Objective:To elucidate the genomic characteristics of the immunoglobulin (IG) heavy-chain variable region and light-chain variable region, the expression of subclones, and the prognostic significance in patients with CLL.Methods:Blood and/or bone marrow specimens were gathered from a cohort of 36 patients with CLL diagnosed at Jiangsu Province Hospital from December 2018 to May 2023, including 12 cases of B cell receptor (BCR) stereotyped patients. IG heavy-chain (IGH) and light-chain (IG Kappa [IGK] and IG lambda [IGL]) gene rearrangements were performed using next-generation sequencing (NGS) technology to analyze the characteristics and prognostic value in CLL.Results:NGS detection of IG variable region (IGHV) demonstrated a significant correlation and superior consistency with Sanger sequencing ( r=0.957, P < 0.001). Among the 36 patients, the IGH variant (IGHV) was observed in 9 (25.0%) but not in 27 (75.0%) participants. The incidence of the MYD88 mutation was higher among patients with mutated IGHV [1/27 (3.7%) vs 4/9 (44.4%), P=0.00]. A high incidence of trisomy 12 was observed in the IGHV #8/#8B subset [4/11 (36.4%) vs 1/25 (4.0%), P=0.023], which were more likely to develop Richter transformation [8/11 (72.7%) vs 4/25 (16.0%), P=0.002]. In the patient cohort, 36 individuals (36/36, 100.0%) used the IGK variable, whereas 15 individuals (15/36, 41.7%) employed the IGL variable (IGLV). IGLV3 - 21 reported the highest utilization rate in IGLV (5/15, 33.3%). Remarkably, patients with CLL with IGLV3-21 fragments were exclusively observed in the Binet C stage and Rai Phase Ⅲ-Ⅳ, with an incidence of del (13) (q14) at 60.0% (3/5). The median time to first treatment (TTFT) of patients with or without IGLV3 - 21 fragments was 5.2 (1.1 - 41.5) and 9.9 (0.1 - 94.4) months, respectively. Using the total reads threshold of 2.5%, 4 (4/36, 11.1%) samples were detected to have two IGHV productive clones. The median TTFT and overall survival (OS) time were 2.8 (0.9-72.7) and 12.8 months in patients with one mutated clone and 57.5 (32.0-120.7) and 51.8 months in those with two mutated clones, respectively. The median TTFT and OS time were 10.9 (0.3-94.4) and 6.3 (0.1 - 12.5) months in patients with one unmutated clone and 49.9 (22.2 - 211.1) and 30.0 (9.6 - 50.3) months in those with multiple unmutated clones, respectively ( P>0.05) . Conclusions:Detection of IG gene rearrangements using NGS technology not only facilitates the analysis of the IGHV mutation status, dominant clones, and prognostic value but also contributes to the exploration of IGK/IGL gene rearrangement fragments and the utilization of subclones. Further, it provides information about the poor prognosis of IGLV3 - 21 CLL. The shortened survival of the two unmutated clone groups in the IGHV unmutated group may indicate a poor prognosis.

Objective:To evaluate the efficacy and long-term outcomes of fludarabine, cyclophosphamide, and rituximab (FCR) in treatment-na?ve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) .Methods:Clinical data from 68 CLL/SLL patients treated with FCR at Jiangsu Province Hospital (August 2008–May 2021) were retrospectively analyzed to assess efficacy, safety, and survival outcomes.Results:Among 68 patients [46 males, 22 females; median age 55 (47, 60) years], 13.1% (8/61) had a complex karyotype, 32.3% (20/62) had immunoglobulin heavy variable region mutated (IGHV-M) type, 6.6% (4/61) had del (17p), and 14.8% (8/54) had del (11q). Patients received a median of 6 (4, 6) FCR cycles. The overall response rate was 88.2% (60/68), including 47.0% (32/68) complete remissions. Over a median follow-up of 82 (59, 98) months, 66.2% (45/68) experienced disease progression. Median progression-free survival was 56 (21, 123) months, while median overall survival was not reached. The 5- and 10-year PFS rates were 42.6% (95% CI: 31.9–56.8% ) and 28.7% (95% CI: 19.0–43.4% ), respectively. Poor PFS was associated with del (17p) ( HR=5.04, 95% CI: 1.72–14.74, P=0.003), del (11q) ( HR=5.27, 95% CI: 2.11–13.15, P<0.001), IGHV unmutated (IGHV-UM) ( HR=4.11, 95% CI: 1.72–9.79, P=0.001), complex karyotype (CK) ( HR=3.53, 95% CI: 1.58–7.85, P=0.002), β 2-microglobulin >3.5 mg/L ( HR=2.87, 95% CI: 1.37–6.01, P=0.005). In multivariate analysis, IGHV-UM remained an independent predictor of PFS ( HR=8.63, 95% CI: 1.09–68.40, P=0.042). Sixteen patients with IGHV-M and lacking del (17p) or CK had a median PFS of 123 (58,123) months and a 5-year PFS rate of 70.7% (95% CI: 49.7–99.1% ), reaching a plateau after 5 years with no recurrences by 10 years. Common grade 3–4 adverse events included hematologic toxicity (44.1%, 30/68), infection (36.7%, 25/68), and liver dysfunction (4.4%, 3/68). Among 25 patients receiving single-agent BTK inhibitors after FCR progression, median follow-up was 45 (26, 64) months; 36% (9/25) experienced disease progression, with a median PFS time of 55 (27, 55) months. Conclusion:First-line FCR provides durable long-term benefits for patients with IGHV-M CLL without del (17p) or CK.

