Abstract
The present study aimed to detect the prevalence of NOTCH1 c.7541-7542delCT mutation in Egyptian CLL patients using HRM assay and to assess its relation to patients’ survival. The study included 50 newly diagnosed treatment-naïve CLL patients and 50 age and sex matched healthy controls. NOTCH1 c.7541-7542delCT mutation was detected using High-resolution melting (HRM) assay and direct Sanger sequencing. Outcome parameters included progression free survival (PFS) and overall survival (OS). NOTCH1 c.7541-7542delCT mutation was detected in 5 (10.0%) of CLL patients. No controls had NOTCH1 c.7541-7542delCT mutation. Similar results were obtained by direct Sanger sequencing yielding a sensitivity and specificity of 100.0% for HRM in detection of NOTCH1 c.7541-7542delCT mutation in the studied patients. In univariate analysis, predictors of OS included Trisomy 12, high LDH, presence of NOTCH1 c.7541-7542delCT mutation and lack of CR. In multivariate analysis, only lack of CR was found as a significant predictor of OS. HRM analysis is a sensitive method for detection of NOTCH1 c.7541-7542delCT mutation in CLL patients. This mutation may be linked to poor disease prognosis.
Keywords: High-resolution melting analysis, DNA sequencing, Chronic lymphocytic leukemia, NOTCH1 mutations, Prognosis
Introduction
The proliferation and aggregation of malignant CD5 + , CD19 + , and CD23 + mature monoclonal B-lymphocytes in the peripheral blood, bone marrow, lymph nodes, and other secondary lymphoid organs define chronic lymphocytic leukemia (CLL) [1].
CLL clinical characteristics and biological habits are extremely diverse. Several factors, including mutations in the immunoglobulin heavy chain variable region (IGHV) gene, zeta-chain-associated protein kinase 70 (ZAP70), CD38 expression and cytogenetic abnormalities help predict prognosis and direct therapeutic decisions. Drug resistance and poor prognosis are associated with recurrent gene mutations in CLL, such as NOTCH1, BIRC3, SF3B1, and MYD88 [2].
NOTCH1 is a non-covalently related heterodimer that encodes a single-pass class I transmembrane protein. It is made up of an extracellular domain that mediates ligand binding and serves as a ligand-activation transcription factor and an intracellular domain mediating signaling [3]. Following binding to the NOTCH receptor, ligands such as Delta-like and Jagged cause the receptor to be proteolytically cleaved, allowing the intracellular domain to be released and translocated to the nucleus, resulting in transcriptional activation of multiple target genes including nuclear factor-κ light-chain-enhancer of activated B cells (NF-κβ). Approximately 80% of all NOTCH1 mutations are found in exon 34, which is chosen to interrupt the PEST domain [4].
NOTCH1 c.7541-7542delCT mutation has been linked to poor prognosis in CLL patients [5]. Techniques used for detection of this mutation include Sanger sequencing, fragment analysis and allele-specific PCR. High-resolution melting (HRM) assay is a more novel technique that was used in detection of CLL related mutations [5, 6].
The present study aimed to detect the prevalence of NOTCH1 c.7541-7542delCT mutation in Egyptian CLL patients using HRM assay and to assess its relation to patients’ survival.
Subjects and Methods
The present study included 50 newly diagnosed treatment-naïve CLL patients and 50 age and sex matched healthy controls. All participants gave informed consent to contribute to this work.
All subjects included in this study were subjected to careful history taking, thorough clinical examination and full laboratory and pathological assessment. CLL was diagnosed according to WHO guidelines [7]. Immunophenotyping was performed to detect CD38 and ZAP70 expression had positive cut-off values of 30 and 20%, respectively. Genotyping was performed using fluorescence in situ hybridization (FISH) analysis to for detection of cytogenetic abnormalities. According to Binet staging system, all patients were classified as stage C.
High-resolution Melting Analysis NOTCH1 c.7541-7542delCT Mutation
CLL cells were removed from bone marrow to obtain somatic DNA. HRM identified the NOTCH1 c.7541-7542delCT mutation, which was confirmed by direct sequencing. DNA was extracted using DNA extraction kits (QIAamp DNA Blood Mini Kits Cat No. /ID: 51,104). The purity of DNA was measured by dividing the absorbance at 260 nm by the absorbance at 280 nm. The A260/A280 ratio of pure DNA was 1.7–2.0.
