Ultra-Deep Sequencing of TP53 in Multiple Myeloma: Double-Hit Prevalence and Clinical Phenotype

Introduction:

The detection of 17p deletion (del(17p)) is recommended as part of the initial risk assessment in newly diagnosed multiple myeloma (MM). Recently, the co-occurrence of a del(17p) and mutation(s) in TP53 gene has led to the definition of Double Hit multiple myeloma (DHMM), which is associated with a worse prognosis. However, TP53 sequencing is not widely performed in the MM workup, primarily due to technical issues regarding sample collection and plasma cell (PC) enrichment. In this project, we aimed to overcome these limitations by employing an ultra-deep TP53 sequencing strategy using NGS on bone marrow immunoselected PCs. We sought to assess the real prevalence of DHMM cases and their clinical impact.

Methods:

Bone marrow aspirates from 185 patients (pts) with MM were subjected to PC immunoselection (CD138+) (Miltenyi, Germany) from 2021 to 2024. DNA was sequenced on an Illumina platform (San Diego, California) covering all TP53 exons with a depth of coverage of 20,000x reads per nucleotide. Only non-synonymous variants with MAF <0.01 and VAF >1% were considered. In all cases, cytogenetic and FISH results, including del(17p), were collected. The cancer clonal fraction (CCF) was defined as the percentage of del(17p) positive cells per enriched sample. Del(17p) cases were categorized into high-risk (CCF >0,55) and low-risk (CCF <0,55) groups. A random analysis of 15 cases showed a mean purity (percentage of CD138+ cell in the enriched sample) of 91.6%.

Results:

The study included 185 pts: 139 at diagnosis and 46 at relapse. Among these cases, we detected 39 TP53 mutations in 35 patients, with 4 patients presenting 2 distinct variants. The mean VAF of TP53 was 21% (range 1,2%-87%). Sixteen variants (41%) showed a VAF between slightly over (<3%) our positive threshold of 1%. Considering the coverage depth of 20,000x, we ensured a conservative minimum of 200 positive reads for a positive result.

In the 139 patients studied at diagnosis, the genomic alterations affecting TP53 were: 11 (8%) pts had a monoallelic mutation, 8 (5,3%) had isolated del(17p), and 8 (5,3%) had both a TP53 monoallelic mutation and del(17p), meeting DHMM criteria. This strategy showed an increased TP53 mutation rate compared to published data (13,3% vs. 3-6% in recent series).

The DHMM group was diagnosed at a significant younger age (65 vs. 73 years old, p=0.037), had higher infiltration of PC in BM (75,5% vs. 30,5%, p<0.0001), lower hemoglobin levels (9,8 g/dL vs. 11,1gr/dL, p=0,035) and a higher percentage of plasmacytoma (50% vs. 19,4%, p=0,046). None of the 8 DHMM were diagnosed as smoldering MM. Among the 8 DHMM patients, 5 (62,5%) were classified as high-risk del(17p), a significantly higher percentage compared to pts with isolated del(17p) (12,5%), further supporting the more aggressive clinical phenotype seen in DHMM patients. With a median follow up of 25,5 months, no significant differences in overall survival (OS) were found based on the type of TP53 abnormality.

In the group of 46 patients sequenced at relapse, we detected 11 (24%) patients with monoallelic TP53 mutation, 7 (15,2%) with del(17p), and 5 (10,8%) with DHMM. At relapse, the percentage of TP53-mutated cases, including DHMM, was significantly higher compared to those sequenced at diagnosis (13,3% vs. 34,8%, p=0.001). Notably, there were no significant differences of DHMM between patients sequenced at diagnosis or relapse (5,3% vs. 10,8%, p=0,49).

Longitudinal samples from 11 patients were sequenced both at diagnosis and at relapse; 3 of these pts acquired a TP53 mutation at relapse that was not detected at diagnosis, and another pt had one TP53 variant at diagnosis, adding two additional variants at relapse.

Conclusions:

The dual strategy of PC enrichment followed by ultra-deep NGS sequencing enhances the detection rate of TP53 mutations in MM both at diagnosis and at relapse, identifying a higher subset of pts meeting DHMM criteria. DHMM cases at diagnosis were associated with an advanced-disease and, interestingly, in significantly younger patients. We did not observe differences in OS, probably due to a limited follow-up. Longer surveillance of the series will help confirm these findings and analyze the impact on therapy responses and survival.