Genetic hallmarks and clinical implications of chromothripsis in childhood T-cell acute lymphoblastic leukemia.

Introduction

T-lineage acute lymphoblastic leukemia (ALL), which is driven by malignant transformation and expansion of immature T-cell progenitors, accounts for 10–15% of pediatric acute leukemias [ 1 ]. The AIEOP-BFM Consensus Guidelines for Flow Cytometric Immunophenotyping of Pediatric Acute Lymphoblastic Leukemia classify T-ALL according to the T-cell development stage into the following: early T-cell precursor (ETP) [CD1a-, CD8-, weak CD5]; pro-T-cell Line T-I (iCD3 + , CD7 + ); pre-T-cell T-II (iCD3 + , CD7 + , and CD5/CD2 + ]; cortical T-cell T-III (iCD3 + , Cd1a + , sCD3 + /−); and mature T-cell T-IV (iCD3 + , sCD3 + , CD1a−) [ 2 ]. These immunological entities, as defined by differentiation arrest, are associated with recurrent molecular abnormalities, including sequence mutations, gene fusions, and structural aberrations. Most frequently, genomic lesions dysregulate genes encoding transcription factors such as LYL1, LMO1/2, TLX1/3, NKX2-1, and TAL1/2, indicating a degree of association with T-cell development stages [ 3 ]. T-ALL may develop in a background of inherited predisposition associated with DNA repair disorders, including constitutional mismatch repair defect (CMMRD), ataxia-teleangiectasia (AT) and Nijmegen breakage syndrome (NBS) [ 4 – 6 ].

Although primary T-ALL is curable for most children given measurable residual disease (MRD)-based risk-adapted intensive chemotherapy, salvage treatment for relapsed T-ALL is usually far less effective. In contrast to the excellently defined prognostic significance of the majority of molecular subtypes of childhood B-cell precursor ALL, relatively few genetic lesions with proven clinical relevance exist in pediatric T-ALL. Recently, such a poor prognostic value was shown for uniformly treated adult patients harboring somatic DNMT3A, N/KRAS, MSH2 and/or U2AF1 mutations [ 4 ]. In addition, the adverse impact of a complex karyotype (defined by the presence of ≥3 cytogenetic alterations) in T-ALL has been reported [ 5 , 6 ].

An extreme form of a complex karyotype is chromothripsis (cth), which is caused by genome instability and leads to massive DNA breakage and repair in cancer cells in a single catastrophic event. In cth, tens to hundreds of genomic rearrangements are acquired in a few cell divisions and remain clustered within the focal chromosomal region [ 7 ]. Shattering of chromosomes and erroneous DNA repair processes result in loss and random rejoining of some genomic fragments, leading to inactivation of tumor-suppressor genes, amplification of protooncogenes and formation of carcinogenic gene fusions [ 8 , 9 ]. Although newly recognized and only partly understood, the phenomenon of the cth remains an alternative to the traditional stepwise model of cancer development. Two essential mechanisms initiating cth have been suggested: telomere attrition and DNA damage in micronuclei [ 10 ]. Telomere shortening and breakage-fusion-bridge cycles which result in the formation of dicentric chromosomes and chromosome bridges, were found to initiate chromothriptic events in cell culture models [ 7 , 11 ]. In addition to the telomere attrition hypothesis, alternative models of micronuclei formation and mitotic errors have been proposed as a mechanistic basis of cth. Micronuclei are small DNA-containing nuclear structures that are spatially isolated from the main nucleus. Due to asynchronous DNA replication and impaired checkpoint arrests, the micronucleus DNA can undergo pulverization without subsequent appropriate DNA repair. Pulverized macronuclei are reintegrated into the main nucleus which leads to massive intrachromosomal rearrangements, shattering chromosomes and their random reconnection generating mosaic chromosomes [ 7 , 12 ].

Using an array-based approach, cth was initially detected in only 2–3% of malignancies [ 13 ]. However, a whole-genome sequencing (WGS) study performed across 37 different adult cancer types showed that cth occurs in more than 50% of malignancies, representing several tumor entities, and significantly contributes to cancer genome evolution [ 14 ]. The prevalence of cth varies across different tumors types and the highest frequency has been noted in liposarcomas and osteosarcomas (100 and 77%, respectively) followed by melanomas, glioblastomas and lung adenocarcinomas which exhibited chromothripsis in more than 50% of cases. Among cancer entities showing the lowest susceptibility to chromothripsis are thyroid adenocarcinomas and pilocytic astrocytomas (3.3 and 0%, respectively) [ 11 ].

Cth has also been detected in various types of hematological neoplasms, with different incidence rates, including multiple myeloma (49%), chronic lymphocytic leukemia (10%), acute myeloid leukemia (8.3%), myelodysplastic syndrome (49%), and lymphoma (18%). However, in the majority of blood malignancies, it has been noted in less than 10% of patients, depending on the cth detection method [ 15 ]. Cth has also been observed in very defined and rare subtypes of ALL, including ETP-ALL, BCP-ALL with internal amplification of chromosome 21 (iAMP21), and T-ALL, which develop in the constitutional background of ataxia telangiectasia [ 8 , 16 , 17 ]. In adult patients with ETP-ALL, somatic mutations and/or deletions of DNA repair/genome stability genes ( BLM, STK11, PTEN, ATM, MUTYH, and ATR ) have been found, and up to one-third of these cth cases harbored NUP214 rearrangements [ 17 ].