Objective:To elucidate the genomic characteristics of the immunoglobulin (IG) heavy-chain variable region and light-chain variable region, the expression of subclones, and the prognostic significance in patients with CLL.Methods:Blood and/or bone marrow specimens were gathered from a cohort of 36 patients with CLL diagnosed at Jiangsu Province Hospital from December 2018 to May 2023, including 12 cases of B cell receptor (BCR) stereotyped patients. IG heavy-chain (IGH) and light-chain (IG Kappa [IGK] and IG lambda [IGL]) gene rearrangements were performed using next-generation sequencing (NGS) technology to analyze the characteristics and prognostic value in CLL.Results:NGS detection of IG variable region (IGHV) demonstrated a significant correlation and superior consistency with Sanger sequencing ( r=0.957, P < 0.001). Among the 36 patients, the IGH variant (IGHV) was observed in 9 (25.0%) but not in 27 (75.0%) participants. The incidence of the MYD88 mutation was higher among patients with mutated IGHV [1/27 (3.7%) vs 4/9 (44.4%), P=0.00]. A high incidence of trisomy 12 was observed in the IGHV #8/#8B subset [4/11 (36.4%) vs 1/25 (4.0%), P=0.023], which were more likely to develop Richter transformation [8/11 (72.7%) vs 4/25 (16.0%), P=0.002]. In the patient cohort, 36 individuals (36/36, 100.0%) used the IGK variable, whereas 15 individuals (15/36, 41.7%) employed the IGL variable (IGLV). IGLV3 - 21 reported the highest utilization rate in IGLV (5/15, 33.3%). Remarkably, patients with CLL with IGLV3-21 fragments were exclusively observed in the Binet C stage and Rai Phase Ⅲ-Ⅳ, with an incidence of del (13) (q14) at 60.0% (3/5). The median time to first treatment (TTFT) of patients with or without IGLV3 - 21 fragments was 5.2 (1.1 - 41.5) and 9.9 (0.1 - 94.4) months, respectively. Using the total reads threshold of 2.5%, 4 (4/36, 11.1%) samples were detected to have two IGHV productive clones. The median TTFT and overall survival (OS) time were 2.8 (0.9-72.7) and 12.8 months in patients with one mutated clone and 57.5 (32.0-120.7) and 51.8 months in those with two mutated clones, respectively. The median TTFT and OS time were 10.9 (0.3-94.4) and 6.3 (0.1 - 12.5) months in patients with one unmutated clone and 49.9 (22.2 - 211.1) and 30.0 (9.6 - 50.3) months in those with multiple unmutated clones, respectively ( P>0.05) . Conclusions:Detection of IG gene rearrangements using NGS technology not only facilitates the analysis of the IGHV mutation status, dominant clones, and prognostic value but also contributes to the exploration of IGK/IGL gene rearrangement fragments and the utilization of subclones. Further, it provides information about the poor prognosis of IGLV3 - 21 CLL. The shortened survival of the two unmutated clone groups in the IGHV unmutated group may indicate a poor prognosis.