The primers of NOTCH1 used in this study are listed in Table 1. The NOTCH1 PEST domain was amplified in two fragments (Ex34a and Ex34b) with 131 and 116 bp product sizes, respectively. On a LightCycler 480 instrument, the HRM assay was performed using Quick EvaGreen® qPCR Master Mix (Biotium, Hayward, CA, USA). In a final volume of 20 μl, the reaction mixture contained 1X Quick EvaGreen qPCR Master Mix, 200 nM forward and reverse primers, 50 ng genomic DNA, and PCR grade water. The following were the cycling and melting conditions: 95 °C for 2 min (95 °C for 5 s, 60 °C for 35 s, 72 °C for 25 s) × 50 cycles; HRM: 95 °C for 1 min, 40 °C for 1 min, 65 °C for 1 s, with a continuous temperature rise from 65 to 95 °C at a rate of 0.02 °C per second, with 25 signal acquisitions per degree; and cooling at 40 °C for 30 s. LightCycler 480 was used to examine the melting profiles of the amplicons.
Table 1.
Primers for direct sequencing and HRM assay of the PEST domain of the NOTCH1 gene
| Method | Primer name | Sequences | Amplicon size |
|---|---|---|---|
| Sequencing | Two forward primers: | 327 bp | |
| 1)specific for the mutant allele | For MUT: 5′-TCCTCACCCCGTCCCGA-3′ | ||
| 2) for both mutant and wild-type alleles | For C: 5′-GTGACCGCAGCCCAGTT-3′ | ||
| 3) common reverse primer | Rev, 5′-AAGGCTTGGGAAAGGAAGC-3′ | ||
| HRM | NOTCH1 Ex34a_F | 5'-ACAGCTACTCCTCGCCTGTG-3' |
131 bp 116 bp |
| NOTCH1 Ex34a_R | 5′-GTCGGAGACGTTGGAATGCG-3′ | ||
| NOTCH1 Ex34b_F | 5′-GTGCACACTATTCTGCCCCAG-3′ | ||
| NOTCH1 Ex34b_R | 5′-GAGTAGCTGTGCTGCGAGG-3′ |
Direct Sanger Sequencing
The following thermal profile was used for PCR amplification: 94 °C for 5 min, followed by 40 periods of (94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s) at 72 °C for 10 min. On 2% agarose gels, the PCR products were examined. Purification of PCR products was accompanied by bi-directional sequencing on an ABI 3730 DNA Analyzer (Applied Biosystems, Inc., Beijing, China). The following thermal profile was used for PCR amplification: 94 °C for 5 min, (94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s) × 40 cycles; 72 °C for 10 min. (Table 1).
Treatment Protocol
Fludarabine, cyclophosphamide, and rituximab (FCR) protocol was given to all patients. Cycles were repeated every 28 days for a total of six cycles.
Follow Up and Outcome Parameters
Patients were followed for a median (range) of 8 (1–18) months. Outcome parameters included (1) Treatment response classified as complete response (CR), partial response (PR) or no response (NR), (2) Progression free survival (PFS) and (3) Overall survival (OS).
Statistical Analysis
Data obtained from the present study are expressed as mean ± standard deviation (SD), median and interquartile range (IQR) or number and percent. Numerical data were compared using t test or Mann–Whitney U test while categorical data were compared using chi-square test or Fisher’s exact test. Cox-hazard regression analysis was used to identify predictors of PFS and OS. All statistical operations were computed using SPSS 26 with p value less than 0.05 considered as statistically significant.
Results
The present study included 50 CLL patients and 50 age and sex matched healthy controls. Using HRM method, NOTCH1 c.7541-7542delCT mutation was detected in 5 (10.0%) of CLL patients. No controls had NOTCH1 c.7541-7542delCT mutation. Similar results were obtained by direct Sanger sequencing yielding a sensitivity and specificity of 100.0% for HRM in detection of NOTCH1 c.7541-7542delCT mutation in the studied patients.
At the end of follow up, 13 patients (26.0%) died. Comparison between survivors and non-survivors revealed that survivors had significantly higher platelets count [median (IQR): 104.0 (97.5–115.0) versus 80.0 (65.0–96.5) × 109/L, p < 0.001] and significantly lower β2M levels [median (IQR): 3.3 (3.0–3.45) versus 3.7 (3.25–4.0) mg/L, p = 0.005]. Interestingly, survivors had no NOTCH1 c.7541-7542delCT mutation while non-survivors included 5 patients (38.5%) with NOTCH1 c.7541-7542delCT mutation (p < 0.001). Survivors had significantly higher CR rate (83.8% versus 23.1%, p < 0.001) and significantly lower relapse rate (5.4% versus 30.8%) (Table 2).