To date, no research is available on the prevalence or molecular and clinical features of cth in childhood T-ALL. Therefore, in our study, we aimed to address the biological background and prognostic significance of cth in pediatric patients with T-ALL.

Materials and methods

Study group

We retrospectively reviewed the medical records of 2034 patients aged 1–18 years with newly diagnosed ALL who were diagnosed in all 16 hemato-oncological centers of the Polish Pediatric Leukemia/Lymphoma Study Group according to the ALLIC BFM 2009 (December 2011 and September 2018; n = 1388) and AEIOP BFM 2017 (October 2018 to June 2021; n = 646) protocols in Poland. We were able to identify 283 children with T-cell ALL (T-ALL) diagnosed and treated between 2011 and 2021, n = 180 patients according to ALLIC BFM 2009 protocol and n = 103 individuals according to AEIOP BFM 2017 protocol. For our current retrospective study germline and leukemic samples were available from 173 (61%) pediatric patients with T- ALL diagnosed in Poland during these ten years, 99 cases out of 103 (96%) enrolled into AEIOP BFM 2017 protocol and n = 74 out of 180 cases (41%) treated according to ALLIC BFM 2009. Bone marrow samples collected from all patients were cytogenetically investigated by G-banding and FISH analysis at the time of ALL diagnosis. All patients were screened for t(9;22)(q34;q11)/ BCR :: ABL1 and 11q23/KMT2A rearrangements using locus-specific probes. Bone marrow samples from all T-ALL patients at diagnosis and on Day 15 of the treatment protocol were analyzed by 8-color flow cytometry according to the protocols of the EuroFlow Consortium at one center (Department of Pediatric Hematology and Oncology, Medical University of Silesia in Zabrze). Bone marrow MRD at the end of the induction phase (Day 33) was assessed using real-time quantitative PCR analysis of clone-specific immunoglobulin ( IG ) and T-cell receptor ( TCR ) gene rearrangements according to the guidelines described by van der Velden et al. [ 18 ]. The study was conducted in accordance with the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of the Medical University of Lodz, and informed consent was obtained from all participants and/or their parents (No. KE-0254/74/03/2023).

Microarray analysis

Copy number alterations (CNA) analysis was performed using a CytoScan HD array (2 670 000 markers, including 750 000 single-nucleotide polymorphisms (SNPs) and 1 900 000 nonpolymorphic copy number variant (CNV) markers ( Applied Biosystems, Thermo Fisher , Waltham, MA, USA), as described previously [ 19 ]. All laboratory procedures were carried out according to manufacturer's protocols and were standardized based on the AIEOP-BFM array screening strategy recommendations for microdeletion and microduplication assessments. Chromosome Analysis Suite v 4.4 software (ChAS, Thermo Fisher Scientific) was subsequently used to convert CEL files to CYCHP files containing information on copy number, loss of heterozygosity (LOH), mosaicism, and genotype. Thresholds of log2 ratios ≥ 0.5 and ≤ 0.5 were used for categorization of altered chromosomal regions as CNV gains and losses, respectively. The experimental data were divided into two categories: genome-wide CNVs > 5 Mbp and leukemia-associated region/gene-specific CNAs (leukemia genes_all_20150505; Fullerton Overlap Map_hg19). Our panel of 1276 genes was created based on comprehensive literature describing genetic alterations and their role in the pathogenesis of ALL, and these genes are called cytoregions. The genes are listed in Supplementary Table S1 . The minimum number of probes applied to determine the CNAs was 50 for duplications (gain) and 25 for deletions (loss). Copy number-neutral loss of heterozygosity (cnLOH) is reported when a gene is greater than 3000 kb in length. Chromothripsis was defined according to the criteria provided by Korbel J and Campbell P [ 20 ].

RNA sequencing

RNA sequencing was performed using the TruSight RNA Pan-Cancer panel ( Illumina , San Diego, CA, USA) with a Next Seq 550 system ( Illumina ) and NextSeq® Reagent Kit v3 (150 cycles) with a PE NextSeq® Flow Cell. Data analysis was performed using the Illumina BaseSpace app TopHat Alignment (version 1.0.0), read mapping to the hg19 reference genome by TopHat21, fusion calling by TopHat-Fusion2, and RNA-seq alignment (version 1.1.0; read mapping to the hg19 reference genome by STAR3; and fusion calling by Manta4 using standard settings ( https://basespace.illumina.com/apps ). Fusion transcripts with a low number of split reads ( < 10) were excluded as likely false-positives. Raw sequence variant data were converted to variant call format (vcf) files and analyzed with Variant Studio software v.4.0.

