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Identification and validation of the optimal reference genes for standardizing the gene expression profiling diagnostic panel of Ph-like B-lineage acute lymphoblastic leukemia

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Abstract

Gene expression profiling is the criterion standard for recognizing Ph-like ALL signatures among B-ALLs. The prerequisite of GEP is the accurate normalization of target genes with stable expression of housekeeping genes in a quantitative PCR. HKGs exhibit differential expression in the different experimental conditions and affect the target genes' expression, leading to imprecise qPCR results. The selection of stable HKGs is crucial in GEP experiments, especially in identifying high-risk Ph-like ALL cases. We have evaluated the expression stability of nine HKGs (GAPDH, ACTB, GUSB, RNA18S, EEF2, PGK1, B2M, TBP and ABL1) in identified Ph-like ALLs and Ph-negative (n = 23 each) using six algorithms, 4 traditional softwares; geNorm, BestKeeper, NormFinder, Delta Cq value method, and two algorithms, RefFinderTM and ComprFinder. Further, we have validated the expression of 8 overexpressed normalized genes in Ph-like ALL cases (JCHAIN, CA6, MUC4, SPATS2L, BMPR1B, CRLF2, ADGRF1 and NRXN3). GeNorm, BestKeeper, NormFinder, Delta Cq value method, RefFinderTM and ComprFinder algorithm analysis revealed that EEF2, GAPDH, and PGK1 form the best representative HKGs in Ph-like ALL cases, while RNA18s, ß-actin, and ABL1 in Ph-negative ALLs. Lastly, we performed a correlation analysis and found that the combination of EEF2, GAPDH, and PGK1 represents the best combination with a very high correlation in Ph-like ALL cases. This is the first report that shows EEF2, GAPDH, and PGK1 are the best HKG genes and can be used in the diagnostic panel of Ph-like ALL cases using qPCR at baseline diagnosis.

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Data availability

The data will be provided upon request, and all the data generated during this study are included in this research article.

Change history

  • 26 August 2023

    In the introduction section, reference citation has been updated from [3, 5–14, 14–21] to [3, 5–21]

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Acknowledgements

We are highly thankful to the Department of Hematology faculty members for providing the necessary infrastructure and facilities for this research study. We also thank the Department of Paediatrics, Internal Medicine and Clinical Hematology and Medical Oncology for providing the pediatric and adult B-ALL samples. Our sincere appreciation to the clerical staff (Mr. Gurpreet) of Hematology for their valuable support and contribution to this research work.

Funding

Supported by the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India (Grant INT/IEC/2019/000611; 19.03.2019).

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Authors and Affiliations

  1. Department of Urology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Chicago, IL, 60611, USA

    Dikshat Gopal Gupta & Sarki Abba Abdulkadir

  2. Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Neelam Varma, Man Updesh Singh Sachdeva, Shano Naseem, Parveen Bose & Jogeshwar Binota

  3. Department of Anatomy, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Parmod Singh

  4. Department of Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA

    Mohammad Rizwan Siddiqui

  5. Department of Clinical Hematology and Medical Oncology, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Pankaj Malhotra & Subhash Varma

  6. Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Alka Khadwal

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  1. Dikshat Gopal Gupta
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Contributions

DGG and NV conceptualized and designed the experimental plan of the research study. DGG, JB, and PB performed the outlined experiments planned in the study; DGG and NV wrote and prepared the manuscript. DGG SAA, and PS, MRS, performed the statistical analysis and analyzed the generated research data. PM, AK, and SV provided the B-ALL samples in this study. All other authors read, approved, and provided the necessary intellectual comments to the submitted manuscript.

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Correspondence to Neelam Varma.

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Post Graduate Institute of Medical Education and Research (PGIMER) constituted Institutional Ethics Committee (IEC) approved this study (vide no. INT/IEC/2019/000611; 19.03.2019). Written consent Performa was obtained from all the enrolled patients, and all the procedures were performed according to the Helsinki Declaration 1975 (Revised 2008).

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Gupta, D.G., Varma, N., Abdulkadir, S.A. et al. Identification and validation of the optimal reference genes for standardizing the gene expression profiling diagnostic panel of Ph-like B-lineage acute lymphoblastic leukemia. Clin Exp Med 23, 4539–4551 (2023). https://doi.org/10.1007/s10238-023-01131-z

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