A total of 138 patients who had been newly diagnosed with CLL, based on the iwCLL criteria (8), were admitted to the Hematology Department of Tongji Hospital from 2019 to 2021. In total, 123 bone marrow and 15 peripheral blood samples were subjected to cytogenetic analyses, including conventional karyotyping and FISH. We also retrospectively analyzed the clinicopathologic features, immunophenotypes, and immunoglobulin heavy variable (IGHV) mutation status. NGS of 70 samples were performed. Samples with positive IGH-CCND1 probe were excluded from the study.

Using the standard Wright-Giemsa staining protocol, peripheral blood smears were prepared for manual 100-cell differential white blood cell counts. Additionally, manual 200-cell differential white blood cell counts were performed using bone marrow aspirate smears. Particular attention was paid to lymphocytic features.

As previously described (9), the panel of monoclonal antibodies used in our laboratory included antibodies against CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, CD45, CD79b, FMC-7, CD2, CD3, CD4, CD7, CD8, CD56, and CD10, as well as the immunoglobulin κ and λ light chains (BD Biosciences). We calculated the Matutes score for each case according to the method proposed by Matutes et al. (10). Samples with the score of 4–5 were considered as typical immunophenotypes, while samples with the score of 3 were considered as atypical immunophenotypes. Only samples with a score above 3 were enrolled in our study.

Cells (10⁶ cells/mL) were cultured with CpG-oligonucleotide DSP30 (2 μmol/L) plus IL-2 (0.2 μg/mL) (11) for 72 h and harvested, following which banding was performed according to standard procedures. Where possible, at least 20 metaphases were analyzed, and description of karyotypes was performed according to the International System for Human Cytogenetic Nomenclature (ISCN 2016). Any abnormal clone with chromosome gains present in at least two different metaphases and chromosome deletions observed in at least three metaphases was defined as a structural aberration. A complex karyotype (CK) was defined as having at least three independent abnormalities (7). CK cases with 3 or 4 aberrations were defined as low-CK or intermediate-CK, respectively. Cases with ≥ 5 aberrations were defined as high-CK (12).

All samples underwent interphase FISH which was performed using a commercial prognostic probe panel (MetaSystems, Altlussheim, Germany), including del(13q14)-D13S19, +12-CEP12, del(11q22)-ATM, and del(17p13)-TP53. In addition to routine panel probes, an IGH break-apart translocation probe was used to identify IGH-t. Based on the presence or absence of IGH-t, the whole patient cohort was divided into two groups: the IGH-t group and the non-IGH-t group. Next, t (13, 14) (q32;q21) IGH-BCL2, t (8, 13) (q24;q32) IGH-CMYC dual color dual fusion translocation probes, and 19q13-BCL3 and 3q27- BCL6 break-apart probes were used to identify IGH partner genes in the IGH-t group. Moreover, the t (11, 13) (q13;q12) IGH-CCND1 dual color dual fusion translocation probe was used to rule out mantle cell lymphoma (MCL). Based on the FISH risk classification, described by Rigolin et al. (15), the patients were categorized into three groups: the favorable group (isolated 13q14 deletion or absence of FISH aberrations); the unfavorable group (11q22 or 17p13 deletions); and the intermediate group (+12). The probe cut-off values were as follows: 5% for the deletion probe; 3% for the trisomy probe; 5% for the break-apart probe; and 1% for the dual color dual fusion translocation probe.

Genomic DNA was isolated from mononuclear cells from bone marrow or blood samples with the QIAamp DNA Blood Kit (Qiagen, Germany) and used for IGHV mutational status assessment and NGS gene panel analysis.

IGHV gene rearrangements were sequenced by Sanger sequencing using an automated genetic analyser (3500 ABI Applied Biosystems, Foster City, CA, USA). Obtained sequences were analyzed using the International ImMunoGeneTics (IMGT) information system and database tools (IMGT/V-Quest, http://imgt.org). The IGHV mutational status was designated as unmutated, if there were ≤ 2% mutations (> 98% identity), or mutated, if there were > 2% mutations (≤ 98% identity), compared to germline sequences.

