Chronic lymphocytic leukemia patients with chromosome 6q deletion as the sole cytogenetic abnormality display a high frequency of <i>RPS15</i> mutations and have a poor prognosis
Authors: Claudia Pérez Carretero, Teresa González, Miguel Quijada Álamo, Gian Matteo Rigolin, Adrian Dubuc, Ángela Villaverde Ramiro, Alberto Rodríguez‐Sánchez, Araceli Rubio, Julio Dávila, Mª. Jesús Vidal, Isabel González Gascón y Marín, José‐Ángel Hernández‐Rivas, Rocío Benito, Virginia Volpe, Matthew S. Davids, Jeremy S. Abramson, Antonio Cuneo, Paola Dal Cin, Ana‐Eugenia Rodríguez‐Vicente, Jesús‐María Hernández‐Rivas
Journal: American Journal of Hematology (2024)

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, with survival times ranging from months to decades, reflecting a great biological diversity. Classical cytogenetical models allow to classify patients in different risk subgroups according to the presence of certain chromosomal aberrations, being the most common the 13q deletion (del(13q)), 17p deletion (del(17p)), 11q deletion (del(11q)), and trisomy 12 (+12).1 By contrast, other less frequent cytogenetic alterations, including 6q deletion (del(6q)), affects a lower but not insignificant percentage of patients (5%). Several studies have suggested an association of del(6q) with inferior outcomes, allocating these CLL cases in an intermediate-risk category,2, 3 although other studies have shown no difference in outcomes.4 The recurrence of del(6q) in other B-cell malignancies strongly suggests that this region contains unidentified tumor-suppressor gene(s). Nevertheless, CLL patients harboring del(6q) remain poorly characterized at the molecular level, partly due to the low incidence of cases, the lack of a FISH-based routinely assessment of del(6q), and the co-occurrence with other abnormalities that masks their clinical and biological significance. Here, we comprehensively characterize for the first time the genetic landscape of CLL patients with del(6q) identified by karyotyping, since we believe that a mutational screening could allow for refinement in predicting overall survival and time to first treatment, as well as provide novel insights into del(6q) CLL pathobiology. To this aim, patients' samples and clinical data were collected from four different institutions: Brigham and Women's Hospital (Boston, MA, USA), Massachusetts General Hospital (Boston, MA, USA), Hospital of Ferrara (Italy), and University Hospital of Salamanca (Spain), diagnosed according to the International Workshop on CLL criteria. The study was approved by the local ethical committees. Conventional cytogenetics were performed to assess the presence of del(6q) in a total of 39 samples, either peripheral bloods or bone marrows (Table S1). In addition, interphase FISH analysis was performed on all samples using the standard CLL FISH panel which include CEP12, D13S319/LSI13q34, ATM, and TP53. Mutational analysis was performed on genomic DNA from mononucleated cells by next-generation sequencing (NGS), and custom-designed libraries of exonic regions were used to assess the mutational status of 54 CLL-related candidate driver genes (Supplementary Methods). A control group was included in the study (n = 317) for survival, clinical, and molecular studies. Control group was representative of the disease in terms of clinical and biological characteristics and CLL-IPI scores, matched by gender and time of follow-up with the study group (Table S2). Statistical analyses were conducted in IBM SPSS v23.0 and R v4.0. Mann–Whitney U test was used for continuous variables, while chi-square and Fisher's exact tests were used to determine associations between categorical variables and patterns of mutual co-occurrence or exclusivity. Overall survival (OS) and time to first treatment (TTFT) were calculated from the date of cytogenetic analysis to the date of death, first treatment, or last follow-up (considering disease-unrelated deaths as competing events). Statistically significant variables related to OS and TTFT were estimated by the Kaplan–Meier method, using the log-rank test to compare the curves of each group. Results were considered statistically significant for values of p < .05. A total of thirty-eight patients with CLL had a del(6q) identified by karyotyping. Del(6q) was observed as the sole abnormality in 15 samples. In addition, all these sample demonstrated a normal CLL FISH panel. In the remaining 23, del(6q) was present either with other cytogenetic alterations, or as additional no-related clone (Table S1). The whole series of 38 patients with del(6q) showed more adverse risk markers including shorter median age at diagnosis (61 vs 72, p < .001), unmutated IGHV (75% vs 45.7%, p = .005), CD38 