A detailed description of all methods can be found in the online Supplementary Materials .

Results

Incidence and characteristics of T-ALL with chromothripsis

Chromothripsis was identified in 10 T-ALL samples (5.78%). In six of them, cth occurred in a constitutional background of cancer-predisposing syndromes, including Li-Fraumeni syndrome [n = 1; NM_000546.5 :c.469 G > T (p.Val157Phe)] or Nijmegen breakage syndrome [NBS; (n = 5)]. There were six patients with NBS enrolled in the study group, they were clinically confirmed (according to the diagnostic criteria for NBS developed by the Clinical Working Party of ESID) to be carriers of the same homozygous, five-base pair founder deletion (c.657_661del5, p.Lys219Asnfs) within the NBN gene. The clinical and biological characteristics of the cth-positive (cth pos) and cth-negative (cth neg) patients treated according to the ALLIC BFM 2009 and AEIOP BFM 2017 protocols are presented in Supplementary Table S2 . The majority of individuals were diagnosed with T-ALL before the age of 9 years, and they tended to have slower blast clearance during the induction phase of the treatment. None of the cth pos leukemia patients exhibited the ETP immunophenotype. The cth pos patients had a lower probability of 5-year overall survival (OS) [0.56 vs. 0.81; hazard ratio (HR) = 4.14 (1.42–12.02) p = 0.017] as did 5-year event-free survival [0.45 vs. 0.74; HR = 3.91 (1.52–10.08); p = 0.012] (Fig. 1A, B ). Unfavorable outcomes in the cth pos group resulted from the high rate of death in individuals with NBS. During the median follow-up of 1.92 (1.003–4.47) years, two NBS patients developed disease progression, and another two died due to treatment-related toxicity. However, there was a clear difference in representativeness between the two patient cohorts treated with different protocols. While we were able to include in the study the vast majority of patients diagnosed with the AEIOP BFM 2017 protocol (96%), 74 out of 180 individuals treated according to ALLIC BFM 2009 protocol and involved in the analysis represent the minority (41%). Therefore, we also performed the survival analysis for each protocol separately (Supplementary Fig. S1 ). We could observe the tendency for decreased EFS but not RFS in cth pos patients in both protocols. Still, it remained significant only in the case of cth pos patients treated according to the AEIOP BFM 2017 protocol. This might be explained by the unequal distribution of syndromic cth pos patients who were predominantly enrolled in the AEIOP BFM 2017 protocol. Fig. 1 Outcomes for pediatric T-ALL patients depending on the incidence of chromothripsis. Kaplan-Meier curves displaying the probability of overall ( A ), event-free ( B ) and relapse-free survivals ( C ) among T-ALL patients with (the blue line) and without (the red line) chromothripsis.

Distribution of numerical aberrations in T-ALL patients according to the presence of chromothripsis

The original karyotypes and revision results based on SNP array analysis of 173 pediatric patients with T-ALL are presented in Supplementary Table S3 . In total, 68 patients (39.2%) had a normal karyotype. Low hyperdiploidy (47–50 chromosomes) was identified in ten patients, including six with isolated trisomy 8; high hyperdiploidy ( ≥ 51 chromosomes) and hypodiploidy ( < 44 chromosomes) occurred in one and two patients, respectively. In total, repeated trisomy occurred in 58 patients, with chromosome 8 being the most common (n = 10). Based on classical cytogenetic testing, we identified complex karyotypes (more than 5 abnormalities) in 12 patients (6.93%). A complex karyotype occurred more frequently in cth pos than in cth neg individuals ( n = 7/10 vs. n = 5/163; p < 10 − 6 ; two-tailed Fisher’s exact test; OR 73.33 (95%CI 14.60–373.30).

Comparison of large structural variants between cth positive and cth negative T-ALL

In the next step, we searched for differences in the frequency and spectrum of structural aberrations exceeding 5 Mbp between the cth pos patients and cth neg individuals. Overall, the SNP array detected 552 events (0–64; 3.19 per case), including 199 losses, 212 gains, and 141 copy-neutral losses of heterozygosity (cnLOH). CNAs were present in all the cth pos T-ALL patients and in half of the cth neg patients ( n = 95; 55%). The number of CNAs per patient was eight times greater in the first group than in the second group [median number of CNAs was 19.2, range: 4–64; range: 2.2, range: 0–17].

A total of 298 segmental gain and losses of chromosomal regions including segmental duplication ( n = 63), segmental deletion ( n = 141), segmental loss of heterozygosity ( n = 94), and loss of heterozygosity of whole chromosomes ( n = 3), were detected in 163 cth neg T-ALL patients. In total, 49 patients had only one large segmental abnormality exceeding 5 Mbp. A total of 192 segmental CNAs, duplications ( n = 13), deletions ( n = 21), and segmental loss of heterozygosity ( n = 1), were observed in 10 cth pos T-ALL cohort. The number of CNAs ranged from 8 to 38 segmental CNAs, with an average of 72.29 (±55.35) Mb.

