Cytogenetic aberrations of lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia in Chinese patients

To the Editor: As a rare indolent B cell non-Hodgkin lymphoma, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/WM) has unique clinical and biological characteristics.[1] However, due to the difficulties in obtaining tumor metaphase for karyotyping and slow cell proliferation, only very few studies have detected the cytogenetic aberration of LPL/WM.[2,3] In addition, 6q deletion is the most common cytogenetic aberration in WM, with an incidence rate of about 50%.[2] Nevertheless, other cytogenetic aberrations remain largely unclear, and the prognostic role of cytogenetic aberrations needs to be further explored. In the present study, we systematically analyzed 305 LPL/WM cases in China, focusing on the characteristics and cytogenetic aberrations in Chinese patients. A total of 305 patients with LPL/WM admitted to the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC) between June 1998 and December 2018 were enrolled in the present study. The diagnosis was confirmed according to the consensus panel definition for WM.[4] Chromosomal karyotypes were detected in each patient with bone marrow involvement at diagnosis prior to initiation of treatment. Bone marrow tumor cells were cultured in Dulbecco modified Eagle medium (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA) for 24 h. Cells were not stimulated with 12-O-tetradecanoyl-phorbol-13-acetate or CpG-oligonucleotide plus interleukin-2, which might make the cytogenetic detection rate slightly lower than that with stimulating factors in other studies.[2,3] Therefore, a minimum of 15–20 metaphase cases is considered evaluable for analysis. The conventional metaphase cytogenetic analysis with R-banding and G-banding techniques were used to identify the karyotypes. Sample preparation and the operational procedure were performed according to the manufacturer's recommendations and as previously described.[5] Patients enrolled in this study provided informed consent, and the study was approved by the Ethics Review Boards of Institute of Hematology and Blood Disease Hospital (No. IIT2021030-EC-1) and performed in accordance with the Declaration of Helsinki. Fluorescence in situ hybridization (FISH) was performed on metaphase and interphase nuclei using the same samples as the karyotype. The probe panel of FISH included the 1q21 (CKS1B), 6q21 (MYB), 11q22 (LSI ATM), loci centromere 12 (CEP12), 13q14.3 (LSI D13S25/RB-1), and 17p13 (LSI TP53) (All probes were purchased from Vysis, Abbott, Downers Grove, IL, USA). Sample preparations and hybridizations were carried out following the standard procedures as previously described.[5,6] Statistical analyses were performed using the SPSS software package (version 13.0, SPSS Inc., Chicago, IL, USA). The chi-squared test or Fisher's exact test was used to determine the statistically significant differences between clinical groups. Kaplan–Meier analyses were adopted to construct the survival curves. For categorical variables, number and percentage were used to present statistical data, and for continuous variables, data were expressed as the mean and standard deviation. The cytogenetic abnormalities were detected by chromosomal karyotypes and FISH. P values <0.05 were considered statistically significant. The median age of the 305 patients was 62 (32–87) years, and the male-to-female ratio was 2.5:1.0. Among these 305 LPL patients, 285 had serum immunoglobulin M (IgM) paraproteins, 17 cases had immunoglobulin G (IgG) paraproteins, and three had immunoglobulin A (IgA) paraproteins. The most common symptoms at diagnosis were fatigue (64.9%, 198/305) and bleeding (14.1%, 43/305). The median IgM level was 3485 mg/dL (range: 120– 14,400 mg/dL), and the IgM level in 41.3% (126/305) of patients was ≥4000 mg/dL. The median hemoglobin (Hb) level was 8.5 g/dL (range: 2.4–18.7 g/dL), with 80.0% (244/305) lower than 11.5 g/dL. According to the International Prognostic Scoring System for WM (ISSWM), the proportion of patients in low-, intermediate-, and high-risk groups was 17.6%(45/255), 36.5%(93/255), and 45.9%(117/255), respectively. The proportion of hepatomegaly, splenomegaly, lymphadenopathy, and