As previously described (13), 68 genomic DNA samples and 2 ctDNA (samples 76 and 125) underwent NGS in our laboratory with a panel targeting 157 genes (Supplementary Table S1) which are frequently altered in B-cell lymphoma, according to several previously published large-scale cohort studies. Using genome build hg19/GRCh37 as a reference, a sequencing panel covering exons and adjacent 5 intronic base pairs flanking exonic regions in 157 genes was designed online (Designstudio Sequencing, Illumina, San Diego, USA). Sequencing libraries were prepared with AmpliSeq™ Library PLUS for Illumina, using 20 ng of input genomic DNA per sample. Using paired-end 150 sequencing, library sequencing was performed to 2000× coverage on a NextSeq™ 550 system using an Illumina NextSeq™ 500/550 High Output v2 Kit (Illumina, San Diego, USA). Alignment and variant calling were performed using a DNA Amplicon workflow with default parameters on BaseSpace Sequence Hub (Illumina). The generated variants were further annotated using Annovar. Variant filtering was performed by the following steps: 1) select nonsynonymous variants and small insertions/deletions (indels) that overlap coding exons, splice sites; 2) exclude variants with population frequency > 0.0001 in the gnomAD database unless variant is included as a somatic variant of lymphoid neoplasm in the COSMIC database; 3) exclude variants present in an in-house curated blacklist. 4) exclude variants with quality less than 30 or read depth less than 20. For activation-induced cytidine deaminase (AID) somatic hypermutation (SHM) analysis, we additionally selected synonymous variants and variants in intron/UTR regions, and each variant also needed to fulfill the aforementioned criteria from step 2 to step 4.

In addition, some low frequent mutations from case 76 and 125 were listed in red mark in Supplementary Table S2 because they were validated by the new NGS methods which increased the depth of sequencing to above 10000× by adopting the unique molecular identifiers (UMI) enrichment and target capture DNA library preparation technology. ctDNA from them was extracted from 10ml of plasma by the QIAamp Circulating Nucleic Acid Kit (Qiagen). The NGS library constructed using NadPrep DNA Library Preparation Kit (IDT). Statistical analysis was performed with Fgbio tools (Version 1.2.0).

Descriptive statistics of quantitative variables included counts and frequency distributions, while whole statistical measurements were based on medians. Chi square tests and Fisher’s exact tests were used to assess associations between categorical variables. Statistical significance was set at P≤ 0.05. Statistical analysis was performed using SPSS 23.0.

FISH analysis was performed on samples from all 138 patients enrolled. 98 (71%) patients were positive for at least one of the four probes that were used, as follows: del(13q14)- D13S19 deletion (28.3%); +12 (21.0%), del(11q22)-ATM deletion (10.9%); and del(17p13)-TP53 deletion (8.7%). Based on the FISH risk classification, 66% of the patients were allocated to the favorable group, 21.0% to the intermediate group, and 19.6% to the unfavorable group (Table 1). IGH-t was observed in 25 samples (18.1%), of which 10 (7.2%) carried IGH-BCL2 translocation and 3 (2.2%) carried IGH-BCL3 translocation, and none were positive for BCL6 or IGH-CMYC (Figure 1), as confirmed by FISH. Based on IGH and partner gene FISH results, the IGH-t patients were divided into three groups: IGH-BCL2 group, IGH-BCL3 group, and other IGH-t partner gene group (IGH-others). The remaining 113 patients were assigned to the non-IGH-t group. In the IGH-t group, concomitant FISH aberrations were detected in 15 cases including del(13q) in 8 cases, +12 in 7 cases, del(17p) in 3 cases, and del(11q) in 1 case. Moreover, in the non-IGH-t group, 83 cases had the concomitant aberrations, including the most frequent abnormalities of del(13q) (31 cases), +12 (22 cases), del(11q) (14 cases), and del(17p) (9 cases). Therefore, there was no significant difference between the concomitant FISH abnormalities seen in the IGH-t and non-IGH-t groups (Table 1).