We further investigated whether cth pos T-ALL patients exhibit a common pattern of structural abnormalities or whether they resemble any specific nonsyndromic T-ALL subgroup. For this purpose, we performed unsupervised hierarchical clustering analysis using information about whole-genome CNAs exceeding 5 Mbp in 173 leukemia samples. As shown in Fig. 2A , all NBS T-ALL patients grouped within Cluster 3, which may reflect their intrinsic genetic resemblance. The remaining cth pos non-NBS patients clustered either in Cluster 3 (two samples) or in Cluster 2 (three samples). Fig. 2 Copy number alteration (CNA) analysis of childhood T-ALL depending on the presence of chromothripsis. A Hierarchical clustering analysis based on CNA data obtained from 173 pediatric T-ALL patients. B Histograms displaying the frequency of gains and losses exceeding 5 Mbp in the study group depending on the cth and NBS status. Gains and losses are displayed as red and blue bars, respectively.

Overall, there was a significant difference in the distribution of large segmental aberrations, both deletions and duplications, between the cth pos and cth neg patients, which are marked in black in the inner circle in Fig. 2B . Segmental duplications involving chromosomal regions 1q, 5p, 8q, 14q, 18p, 18q, 19q, and 22q were more frequent in cth pos T-ALL than in cth neg patients. Similarly, segmental deletions affecting 1q, 5q, 7q, 8p, 12p, and 13q were significantly more common in T-ALL patients with cth.

We also investigated the pattern of large structural CNAs according to T-cell immunophenotype (Supplementary Figure S2 ) and found that duplication of chromosome 8 was significantly common among pre-T-ALL patients. Although ETP-ALL was underrepresented in our study group, we observed a frequent incidence of deletions involving chromosomes 5q, 6q, and 11q in T-ALL samples representing this leukemic subtype.

As expected, the most recurrent copy number losses ( n = 37; 22.15%) in nonsyndromic T-ALL were found on the short arm of chromosome 9 and encompassed the common 9p22.2p21.1 (17262395_28275543) region, including the SH3GL2, MLLT3, IFNA1, MTAP, CDKN2A/B , and TEK genes. Another frequent structural deletion ( n = 20; 11.97%) was identified on the long arm of chromosome 5q and involved the 5q33.3q35.1 (157861361_172589313) region; this deletion included the EBF1, PTTG1, RANBP17, TLX3 , and NPM1 genes. Moreover, we also observed recurrent deletions on the long arm of chromosome 6 ( n = 19; 11.37%) encompassing the 6q14.1q16.3 (79658603_104885348) region, leading to loss of the CASP8AP2, BACH2, MAP3K7, EPHA7 , and CCNC genes. Other less common deletions were observed on the short arm of chromosome 12p13.2p12.1 (11256830_22116241) ( n = 12; 7.18%), including ETV6, BCL2L14, CDKN1B, ATF7IP, GUCY2C, EPS8 and 1p36.33p36.22 (854277_10485206) ( n = 11; 6.58%), encompassing the DVL1, MIB2, PRDM16, RPL22, CAMTA1, ERRFI1, ENO1 , and PIK3CD genes. The most recurrent duplications were observed on the long arm of chromosome 4q32.1q35.2 (159407548_190957473) ( n = 7; 4.2%), which included FBXW7, NPY1R, ING2, SORBS2, FAT1 , and DUX4 ; on the long arm of chromosome 8q23.3q24.3 (116593872_146295771) ( n = 6; 3.6%), which included TNFRSF11, ENPP2 , and RNF139 ; and on the long arm of chromosome 9q33.1q34.3 (118514469_141020389) ( n = 6; 3.6%), which included TSC1, GFI1B, PAEP , and MIR126 .

Interestingly, the most characteristic structural aberrations for NBS were those affecting chromosome 8, such as loss of the short arm and duplication of the long arm. In two patients, we cytogenetically confirmed the presence of isochromosome 8 [i(8)(q10)]. Segmental LOH involving the NBN gene at 8q21 was present in the entire group of NBS patients. Apart from the short arm of chromosome 8, the most frequent deletion was located at the 12p13.31p12.1 (9910041_23561624) region and involved GABARAPL1, KLRK1, ETV6, BCL2L14, CDKN1B, ATF7IP, GUCY2C , and EPS8 ( n = 4). The most recurrent duplications in syndromic T-ALL occurred on the short arm chromosome 5p15.33p12 (113577_42844121) region and encompassed IL7R, PRLR, LIFR, PDCD6, TRIO, DAB2 , and SEPP1 . In two NBS patients, we also noted coincidence of CNAs involving chromosomes 7 and 14, suggesting reciprocal translocations, which could not be confirmed due to the lack of biological sample for cytogenetic studies.