positivity (50% vs 28.3%, p = .028), and ZAP70 positivity (61.9% vs 15.9%, p < .001) (Table S2). Del(6q) was detected at or near diagnosis in 23/38 cases (cutoff: 2 years from diagnosis). A total of 11 out of 38 patients received treatment before sampling date, and 20 out of 27 were treated during follow-up. TTFT was assessed in cases where del(6q) was identified before therapy. The presence of del(6q) was associated with shorter TTFT when compared to the control group (N = 317) (median: 7 vs 36 months, p < .001) (Figure 1A). In addition, the TTFT from the cytogenetic analysis of patients with del(6q) as the sole abnormality was also significantly shorter than that of control group (1 vs 36, p < .001) (Figure 1B), and not significantly different from that of patients carrying del(6q) and other cytogenetic abnormalities (Figure S1). No statistically significant differences were noted in overall survival (data not shown). According to the mutational analyses, 92% of del(6q) samples exhibited at least one mutation in one of the 54 genes analyzed, and the most frequently mutated genes were TP53 (26%), RPS15 (26%), and NFKBIE (16%) (Figure 1C). Notably, the mutational landscape varies depending on the presence of additional cytogenetic alterations (Figure 1D). Samples with del(6q) as the sole abnormality showed higher mutational frequencies in RPS15 (6/15, 40%), XPO1, and ATM (3/15, 20% each). The analysis of patterns of co-occurrence showed that certain combinations of gene mutations are significantly rare events within this cohort, highlighting the mutual exclusivity of RPS15 and TP53 mutations (p < .001) (Figure S2). In samples with del(6q) and additional cytogenetic alterations, the most frequently mutated gene was TP53 (8/23, 35%), followed by NOTCH1 (4/23, 17%) and NFKBIE (4/23, 17%) (Figure 1D). Most samples showing TP53 mutations in the entire del(6q) cohort present other mutated genes and, as expected, 5/10 exhibited del(17p) and 5/10 complex karyotype (Figure 1C). Given the high incidence of RPS15 and TP53 mutations within del(6q) cohort, we next assessed their clinical impact. Strikingly, only RPS15 mutations contributed to a worse outcome in del(6q) subgroup in terms of TTFT (3 vs. 13 months, p = .021) (Figure 1E) (Figure S3), with an even worse prognosis than patients without del(6q) harboring RPS15 mutations (3 vs. 12 months, p = .037) (Figure S4). Conversely, the presence of TP53 mutations was associated with a significant shorter OS in del(6q) patients (22 months vs. median not reached, p = .028) (Figure 1F), while RPS15 mutations did not impact OS (p = .498) (Figure S5). It is noteworthy to mention that while most RPS15 mutations (8/10) were detected in untreated patients and located in different codons (Figure 1C) (Table S3), two of them were detected at relapse in two patients in the same codon (S139, NM_001018), after anti-CD20 (P7) and chemotherapeutic (P38) regimens. Interestingly, the patient treated with chemotherapy (P38) exhibited the del(6q) as the sole cytogenetic abnormality, and both of them (P7, P38) were refractory to ibrutinib (Figure S6). In this study, we demonstrate that del(6q) in CLL is not only acquired as a secondary abnormality, but also may occur as the first and only cytogenetic alteration. Furthermore, patients showing del(6q) as the sole abnormality had significantly shorter TTFT than the control group, similar to that of patients harboring additional cytogenetic abnormalities, supporting that the intermediate outcome previously described is not just due to genomic instability or the accumulation of different genetic alterations.3, 4 NGS studies allowed us to identify that CLL patients with del(6q) had a distinct mutational profile depending on whether the del(6q) appears as the sole abnormality or in combination with other cytogenetic alterations. Surprisingly, the most frequently mutated gene in del(6q)-only CLL patients was RPS15 (40%). RPS15 gene encodes for the ribosomal subunit S15, located in the short arm of chromosome 19. In treatment naïve CLL patients, RPS15 mutations are infrequent (∼4%), but enriched at relapse or in high-risk subgroups such as del17p (∼20%).5, 6 Within the whole del(6q) cohort (N = 38), TTFT of patients harboring RPS15 mutations was shorter than the rest of del(6q) cases without this gene mutated and shorter than patients harboring RPS15 mutations in the control group, demonstrating the prognostic significance of the co-occurrence of del(6q) and RPS15 mutations. On the other hand, the most frequent alterations in samples carrying del(6q) with other abnormalities were TP53 mutations, del(17p)/TP53, and complex karyotype, indicating a higher genomic instability than in samples with del(6q) as the