Table 2.
Clinical, laboratory and outcome data in survivors and non-survivors
| All patients N = 50 |
Survivors n = 37 |
Non-survivors n = 13 |
p value | |
|---|---|---|---|---|
| Age at diagnosis (years) mean ± SD | 53.6 ± 5.7 | 53.7 ± 5.8 | 54.8 ± 6.8 | 0.57 |
| Male/female n | 29/21 | 23/14 | 6/7 | 0.31 |
| Clinical findings n (%) | ||||
| Splenomegaly | 36 (72.0) | 25 (67.6) | 11 (84.6) | 0.24 |
| Lymphadenopathy | 43 (86.0) | 30 (81.1) | 13 (100.01) | 0.09 |
| Laboratory data median (IQR) | ||||
| Hemoglobin concentration (g/dL) | 10.0 (8.8–12.5) | 9.5 (8.4–10.2) | 9.0 (8.0–9.6) | 0.2 |
| Total leucocytic count (× 109/L) | 63.0 (10.0–80.0) | 70.0 (61.0–84.5) | 89.0 (64.0–97.0) | 0.13 |
| Absolute lymphocytic count (× 109/L) | 42.0 (40.0–55.0) | 44.0 (39.5–56.0) | 54.0 (42.5–75.6) | 0.14 |
| Relative lymphocytic count (%) | 63.0 (45.0–71.0) | 66.0 (61.5–72.0) | 68.0 (61.5–76.5) | 0.6 |
| Platelet count (× 109/L) | 108.0 (96.0–180.0) | 104.0 (97.5–115.0) | 80.0 (65.0–96.5) | < 0.001 |
| LDH (U/L) | 730.0 (280.0–820.0) | 756.0 (690.0–820.0) | 820.0 (752.5–990.0) | 0.07 |
| β2M (mg/L) | 3.1 (2.0–3.5) | 3.3 (3.0–3.45) | 3.7 (3.25–4.0) | 0.005 |
| Trisomy 12 | 7 (14.0) | 1 (2.7) | 6 (46.2) | < 0.001 |
| NOTCH1 c.7541-7542delCT mutation n (%) | 5 (10.0) | – | 5 (38.5) | < 0.001 |
| Treatment response n (%) | ||||
| CR | 34 (68.0) | 31 (83.8) | 3 (23.1) | < 0.001 |
| PR | 4 (8.0) | 4 (10.8) | – | |
| NR | 12 (24.0) | 2 (5.4) | 10 (76.9) | |
| Relapse n (%) | 6 (12.0) | 2 (5.4) | 4 (30.8) | 0.015 |
Cox-hazard regression analysis identified trisomy 12 genotype, high LDH levels, presence of NOTCH1 c.7541-7542delCT mutation and lack of CR as predictors of PFS in univariate analysis. However, in multivariate analysis only lack of CR was significant predictor of PFS [HR (95% CI): 0.11 (0.02–0.55), p = 0.007] (Table 3). In univariate analysis, predictors of OS included Trisomy 12, high LDH, presence of NOTCH1 c.7541-7542delCT mutation and lack of CR. In multivariate analysis, only lack of CR was found as a significant predictor of OS [HR (95% CI): 6.24 (1.26–36.64), p = 0.025] (Table 3).
Table 3.