We also investigated the frequency of UPDs, which are relatively rare events in T-ALL ( < 1%) [ 21 ], in contrast to pediatric BCP-ALL, in which UPDs occur in 5 to 10% of patients [ 22 ]. Loss of heterozygosity involving chromosomes 8, 11, and 18 was identified in a single cth neg patients with low hyperdiploid karyotypes. However, common segmental LOH affects the short arm of chromosome 9p24.3p13.3 (192128_21894495), with a concomitant deletion occurring prior to sUPD9p in all instances [ 23 ]. The presence of multiple regions of homozygosity (ROH) exceeding ≥ 10% of the genome using a threshold of 3–5 Mb or larger was identified in one NBS patient (M38). According to ACMG guidelines [ 1 ], a possible first- or second-degree relationship between the patient’s parents may be suspected, but this was not reported by the care givers. (Supplementary Figure S3 ).

Profile of microaberrations in T-ALL patients with the chromothripsis pattern

The total number of microdeletions and microduplications in the study group was 2953 in 173 patients (17.17/per sample). The median number of microCNAs did not significantly differ between cth pos and cth neg individuals [ n = 13 (range: 8–20.5) and n = 17 (range: 11–21.50), respectively]. In both groups, the most recurrent CNAs affected genes encoding lymphocyte T receptors ( TRA, TRD, TRB, and TRG ) and immunoglobulin heavy chain ( IGH) (Supplementary Table S4 ). CNAs involving IG and TR probably represent the clonality tag of IG/TR somatic rearrangements in leukemic cells. Moreover, there was no specific pattern of microaberrations in the cth pos T-ALL patients (Table 1 ). Table 1 The detailed distribution of microdeletions and microduplications in cth pos T-ALL patients. Deletions and duplications are displayed in dark grey and light grey boxes, respectively. The numbers reflect the copy numbers of the genes. Gene function Gene Localisation Number of deletions %del Number of duplications %dup cth positive T-ALL ( n = 10) M6 M13 M38 M55 M94 M97 M130 M150 M162 M171 transcription factors PRDM16 1p36.32 0 0 1 10 2~3 PAX5 9p13.2 1 10 0 0 1 FLI1 11q24.3 1 10 0 0 ex10 ex 7 ETV6 12p13.2 1 10 0 0 1 SOX5 12p12.1 0 0 1 10 ex1-7 HOXC11 12q13.13 0 0 1 10 CEBPE 14q11.2 1 10 0 0 1~2 CBFB 16q22.1 1 10 0 0 1~2 JUNB 19p13.2 1 10 0 0 1 BCL3 19q13.32 0 0 1 10 2~3 RELB 19q13.32 0 0 1 10 2~3 signaling pathways regulators, kinase DVL1 1p36.33 0 0 1 10 2~3 ERRFI1 1p36.23 0 0 1 10 2~3 BCL9 1q21.2 1 10 0 0 1~2 SORBS2 4q35.1 3 30 0 0 ex 14-16 ex6-7 ex6-7 POM121 7q11.23 1 10 0 0 ex1-3 SH3GL2 9p22.2 0 0 1 10 2~3 NTRK2 9p21.33 0 0 1 10 2~3 ABL1 9q34.12 0 0 1 10 ex 2-11 VWF 12p13.31 1 10 0 0 ex39-42 GUCY2C 12p13.1p12.3 1 10 0 0 1 KRAS 12p12.1 0 0 1 10 2~3 NDRG2 14q11.2 3 30 0 0 1 1~2 1~2 MMP14 14q11.2 1 10 0 0 1~2 TCL1B 14q32.13 0 0 1 10 3 TCL1A 14q32.13 0 0 1 10 3 AXIN1 16p13.3 1 10 0 0 1~2 TSC2 16p13.3 1 10 0 0 1~2 PKD1 16p13.3 1 10 0 0 1~2 FAM65A 16q22.1 1 10 0 0 1 CTCF 16q22.1 1 10 0 0 1 RANBP10 16q22.1 1 10 0 0 1 CDH1 16q22.1 1 10 0 0 1~2 CBFA2T3 16q24.3 0 0 1 10 ex 2-12 CLDN7 17q13.1 1 10 0 0 1 ERBB2 17q12 1 10 0 0 ex4 TYK2 19p13.2 1 10 0 0 ex 1-3 DNM2 19p13.2 1 10 0 0 ex 1-2 CYP2A6 19q13.2 1 10 0 0 ex 1-5 CEACAM1 19q13.2 1 10 0 0 1 PLAUR 19q13.31 0 0 1 10 2~3 MARK4 19q13.32 0 0 1 10 2~3 TNFRSF6B 20q13.33 0 0 1 10 2~3 DYRK1A 21q22.13 1 10 0 0 ex5-6 S100B 21q22.3 0 0 1 10 ex 1 IGL@ 22q11.22 0 0 4 40 3 3 3 3 IL3RA Xp22.33 2 20 1 10 /3 ex1-2 CRLF2 Xp22.33 0 0 1 10 3 L1CAM Xq28 0 0 3 30 3 3 3 cell cycle regulators PDCD6 5p15.33 0 0 1 10 4 CDKN2A 9p21.3 1 10 0 0 CDKN2B 9p21.3 1 10 0 0 HTATIP2 11p15.1 1 10 0 0 1~2 FANCF 11p14.3 1 10 0 0 1~2 BCL2L14 12p13.2 1 10 0 0 1 CDKN1B 12p13.1 1 10 0 0 1 ATF7IP 12p13.1 1 10 0 0 1 NOL3 16q22.1 1 10 0 0 1 ACD 16q22.1 1 10 0 0 1 TSNAXIP1 16q22.1 1 10 0 0 1 CENPT 16q22.1 1 10 0 0 1 TERF2 16q22.1 1 10 0 0 1 FBXO31 16q24.2 1 10 0 0 1 CDT1 16q24.3 0 0 1 10 3 GADD45GIP1 19p13.2 1 10 0 0 1 ERCC2 19q13.32 0 0 1 10 2~3 PPP1R13L 19q13.32 0 0 1 10 2~3 ERCC1 19q13.32 0 0 1 10 2~3 epigenetic regulators KAT6A 8p11.21 1 10 0 0 1 EED 11q14.2 1 10 0 0 CREBBP 16p13.3 1 10 0 0 ex 3-31 RBL2 16q12.2 1 10 0 0 1 translation and RNA stability FIP1L1 4q12 2 20 0 0 ex 5-8 ex 3 ANG 14q11.2 3 30 0 0 1 1~2 1~2 EEF1A2 20q13.33 0 0 1 10 2~3 ZRSR2 Xp22.2 0 0 2 20 ex 6-7 ex 6-7 RPL10 Xq28 0 0 1 10 3 DKC1 Xq28 0 0 1 10 3 cell metabolism, ubiquitination MIB2 1p36.33 0 0 1 10 2~3 FH 1q43 1 10 0 0 ex 8-10 NUP214 9q34.13 0 0 1 10 ex1-32 RAB39 11q22.3 1 10 0 0 ex1 OPCML 11q25 1 10 0 0 ex1-5 TRAP1 16p13.3 1 10 0 0 1~2 MMP2 16q12.2 1 10 0 0 1 AMFR 16q12.2 1 10 0 0 1~2 GFOD2 16q22.1 1 10 0 0 1 NQO1 16q22.1 1 10 0 0 1~2 WFDC1 16q24.1 1 10 0 0 1~2 CALR 19p13.2 1 10 0 0 1 LYPD3 19q13.31 0 0 1 10 2~3 CBLC 19q13.32 0 0 1 10 2~3 SS18L1 20q13.33 0 0 1 10 2~3 SEPT6 Xq24 0 0 1 10 ex 1 lymphocyte specific genes TRG@ 7p14.1 8 80 1 10 1 0/1 0 1 1 1 0 TRB 7q34 3 30 0 0 0 1 1 RAG2 11p12 1 10 0 0 1 TRA 14q11.2 6 60 0 0 1 1/0 0 0 1 TRD 14q11.2 4 40 0 0 1 0 0 1 TRD@ 14q11.2 3 30 0 0 0 0 1 IGH 14q32.33 4 40 6 60 3 3 1 3 3 1 3 0 0 IGH@ 14q32.33 0 0 8 80 3 3 3 3 3 3 3 3 RLTPR 16q22.1 1 10 0 0 1 MTCP1 Xq28 0 0 1 10 3