B symptoms was 16.3%(49/300), 37.9%(113/298), 38.2% (112/293), and 29.7% (87/293), respectively. The degree of splenic involvement was always mild or moderate. In addition, 28.3% (32/113) of patients had a giant spleen. The tumor burden was relatively low in LPL/WM patients. The median proportion of lymphoplasmacytic cells, calculated on bone marrow smears, was 45.0% (range: 2.0–97.0%), and the median proportion of clonal lymphocytic cells detected by flow cytometry was 16.4% (range: 0.2–92.9%) [Supplementary Table 1, https://links.lww.com/CM9/B502]. Two hundred and six patients had detected the conventional karyotyping, and it was successfully performed in 194 cases (194/206, 94.2%), of which 20 patients showed clonal abnormalities. Eight of 20 cases with abnormal conventional cytogenetics had two chromosomal abnormalities, and four patients had complex karyotypes. Most of the karyotype abnormalities were caused by chromosomal amplification and deletion. Only one patient had t(1,12)(p22,q12) translocation. The most common conventional cytogenetic abnormalities were 6 or 6q deletion (5/20) and Y deletion (4/20). Other recurrent abnormalities included trisomy 12, 7 deletion, 20q deletion, trisomy 3, trisomy 4, 8q amplification, and 17p deletion. All the patients with 6 or 6q deletion and three patients with Y deletion were accompanied by other karyotype abnormalities. In addition, patients with abnormal karyotypes were also susceptible to other cytogenetic abnormalities detected by FISH (5/20), such as trisomy 12. We then analyzed the survival of patients with or without karyotype aberration. We found that the median progression-free survival (PFS) was 46 months in the normal karyotype group, which was similar to that in the abnormal karyotype group (46 vs. 62 months, P =0.775). The median overall survival (OS) was slightly higher in the normal karyotype group compared with the abnormal karyotype group (114 vs. 75 months, P =0.180). In addition, the survival of patients with two or more karyotypes had no statistically significant difference than those with a single abnormal or normal karyotype. Among the 305 patients, 194 had FISH results, and 106 (54.6%) patients had three or more FISH probes. The estimated median proportion of tumor cells detected by flow cytometry was 16.0% (range: 0.2–92.9%). The incidence rate of each probe was as follows: 6q deletion in 21.4% (6/28) cases, trisomy 12 in 7.6% (5/66) cases, 17p13 deletion in 6.0% (11/184) cases, 13q14 deletion in 5.3% (9/171) cases, 1q21 application in 2.8% (1/36) cases, and 11q22 deletion in 2.0% (2/100) cases. Of the 194 patients, 28 (14.4%) had FISH abnormalities. Among these 28 patients, 23 had a single abnormality, four had two abnormalities, and one had three. Among the five patients with two or more abnormalities, four patients had 17p13 deletion, and four had 13q14 deletion. The mean number of genetic abnormalities was two (range: 1–8) when combining the FISH and conventional karyotyping [Supplementary Table 2, https://links.lww.com/CM9/B502]. We then analyzed the impact of different FISH abnormalities on PFS and OS [Table 1]. Our data showed that the 11 patients with 17p13 deletion had shorter median PFS (28 vs. 54 months, P =0.054) and OS (28 vs. 114 months, P =0.001) compared with those without 17p13 deletion. The patients with a single 17p13 deletion or accompanied by other abnormalities had similar PFS and OS. Among 194 patients, only two had 11q22 deletion, and one had 1q21 application. The two patients with 11q22 deletion progressed and died at 34 and 75 months, respectively. Other cytogenetic aberrations did not impact PFS and OS [Table 1]. When conventional cytogenetics was combined with FISH, the 17p13 deletion was also a poor prognostic factor for PFS and OS. Besides, patients with 11q22 deletion had worse OS than those without this aberration (34 vs. 114 months, P =0.024) [Supplementary Table 3, https://links.lww.com/CM9/B502]. Table 1 - Median PFS and OS of LPL/WM patients according to FISH abnormalities. Cytogenetic abnormalities* Number Median PFS (months, 95% CI) P values Median OS (months, 95% CI) P values 13q14 deletion 0.867 0.298 Positive 9 34.0 (6.8–61.2) 34.0 (10.0–57.9) Negative 162 50.0 (40.5–59.4) 114.0 (89.4–138.6) Trisomy 12 0.165 0.102 Positive 5 22.0 (17.7–26.3) 41.0 (14.5–67.5) Negative 61 50.0 (34.4–65.6) 135.0 (66.1–204.0) 6q deletion 0.383 0.113 Positive 6 108.0† NR Negative 22 46.0 (22.9–69.1) 109.0† 17p13 deletion 0.054 0.001 Positive 11 28.0 (20.8–35.2) 28.0 (20.8–35.2) Negative 173 54.0 (44.9–63.1) 114.0 (89.4–138.6) *Not all the patients had the entire FISH probes: 13q14 deletion had been detected in 171 patients, Trisomy 12 in 66 patients, 6q deletion in 28 patients and 17p13 deletion in 184 patients. †The 95% CI values of PFS and OS were not available. CI: Confidential interval; FISH: Fluorescence in situ hybridization; LPL/WM: Lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia; NR: Not reached; OS: Overall survival; PFS: Progression-free survival. Among the cytogenetic aberrations, trisomy 12 was statistically significantly associated with neutrophil count ≤1.5 × 109/L (60.0% [3/5] vs. 16.4% [10/61], P =0.018) and albumin <35 g/dL (100.0% [5/5] vs. 56.7% [34/60], P =0.057). Moreover, 13q14 deletion was associated with lactate dehydrogenase (LDH) >250 U/L (33.3% [3/9] vs. 8.1% [12/148], P =0.012), and 6q deletion was associated with platelet count >100 × 109/L (100% [6/6] vs. 59.1% [13/22], P =0.057) and LDH >250 U/L (1/5 vs. 0, P =0.059). Cytogenetic aberrations were comparable across the low-, mediate-, and high-risk groups defined by the ISSWM. Supplementary Table 4 (https://links.lww.com/CM9/B502) also reveals the association of other factors. Currently, the clinical characteristics and treatment outcomes of WM patients from multicenters in China have been reported,[7] while the cytogenetic landscape of Chinese patients still remains largely unexplored. Our study provided a comprehensive landscape on cytogenetic aberration in Chinese patients with LPL/WM, and it might well complement the understanding of LPL/WM in Chinese patients. It is well known that 6q deletion is one of the most common cytogenetic abnormalities in WM, whereas its impact on prognosis remains unclear. Recently, a Spanish study has shown that asymptomatic patients with 6q deletion require therapies more often and have a shorter time to transform into the symptomatic disease, ultimately leading to a shorter OS.[3] However, other studies have confirmed that 6q deletion is only related to complex karyotypes and adverse characteristics but has no adverse impact on PFS and OS.[2] These results are similar to our findings. In the present study, we showed that the 6q deletion had no statistically significant impact on PFS or OS, while it was associated with a higher level of LDH. However, due to the limited sample size and lack of stimulating factors, the correlation between 6q deletion and prognosis remained to be further confirmed. In addition, we also showed that 17p13 deletion aggravated adverse outcomes in our cohort, while other cytogenetic aberrations had little impact on survival. Besides being related to survival, cytogenetic aberrations were also significantly associated with adverse clinical and biological features. There are a few limitations in this study that might have led to bias. First, as a respective study, the baseline characteristics of the patients were not entirely consistent. Second, in this study, the tumor cells undergoing karyotyping and FISH were not stimulated by stimulating factors, which might make the cytogenetic detection rate slightly lower than other studies. Third, the sample size of patients with genetic abnormalities were limited, so the correlation between genetic abnormalities and prognosis factors remained to be further confirmed. We hope to conduct a larger series of prospective study in the future to overcome the bias. In conclusion, despite the low incidence and cytogenetic detection rate of LPL/WM, the clinical and genetic features are unique in Chinese patients, and it also plays a vital role in prognostic prediction. Funding This work was supported by grants from the National Nature Science Foundation of China (Nos. 81900203, 81970187, 82170193, and 81920108006), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Nos. 2022-12M-1-022, 2021-I2M-C & T-B-081). Conflicts of interest None.