Outcome predictors in the studied patients
| PFS | |||||||
|---|---|---|---|---|---|---|---|
| Univariate analysis | Multivariate analysis | ||||||
| HR | 95% CI | p | HR | 95% CI | p | ||
| Trisomy 12 | 0.11 | 0.04–0.34 | < 0.001 | 0.29 | 0.05–1.79 | 0.18 | |
| LDH | 1.0 | 1.0–1.1 | 0.03 | 1.0 | 0.99–1.01 | 0.2 | |
| NOTCH1 c.7541-7542delCT mutation | 0.16 | 0.05–0.48 | 0.001 | 4.39 | 0.33–59.1 | 0.27 | |
| CR | 0.09 | 0.025–0.32 | < 0.001 | 0.11 | 0.02–0.55 | 0.007 | |
| OS | |||||||
| Trisomy 12 | 4.9 | 1.62–14.73 | 0.005 | 1.34 | 0.14–12.8 | 0.81 | |
| LDH | 1.0 | 1.0–1.01 | 0.007 | 1.0 | 0.99–1.01 | 0.18 | |
| NOTCH1 c.7541-7542delCT mutation | 0.13 | 0.04–0.42 | 0.001 | 0.81 | 0.04–16.6 | 0.89 | |
| CR | 7.4 | 2.01–27.26 | 0.003 | 6.24 | 1.26–36.64 | 0.025 | |
Discussion
The present study assessed the sensitivity of HRM in detection of NOTCH1 c.7541-7542delCT mutation in CLL patients. In this regard, we found perfect sensitivity and specificity of HRM in detection of this mutation considering direct Sanger sequencing as the reference method. Similar perfect performance of HRM assay was reported by the study of Xu et al. [5]. The concordance between HRM and direct Sanger sequencing methods was also reported in identification of other mutations in other malignant conditions [8, 9]. In addition, HRM has the added advantages of being less costly and quicker. Another benefit of HRM is that it is performed in a "closed-tube" device, eliminating the possibility of post-PCR product contamination [10].
NOTCH1 c.7541-7542delCT mutation was detected in 5 patients (10.0%) in our work. In line with our findings, the Italian study of Pozzo et al. [11] found c.7541-7542delCT mutation in 12.0% of 692 CLL patients using amplification refractory mutation system. In contrast, another Italian study reported c.7541-7542delCT mutation in a much higher percentage (53.4%) of their study on 88 CLL patients [12] using droplet digital PCR. In the study of Vavrova et al. [6] from the Czech Republic, the authors found the mutation in 15% of patients using the fragment analysis method. In comparison, the Indian study of Srinivasan et al. [13] identified this mutation in only 1 patient (1.9%) of their 52-patient series. These findings reveal a wide variation between different patients’ populations regarding the prevalence and methods of detection of NOTCH1 c.7541-7542delCT mutation in CLL patients and highlights the need for more studies to uncover the prevalence of this mutation among different ethnic groups.
In our study, all patients with NOTCH1 c.7541-7542delCT mutation died at the end of follow up. While NOTCH1 c.7541-7542delCT mutation was a significant predictor of PFS and OS in univariate cox-hazard regression analysis, only lack of CR was a significant predictor of PFS and OS in multivariate analysis.
In accordance with our conclusions, Rossi et al. [14] reported an association between NOTCH1 mutations and CLL prognosis. Also, Del Poeta et al. [15] suggested a link between NOTCH1 mutations and deficient apoptosis, lower redistribution lymphocytosis, and shorter PFS and OS in CLL patients.
The link between NOTCH1 mutations and CLL prognosis has many explanations. NOTCH1 mutations cause a progressive clinical phenotype that necessitates treatment soon after the onset of symptoms [16]. Also, NOTCH1 mutations are prevalent among chemo-refractory CLL patients [17]. Activation of NOTCH1 in vitro confers resistance to apoptosis via NF-B pathway activation. C-terminal PEST domain in normal conditions is required to limit the intensity and duration of NOTCH1 activation. As a result of NOTCH1 mutation, the PEST domain is removed from NOTCH1, resulting in impaired degradation and accumulation of an active NOTCH1 isoform, resulting in deregulated signaling [18].
Targeting NOTCH1 is a promising therapeutic field in CLL patients. Gamma secretase inhibitors (GSIs) are the most thoroughly investigated anti-NOTCH1 molecules, and their combination with fludarabine has shown anti-tumor effects in primary CLL with NOTCH1 mutation [19]. In relapsed/refractory lymphoid malignancies, a humanized antibody targeting NOTCH1 (OMP-52M51) has entered phase I testing. However, the combination of small molecule inhibitors targeting both the BCR pathway and the anti-apoptotic Bcl-2 protein is needed for future treatment of CLL with NOTCH1 mutation [20, 21].
In conclusion, HRM analysis is a sensitive method for detection of NOTCH1 c.7541-7542delCT mutation in CLL patients. This mutation may be linked to poor disease prognosis.
Acknowledgements
None.
Funding
The research is self-funded from the authors.
Declarations
Competing interests
Authors state no conflict of interest.
Ethical Approval
The local Institutional Review Board deemed the study exempt from review.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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