In contrast, cth neg T-ALL patients presented recurrent microdeletions in the following genes: LEF1, HOXA11 (TLX1), FLI1, CEBPE, RB1, SORBS2, IFNA1, PTEN, NDRG2, MMP14, CTCF, NF1, DCC, STIL, CDKN2A, CDKN2B, FIP1L1, MLLT3 (AF9), ANG, MTAP , and ST13P4 . The most frequent microduplications involved MYB, AHI1, TSC1, IGL@, IL3RA, L1CAM, AIFM1, GPC3, PHF6, ZRSR2 , and SEPT6 (Supplementary Table S4 ). Analysis of the frequency of microalterations according to gene function revealed no significant differences between the cth pos and cth neg T-ALL patients, except for cell cycle regulators (Supplementary Figure S4 ). This difference resulted from the common incidence of microdeletions affecting CDKN2A, CDKN2B , and STIL in cth neg T-ALL patients (263 deletions in 118 patients).

The incidence of the chromothriptic pattern

According to the criteria for chromothripsis (cth) provided by Korbel J and Campbell P [ 20 ], we identified chromothriptic phenomena in 10 (5.7%) T-ALL patients; chromothripsis was more common in patients with an inherited cancer predisposition than in nonsyndromic individuals (5/6 vs. 5/167 p < 10 − 5 ; two-tailed Fisher’s exact test; OR 162.00 (95%CI 15.85-1655.41) (Fig. 3 ). In all but one patient, the cth involved only one chromosome, but there was one T-ALL in the NBS patient, whose cth occurred on three chromosomes. Chromosomes 7, 9 and 19 were recurrently affected by cth. Fig. 3 Microarray data plots showing chromothriptic pattern of copy number alterations in T-ALL samples. The gains and losses are displayed as red and blue bars, respectively.