Objective:To evaluate the efficacy and long-term outcomes of fludarabine, cyclophosphamide, and rituximab (FCR) in treatment-na?ve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) .Methods:Clinical data from 68 CLL/SLL patients treated with FCR at Jiangsu Province Hospital (August 2008–May 2021) were retrospectively analyzed to assess efficacy, safety, and survival outcomes.Results:Among 68 patients [46 males, 22 females; median age 55 (47, 60) years], 13.1% (8/61) had a complex karyotype, 32.3% (20/62) had immunoglobulin heavy variable region mutated (IGHV-M) type, 6.6% (4/61) had del (17p), and 14.8% (8/54) had del (11q). Patients received a median of 6 (4, 6) FCR cycles. The overall response rate was 88.2% (60/68), including 47.0% (32/68) complete remissions. Over a median follow-up of 82 (59, 98) months, 66.2% (45/68) experienced disease progression. Median progression-free survival was 56 (21, 123) months, while median overall survival was not reached. The 5- and 10-year PFS rates were 42.6% (95% CI: 31.9–56.8% ) and 28.7% (95% CI: 19.0–43.4% ), respectively. Poor PFS was associated with del (17p) ( HR=5.04, 95% CI: 1.72–14.74, P=0.003), del (11q) ( HR=5.27, 95% CI: 2.11–13.15, P<0.001), IGHV unmutated (IGHV-UM) ( HR=4.11, 95% CI: 1.72–9.79, P=0.001), complex karyotype (CK) ( HR=3.53, 95% CI: 1.58–7.85, P=0.002), β 2-microglobulin >3.5 mg/L ( HR=2.87, 95% CI: 1.37–6.01, P=0.005). In multivariate analysis, IGHV-UM remained an independent predictor of PFS ( HR=8.63, 95% CI: 1.09–68.40, P=0.042). Sixteen patients with IGHV-M and lacking del (17p) or CK had a median PFS of 123 (58,123) months and a 5-year PFS rate of 70.7% (95% CI: 49.7–99.1% ), reaching a plateau after 5 years with no recurrences by 10 years. Common grade 3–4 adverse events included hematologic toxicity (44.1%, 30/68), infection (36.7%, 25/68), and liver dysfunction (4.4%, 3/68). Among 25 patients receiving single-agent BTK inhibitors after FCR progression, median follow-up was 45 (26, 64) months; 36% (9/25) experienced disease progression, with a median PFS time of 55 (27, 55) months. Conclusion:First-line FCR provides durable long-term benefits for patients with IGHV-M CLL without del (17p) or CK.

Objective:To analyze the distribution of different SF3B1 genotypes in patients with myelodysplastic syndromes (MDS) and its prognostic value.Methods:Totally, 377MDS patients who were initially diagnosed in the First Affiliated Hospital of Nanjing Medical University from January 2014 to January 2022 were included in the retrospective analysis.The patients were divided into three different groups according to mutation stcote of SF3B1, including 317 patients with SF3B1 wild type (SF3B1 WT) (214 males and 103 females, 63(49, 71) years old),39 patients with SF3B1 K700E mutation(SF3B1 K700E(17 males and 22 females, 65(52, 73)years old)) and 21 patients with SF3B1 non-K700E mutation(SF3B1 non-K700E)(13 males and 8 females, 67(63, 73) years old). MDS-related 20 gene mutations were detected using targeted sequencing technology; Survival curves were constructed by the Kaplan-Meier method; Cox proportional hazards model was established to evaluate different factors at diagnosis on survival by univariate and multivariate analyses.. Results:Compared with SF3B1 non-K700E patients, SF3B1 K700E patients had a higher median absolute neutrophil count ( P=0.002) and were likely to be in the low/int-1 International Prognostic Scoring System (IPSS) categories ( P=0.023). A 20-gene targeted sequencing analysis showed that, compared with SF3B1 WT patients, SF3B1 K700E patients were associated with lower frequency of ASXL1 and U2AF1 mutations ( P=0.018 and P=0.003); while compared with SF3B1 non-K700E patients, the frequency of ASXL1 mutation was significantly lower in SF3B1 K700E cases ( P=0.029). Patients with SF3B1 K700E had better overall survival (OS) in comparison with SF3B1 WT and SF3B1 non-K700E in MDS patients ( P<0.001 and P=0.045, respectively). In comparison with SF3B1 WT patients, SF3B1 MUT patients had more favorable OS and progression-free survival (PFS) in MDS without excess blasts ( P<0.001 and P<0.001, respectively), but no significant difference was found in MDS with excess blasts ( P>0.05). Compared with SF3B1 WT patients, SF3B1 K700E patients had superior OS and PFS in the int-1 IPSS category ( P=0.010 and P=0.013, respectively). By multivariable analysis, the presence of SF3B1 K700Ewas an independent predictor of superior OS ( HR=0.461,95% CI 0.262-0.811, P=0.007). Conclusion:SF3B1 K700E and SF3B1 non-K700E patients had significantly improved OS in comparison with SF3B1 WT MDS patients. Furthermore, SF3B1 K700E patients were associated with a better OS compared with SF3B1 non-K700E MDS patients. SF3B1 mutation could not overcome the poor prognostic effect of excess blasts, which highlights the importance of the SF3B1 mutation subtype in risk assessment of MDS without excess blasts.

