roberts, et al., “Truncating Erythropoietin Receptor Rearrangements in Acute Lymphoblastic Leukemia,” Cancer Cell 29, no. 2 (2016): 186-200.

[25] Y. E. Jang, I. Jang, S. Kim, et al., “ChimerDB 4.0: An Updated and Expanded Database of Fusion Genes,” Nucleic Acids Research 48, no. D1 (2020): D817-D824.

[26] C. A. Maher, C. Kumar-Sinha, X. Cao, et al., “Transcriptome Sequencing to Detect Gene Fusions in Cancer,” Nature 458, no. 7234 (2009): 97-101.

[27] J. S. Reis-Filho, “Next-generation Sequencing,” Breast Cancer Research 11, no. supplement S3 (2009): S12.

[28] J. I. Davila, N. M. Fadra, X. Wang, et al., “Impact of RNA Degradation on Fusion Detection by RNA-seq,” BMC Genomics 17, no. 1 (2016): 814.

[29] S. Konoplev, X. Lu, M. Konopleva, et al., “CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients: A Single-Institution Experience,” American Journal of Clinical Pathology 147, no. 4 (2017): 357-363.

[30] R. C. Harvey and S. K. Tasian, “Clinical Diagnostics and Treatment Strategies for Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia,” Blood Advances 4, no. 1 (2020): 218-228.

[31] K. G. Roberts and C. G. Mullighan, “Genomics in Acute Lymphoblastic Leukaemia: Insights and Treatment Implications,” Nature Reviews Clinical Oncology 12, no. 6 (2015): 344-357.

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