Chromothripsis generated alterations in genes that are associated with leukemogenesis, including deletions of CDKN2A/B and EZH2 and amplifications of CDK6 , also gave rise to gene fusions noted in NBS individuals, such as NUP214 :: ABL1 and TFG :: GPR128 . NonNBS leukemias with cth represented all the immunophenotypes excluding ETP. Three of these patients were assigned to the high-risk group due to poor treatment response during induction (Supplementary Table S5 ). One patient from this group developed T-ALL as a secondary malignancy following choroid plexus carcinoma and carried the germline TP53 pathogenic variant c.469 G > T, p.Val157Phe. In this case, the cth was observed at chromosome 17p, which led to TP53 deletion. In order to search for genetic background of chromothripsis in cth pos nonNBS T-ALL cases, we performed DNA sequencing using TruSight One Expanded Sequencing Panel (Illumina, US). Pathogenic variants that were identified in the leukemic genomes are presented in the Supplementary Table S5 . Interestingly, two cth pos nonsyndromic patients carried somatic heterozygous variants in the MED12 tumor supressor gene [( NM_005120.2 ): c.112_113insGCCTAGGGCAGTCCGG (p.Ala38GlyfsTer15) and c.3920 T > C (p.Leu1307Pro)]. Based on allele frequencies, we did not select any variants in genes from the cancer predisposition gene panel (Supplementary Table 6 ) for further germline testing. In addition, family history of cancer was irrelevant in cth pos nonsyndromic patients.

Discussion

Chromothripis has recently been indicated to be an important mechanism involved in the development and progression of hematological malignancies [ 20 ]. In this study, we aimed to investigate the molecular background and clinical characteristics of childhood T-ALL patients. Although a high-density SNP array is not a perfect tool for detecting chromothripsis, we identified 10 pediatric T-ALL patients (5.78%) with a chromothriptic pattern of CNA [ 20 ]. Interestingly, half of them were found in patients with Nijmegen breakage syndrome (NBS), which is an inherited defect of DNA double-strand break repair resulting from biallelic mutations of the NBN gene located at chromosome 8q21 [ 7 ]. Loss of NBN gene function reduces the activity of the Mre11-Rad50-Nbs1 complex, which maintains genome stability and activates cell cycle checkpoints. Approximately 90% of all NBS patients are of central and Eastern European descent and carry a homozygous five-base pair founder deletion (c.657_661del5, p.Lys219Asnfs) within the NBN gene [ 7 ]. Individuals with NBS are usually diagnosed with hematological malignancies of lymphoid origin during childhood, and their lifetime accumulated risk of cancer is estimated to reach 80% [ 8 ]. While they predominantly develop both B-cell or T-cell non-Hodgkin’s lymphomas (NHLs), ALL occurs in only 10% of NBS patients and derives almost exclusively from the T-cell lineage at different maturation stages. In addition, T-cell leukemias/lymphomas are half of the secondary tumors diagnosed among individuals with NBS [ 8 ]. Our results indicate that being the carrier of a biallelic defect in the NBN gene is an important risk factor for chromothripsis in T-ALL. This observation provides further evidence supporting the link between defective DNA repair and the presence of cth in leukemic cells, which has already been reported for other inherited DNA double-strand repair disorders biologically similar to NBS, including Fanconi anemia and ataxia telangiectasia [ 9 , 16 ].

Cth has also been described in nonsyndromic T-ALL patients with the ETP immunophenotype, but in our cohort, cth was exclusively present in more mature T-cell leukemias [ 17 ]. Although the clinical features of children with cth pos T-ALL did not substantially differ from those of cth neg ALL, apart from the tendency toward a slower initial response to treatment, 40% of these patients died within a short follow-up period. However, this unfavorable outcome did not occur in non-syndromic individuals and in the cth positive NBS group resulted from disease progression or fatal toxic complications, which were both equally common in our study. Cancer progression considerably affects the outcome of NBS patients diagnosed with lymphoma [ 24 ]. In addition, due to the significant risk of excessive toxicity, NBS patients usually need a modified and individualized approach during ALL treatment [ 25 ]. In addition to specific and rapidly applied supportive care, accurate insight into ALL driver aberrations may contribute to the identification of possible targets for individualized therapy. In one NBS patient from our cohort who harbored the cth-induced gene fusion NUP214 :: ABL1 , a potential target for treatment using a tyrosine kinase inhibitor was found. This specific patient exemplifies how the knowledge about causes and consequences of chromothripsis may substantially contribute to development of personalized treatment. Several studies have shown a significant association between homologous recombination deficiency and the presence of chromothripsis in human cancer cells [ 26 ]. Therefore, targeting DNA repair deficiencies by synthetic lethality approach may become beneficial therapeutical approach in chromothriptic cancer. Apart from the possible use of PARP inhibitors, synthetic lethality may also concerns defective DNA damage response and mitotic lesions which are both common in case of chromothripsis [ 26 ].