sole aberration. Despite the previously reported association between RPS15 and TP53 mutations,5, 6 they are not significantly concurrent events in our study, further suggesting the presence of different driving forces of tumor development within del(6q) CLL patients. Nevertheless, to confirm the pathogenic impact and clonality of these alterations, it would be necessary to perform further functional analysis and validate these results in larger del(6q) CLL cohorts by applying single-cell sequencing approaches at different time points in the disease. Regarding minimal deleted regions (MDRs), those of 6q21 and 6q23 have been the most frequent described in CLL, as occur in other B-cell lymphoproliferative malignancies. However, there is still some controversy, as the genes encompassed in these potential MDRs vary among studies, and other regions are also frequently affected. As conventional cytogenetics does not allow us to assess the precisely size of the deleted region, further investigations would be needed to establish correlations between the size of the deletion and clinical and mutational features of del(6q)-CLL patients. In summary, our work demonstrates the genetic complexity of patients carrying 6q deletion and their association with mutations previously related to relapse (RPS15 and TP53), highlighting the presence of higher mutational frequencies of RPS15 in patients with del(6q) as the sole abnormality. Moreover, the co-occurrence of these genetic alterations with 6q deletion contributed to a worse clinical outcome and disease progression, demonstrating the importance of assessing their mutational status and considering all of them in the CLL genetic context to improve treatment decision-making. CPC designed research, performed sample selection, carried out NGS and statistical analyses and drafted the manuscript. TG performed sample selection and FISH analyses. AVR performed data analysis and visualization and interpretated the results. MQA and ARS performed NGS studies and data analysis and critically reviewed the manuscript. AR, JD, MJV, LG, and IGGM provided patients' clinical data. JAHR, AD, VV, MD, JA, GMR, AC, and PDC provided patients' samples and clinical data and critically reviewed the manuscript. RB designed sequencing studies and contributed to data analysis. AERV performed data analysis and together with JMHR conceived the study, designed and supervised the research, and critically reviewed and approved the final version of the manuscript. All authors discussed the results and revised the manuscript. We are grateful to I. Rodríguez, S. González, M.Á. Ramos, A. Martín, A. Díaz, A. Simón, M. del Pozo, V. Gutiérrez, and S. Pujante from the Centro de Investigación del Cáncer, Salamanca, for their technical assistance. This work was supported by grants from Instituto de Salud Carlos III (ISCIII) & FEDER (PI21/00983) (co-funded by the European Union), Consejería de Educación-Junta de Castilla y León (SA118P20), Gerencia Regional de Salud-SACYL (GRS2140/A/20, GRS2385/A/21), Fundación Mutua Madrileña (FMM, AP176752021), Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD12/0036/0069), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233) and by AIL-FE, BEAT leukemia onlus and MIUR PRIN to AC. CPC is supported by a research grant from FEHH ("Fundación Española de Hematología y Hemoterapia"; Beca de Investigación FEHH 2022). ARS is fully supported by an "Ayuda predoctoral de la Junta de Castilla y León" by the Fondo Social Europeo Plus (FSE+) (JCYL- EDU/1868/2022 PhD scholarship). MSD is supported by an NIH 1R01CA266298-01A1 Award. M.S. Davids has received institutional research funding from AbbVie, AstraZeneca, Ascentage Pharma, Genentech, MEI Pharma, Novartis, Surface Oncology, TG Therapeutics, and personal consulting income from AbbVie, Adaptive Biosciences, Ascentage Pharma, AstraZeneca, BeiGene, BMS, Eli Lilly, Genentech, Genmab, Janssen, Merck, Mingsight Pharmaceuticals, ONO Pharmaceuticals, Secura Bio, TG Therapeutics, and Takeda. J.A Hernández M.S. has received research support from Janssen and Roche (Consultant/Speaker's Bureau: Janssen, Roche, Abbvie, Gilead, AstraZeneca, Beigene, Lilly). The remaining authors declare no competing interests. Written informed consent was obtained from all participants before they entered the study. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Supporting Information. Supplementary Table S1. Karyotype and FISH information of 38 chronic lymphocytic leukemia patients with del(6q). Median time from diagnosis to karyotyping was 2 years (range: 0–11). Supplementary Table S3. List of mutations detected by NGS in CLL patients with 6q deletion (N = 38). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.