# From Sample to Sequencing: The Importance of Pre-Analytical Sample Treatment in NGS Analysis of Patients with Chronic Lymphocytic Leukemia

## TL;DR

This study shows that using purified CD19+ cells instead of mononuclear cells improves the detection of TP53 mutations in chronic lymphocytic leukemia patients.

## Contribution

The study demonstrates that sample preparation significantly affects the accuracy of TP53 mutation detection in CLL.

## Key findings

- TP53 mutation c.626_627del was detected in both sample types, but c.825_826del was only found in CD19+ cells.
- Using CD19+ cells increased the variant allele frequency of the detected mutation.
- Inadequate sample material can lead to false-negative results in TP53 mutation testing.

## Abstract

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by uncontrolled accumulation of B lymphocytes, frequently associated with genetic abnormalities. Among these, alterations of chromosome 17 and TP53 mutations are the most clinically relevant for therapeutic decision-making. Therefore, assessment of TP53 mutational status is strongly recommended in routine diagnostics. This study evaluated the reliability of sequencing results depending on the type of sample used for DNA isolation. Two samples from the same patient were analyzed: DNA from mononuclear cells and DNA from purified CD19+ cells. Next-generation sequencing (NGS) revealed the c.626_627del mutation in both samples, while an additional mutation (c.825_826del) was detected only in sample of DNA isolated from CD19+ cells. These findings highlight that inappropriate starting material may lead to false-negative results, particularly when mutation frequency is low. Ensuring sufficient CD19+ cell content prior to sequencing is essential for accurate detection, enabling precise diagnostics and supporting personalized therapy in CLL patients.

Background/Objectives: Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by uncontrolled accumulation of B lymphocytes. A key feature of CLL is the presence of genetic aberrations, particularly alterations of chromosome 17, such as deletion of 17q and/or mutations in the TP53 gene. Since these abnormalities are highly relevant for therapeutic decision-making, assessment of TP53 mutational status is strongly recommended in routine diagnostics. This study aimed to evaluate the reliability of TP53 sequencing results depending on the type of DNA sample analyzed. Methods: DNA was isolated from two different sample types of the same patient: mononuclear cells (CLL1) and purified CD19+ cells (CLL2). The entire coding region of TP53 (exons 2–11), including splice sites (+/− 10 bp), was analyzed using capture-based next-generation sequencing (NGS). Reads were aligned to the GRCh37/hg19 reference genome, and variants were interpreted using DRAGEN Enrichment (Illumina) and Franklin (QIAGEN). Results: In sample CLL1, the NM_000546.6:c.626_627del mutation (Tier I) was identified with a variant allele frequency (VAF) of 57.06%. The same mutation was confirmed in CLL2, but with a higher VAF of 94.78%. Importantly, an additional Tier I mutation (NM_000546.6:c.825_826del) was detected exclusively in CLL2 at a VAF of 1.59%. Both findings met the required sequencing depth as well as coverage per sample, confirming their validity. Conclusions: The study demonstrates that inadequate starting material for DNA isolation may mask low-frequency TP53 mutations, resulting in false-negative results. Accurate detection requires ensuring sufficient CD19+ cell content, which is critical for reliable diagnostics and supports personalized treatment approaches in CLL.