In addition to genomic alterations that are directly generated by the cth, we compared the whole genome profile of CNAs between the cth pos and cth neg T-ALL. The first important difference for leukemic karyotypes was the greater incidence of numerical aberrations and complex karyotypes in patients with cth pos leukemia than in those with cth neg T-ALL. The complex karyotype reflects genomic instability, which is an essential biological feature of NBS, but the prevalence of cth was not restricted only to syndromic patients. Genesca et al. described the link between the complex karyotype of T-ALL and a decreased probability of event-free survival (EFS) and overall survival (OS) and a high cumulative incidence of relapse. This association was independent of other clinical characteristics and the level of MRD at the end of induction [ 5 ].

To further investigate the genetic resemblance of cth pos T-ALL patients, we performed whole-genome profiling of segmental alterations and unsupervised hierarchical clustering analysis. We observed that NBS T-ALL grouped together within Cluster 3 with two other nonsyndromic cth pos patients. Moreover, they exhibited common segmental deletions affecting the short arm of chromosome 12, always encompassing the ETV6, BCL2L14, CDKN1B, ATF7IP, GUCY2C , and EPS8 genes. Only 7.18% of the cth-negative T-ALL patients, mainly those with a cortical immunophenotype, showed this aberration. Heterozygous deletion of CDKN1B , which encodes the tumor-suppressor p27kip1, is one of the most common CNAs and is described in approximately 12% of pediatric T-ALL patients [ 27 , 28 ]. In all of the cases reported by La Starza et al., it cooccurred with ETV6 deletions [ 29 ]. Interestingly, four of the six NBS patients with cth also exhibited loss of chromosome 5q33.3q35.1 (including the EBF1, PTTG1, RANBP17, TLX3 , and NPM1 genes), which was found in approximately 12% of the cth neg T-ALL patients. Although deletion of 5q is a characteristic abnormality for myeloid malignancies, it has rarely been reported in B and T-ALL leukemias [ 30 ]. In contrast, the most common deletions involving the 9p21 region within the CDKN2A/B gene were detected in approximately 70% of cth neg T-ALL patients but occurred significantly less often in cth pos patients [ 31 ]. Regarding the incidence of microaberrations, the cth pos T-ALL patients displayed an increased frequency of TP53 and RB1 deletions, which we previously observed in B-ALL patients with chromothripsis [ 19 ].

Interestingly, two cth pos nonsyndromic leukemia patients harbored somatic pathogenic variants in the tumor-suppressor gene MED12 , which is a member and activator of the CDK8 subcomplex responsible for initiation of transcription. In the literature, MED12 loss-of-function variants have been described as very rare but recurrent X-linked drivers of T-ALL development cooccurring with PHF6 mutation [ 32 ]. Furthermore, MED12 mutations are present in most uterine leiomyomas, which exhibit a high incidence of complex chromosomal rearrangements resembling chromothripsis [ 33 ]. This observation raises the question of the possible link between genomic instability and MED12 dysfunction in leukemic cells, which needs to be further investigated.

To the best of our knowledge, this is the first study providing a comprehensive analysis of the molecular features of childhood T-ALL patients displaying chromothripsis. Nonetheless, interpretation of our results has several limitations, especially considering the frequency of chromothripsis in pediatric T-ALL. Our study was performed in Slavic origin population with increased number of germline NBN gene mutation and Nijmegen breakage syndrome which might overestimate the frequency of cth. On the other hand, we are also aware that using high-density microarrays as a technique for detection of cth, the frequency of the cth in T-ALL patients was likely underestimated. Thus, definite frequency of chromothripsis in pediatric T-ALL should be confirmed in other independent cohorts. Moreover, sequence mutations and gene fusions were only analyzed in patients who had a chromothripsis pattern of CNAs. Chromothripsis is an infrequent genomic phenomenon in pediatric T-ALL patients but is significantly associated with cancer-predisposing syndromes. Moreover, somatic CNAs affecting tumor-suppressor genes ( TP53, RB1 , and MED12 ) may contribute to the occurrence of cth in childhood T-ALL patients. Since we observed predominantly inferior outcome of T-ALL in cth positive syndromic individuals we cannot clearly attribute prognostic significance of cth in T-ALL itself.

Supplementary information

Supplemental materials Supplementary Table S4

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These authors contributed equally: Wojciech Młynarski, Monika Lejman.

Supplementary information

The online version contains supplementary material available at 10.1038/s41375-024-02370-z.

Author contributions

AP, WM, and ML designed the research, analyzed the data and wrote the paper. ZU, ML, and BS performed the classical karyotyping and SNP arrays. KM-W performed the RNA sequencing experiments. LS and TS provided the results of MRD assessment using flow cytometry. MS, ZU, and WF supported the data analysis and visualization. AW and JK provided the clinical data. WM supervised the project. All the authors have read and agreed to the published version of the manuscript.

Funding

AP, WM, and ZU were supported by the Team-Net program (POIR.04.04.00-00-16ED/18-00) of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. ML was supported by the Medical University of Lublin (project: Innovation Grant GI/13). ZN was supported by the Foundation for Polish Science (FNP).

Data availability

All the source microarray data are deposited in the Gene Expression Omnibus (GEO) Database (Accession number: GSE267970 ).

Competing interests

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

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