Baseline clinical characteristics and groupwise comparison for those with and without IGH translocation are shown in Table 2. The median age was 61 years (range, 27–84 years). The sex ratio was 2.29:1, with 96 males and 42 females. Patients were divided into two major categories: IGH-t group and non-IGH-t group. The IGH-t group contained three subgroups: IGH-BCL2 group, IGH-BCL3 group, other IGH-t partner gene group (IGH-others) as mentioned above. Of the ten IGH-BCL2-positive cases, all but one had typical morphological characteristics, including small lymphocytes with scant cytoplasm and clumped chromatin. Moreover, all cases with IGH-BCL3 exhibited an atypical CLL morphology characterized by a heterogeneous mixture of small to medium cells, abundant cytoplasm, and indented nuclei, as indicated in a previous study of ours (9). Approximately half the cases with IGH-others displayed atypical CLL morphology. A total of 29 samples had unmutated IGHV status, including 4 in the IGH-t group and 25 in the non-IGH-t group. There was no significant difference between the sex, clinical stage, elevated β2-microglobulin status, or IGHV status readings of the IGH-t and non-IGH-t groups. Furthermore, the flow score (P=0.032) of IGH-t group was lower than that of the non-IGH-t group.

Using a CpG stimulant, we successfully analyzed the metaphase karyotypes in 125 (90.6%) samples including 23 in the IGH-t group (Table 3) and 102 in the non-IGH-t group. Among the 125 samples, 60 (48%) exhibited an abnormal karyotype involving numerical and structural abnormalities with respect to almost all chromosomes; the abnormalities included translocations, derivative or marker chromosomes, isochromosomes, deletions, insertions, and additions. Among these, balanced and unbalanced chromosome translocations were detected in 24.8% (31/125) of the samples. Overall, the most frequent translocation break point was 14q32, followed by 18q21. Other chromosomal translocations that were identified included three cases of whole arm translocation der (7, 8)(q10;p10), der(10;17)(q10;q10), and der(17;18)(q10;q10); two cases of robertsonian translocation rob(13;21)(q10;q10) and rob(15;22)(q10;q10); and other translocations, including t(1;3)(q32;q27), t(1;12)(q21;p13), t(6;8)(q13;p21), and der (20)t(1;20)(q21;p13). The most frequent numerical aberration detected via karyotyping was +12, which was found in 23 samples. Other recurrent aberrations involving chromosome loci hotspots were as follows: duplication of 1q21; deletions of 6q, 11q22, and 13q14; and translocation involving 12p13, and 20p13. Notably, 22 (17.6%) had a complex karyotype (CK) showing more than three chromosomal aberrations. In the IGH-t group, five samples (21.7%) exhibited CKs, including one case of low CK and four of high-CKs. In the non-IGH-t group, seventeen samples (16.7%) exhibited CKs, including five cases of low-CKs, one case of intermediate-CK, and eleven cases of high-CKs. There was no significant difference on aspect of CK in the two groups (P=0.540); (Table 2).

NGS of 70 samples was conducted. Of these, 44 (62.9%) carried at least one mutation and 30 (42.9%) had ≥2 mutations in the targeted NGS region. Detailed lists of mutation detected in 70 samples are shown in Supplementary Table S2. The most frequent mutations listed involved the genes KMT2D (15.7%), TP53 (12.9%), SF3B1 (8.6%), IGLL5 (8.6%), NOTCH1 (7.1%), MYD88 (7.1%), and FBXW7 (5.7%). 12 of the 25 IGH-t samples underwent NGS, and 11 (91.7%) showed positive results. The most frequent mutations in the IGH-t group involved TP53, KMT2D, NOTCH1, and IGLL5. Other relatively rare mutations were detected in the genes, CCND3 (2 cases), MYD88 (1 case), HIST1H1E (1 case), and CD79B (1 case). In total, 58 of the 113 non-IGH-t samples underwent NGS, and 33 (56.9%) were positive and the most frequent mutations in this group were found in KMT2D and TP53. However, no significant differences were found between the two genes in the IGH-t and non-IGH-t groups (P=0.921; P=0.064, respectively). The IGH-t group showed higher FBXW7 (P=0.014) and ATM (P=0.004) mutations than the non-IGH-t group, and the difference was statistically significant (Table 4). Mutational details are shown in Figure 1.