False-Negative TP53 Results Linked to Inadequate B-Cell Content in CLL Samples

A new study published in Cancers shows that something as seemingly straightforward as sample preparation can make the difference between detecting a critical mutation and missing it entirely in patients with chronic lymphocytic leukemia (CLL).¹ The findings suggest that the type of biological sample used for DNA extraction has a significant impact on whether clinicians can identify TP53 mutations that directly influence treatment decisions.

Researchers at the University Hospital Center Osijek in Croatia analyzed peripheral blood samples from an 81-year-old male patient being evaluated for suspected CLL. Laboratory analysis showed leukocytosis with lymphocytosis, with white blood cell counts reaching 51.56 × 10⁹/L and absolute lymphocyte counts of 40.51 × 10⁹/L. The patient also presented with normocytic anemia, with hemoglobin levels at 104 g/L. Fluorescence in situ hybridization analysis revealed biallelic deletion of the Rb1 gene in 86% of interphase nuclei, though no deletions of 11q, 17p, and 13q34 or trisomy of chromosome 12 were detected.

DNA was isolated in parallel from 2 different sample types from this patient: mononuclear cells separated using density gradient centrifugation (designated CLL1) and purified CD19-positive cells obtained through magnetic bead separation (designated CLL2). Both samples underwent comprehensive NGS analysis targeting the entire coding region of the TP53 gene, including exons 2 through 11 and splice sites. Sequencing performance exceeded recommended quality thresholds in both preparations.

The NGS analysis uncovered significant differences between how the 2 sample types performed. Flow cytometry analysis showed that CLL B cells made up 53% of the total cell population in the peripheral blood sample. In the CLL1 sample derived from mononuclear cells, researchers identified the mutation c.626_627del with a variant allele frequency (VAF) of 57.06%. This mutation, located in exon 6 of the TP53 gene, is classified as pathogenic with tier I clinical significance.

The same mutation was confirmed in the CLL2 sample from purified CD19-positive cells, but with a substantially higher VAF of 94.78%. More importantly, an additional tier I pathogenic mutation, c.825_826del, was detected exclusively in the CLL2 sample at a VAF of 1.59%. This second mutation, located in exon 8, would have been completely missed if only the mononuclear cell sample had been analyzed.

The authors note that “the updated 2024 ERIC guidelines recommend reporting all clinically relevant tier I-III variants (pathogenic, likely pathogenic, or of uncertain significance) regardless of VAF, aligning with treatment recommendations to avoid CIT in any patient with TP53 aberrations.” This is based on multiple retrospective studies showing that low VAF TP53 mutations can expand under treatment pressure and confer resistance similar to dominant TP53 clones.² These patients should instead receive continuous BTK inhibitor monotherapy without a predetermined cessation endpoint, as subclonal TP53 aberrations confer a similarly poor prognosis as dominant clones.

The current study connects these evolving clinical standards directly to laboratory practice.¹ While a minimum overall lymphocyte fraction of 60% to 70% has been commonly used as a precondition for NGS testing, the authors point out that meeting this threshold does not necessarily indicate that there are sufficient malignant B cells to ensure accurate results. “Our experience highlights this challenge. We frequently received samples with a high overall lymphocyte count, yet with <50% CLL B cells confirmed by flow cytometry. In such cases, the reliability of detecting low-VAF TP53 variants becomes questionable. This was exemplified in our study by the variant c.825_826del (p.(A276Lfs*29)), detected at a VAF of 1.59%,” the authors explain.

Although the analysis was performed in a single patient due to logistical difficulties in obtaining paired low- and high-VAF TP53 mutation samples, the technical validation, sequencing depth, and reproducibility testing provide strong proof of principle. The authors concluded that pre-analytical sample handling must be viewed as an integral determinant of diagnostic accuracy. Failure to enrich for the malignant B cell population may obscure clinically decisive genomic information and lead to inappropriate front-line therapy choices.

References

1. Suver Stević M, Holik H, Periša V, Marczi S, Kolobarić N, Samardžija M. From sample to sequencing: the importance of pre-analytical sample treatment in NGS analysis of patients with chronic lymphocytic leukemia. Cancers. 2025;17(22):3668. doi:10.3390/cancers17223668

2. Malcikova J, Pavlova S, Baliakas P, et al. ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia. 2024 update. Leukemia. 2024;38:1455-1468. doi:10.1038/s41375-024-02214-6