Objective:To explore the correlation of CD49d expression patterns with molecular genetics and hotspot gene mutants in patients with chronic lymphocytic leukemia.Methods:The expression of CD49d was detected by flow cytometry and grouped into homogeneous, bimodal, negative and positive expression. Panel fluorescence in situ hybridization (FISH) was used for molecular genetics analysis and next-generation sequencing (NGS) was conducted for gene mutation detection.Results:There were 43 patients (23.89% ) with positive CD49d expression, 137 patients (76.11% ) with negative CD49d expression, 96 patients (53.33% ) with homogeneous CD49d expression and 84 patients (46.67% ) with bimodal CD49d expression. Compared with patients in the CD49d negative group, patients in the CD49d positive group had higher Rai stage ( P=0.048) and higher proportion of spleen enlargement ( P=0.030) . Compared with patients with homogeneous expression of CD49d, patients with bimodal expression of CD49d had a higher proportion of spleen enlargement ( P=0.009) . The expression rate of 11q22- in bimodal CD49d - group was significantly higher than that in homogeneous CD49d - group (24.29% vs 10.45% , P=0.043) . The incidence of +12 in homogeneous CD49d group was higher than that in bimodal CD49d group (16.67% vs 5.95% , P=0.035) . The incidence of +12 in homogeneous CD49d + group was higher than that in bimodal CD49d - group (17.24% vs 4.29% , P=0.045) . The incidence of +12 in homogeneous CD49d - group was higher than that in bimodal CD49d - group (16.42% vs 4.29% , P=0.024) . BIRC3 mutation rate in CD49d positive group was higher than that in CD49d negative group (11.63% vs 2.92% , P=0.037) . Conclusion:There were significant correlations between CD49d and 11q22-, +12 and BIRC3 gene mutation. Patients with bimodal CD49d were more correlated with poor prognosis indexes.

Objective:To analyze the differences in immunophenotype, cytogenetics, and molecular biology between typical and atypical immunophenotype chronic lymphocytic leukemia (CLL) , and explore the correlation of cytogenetic anomalies with gene mutations.Methods:This study included 488 patients diagnosed in the First Affiliated Hospital of Nanjing Medical University between November 2014 and May 2021. Of these, 382 patients scored 4-5 points, which was typical CLL (tCLL) , and 106 scored 3 points, which was atypical CLL (aCLL) as per the Royal Marsden Hospital Immunomarker Integral System. Peripheral blood cells were collected for immunophenotype by multiparameter flow cytometry in 488 patients, fluorescence in situ hybridization (FISH) was employed to detect cytogenetic anomalies in 359 patients, and gene mutations were detected by next-generation sequencing (NGS) in 330 patients.Results:The positive rates of CD10, CD22, CD49d, CD81, and FMC7 were significantly higher in the aCLL compared with the tCLL group ( P=0.020, P<0.001, P<0.001, P=0.027, and P<0.001, respectively) , while the positive rates of CD5, CD23, CD148, and CD200 were lower in the former compared to the latter ( P<0.001, P=0.017, P=0.041, and P<0.001, respectively) . aCLL exhibited a higher frequency of trisomy 12 and lower frequency of del (13q14) compared to the tCLL group ( P<0.001 and P<0.001, respectively) . Moreover, aCLL patients also showed a higher incidence of NOTCH1 mutations than the tCLL patients ( P=0.038) , while no statistically significant differences in other gene mutations occurred between the two groups. No significant differences in overall survival (OS) and treatment-free survival (TFS) occurred between aCLL and tCLL using Kaplan-Meier analysis ( P>0.05) . Conclusion:aCLL has characteristic immunophenotype, cytogenetic, and somatic mutation that differ from tCLL, and this can provide reliable information for the diagnosis and differential diagnosis between the two groups.

Objective:To investigate the expression characteristics of TRBC1 protein in mature T-cell lymphoma (TCL) , and compare with T-cell receptor (TCR) -Vβ repertoire analysis and TCR gene rearrangement results, to explore the value of TRBC1 in the diagnosis of TCL.Methods:The expression of TRBC1 was detected by multi-parameter flow cytometry in 30 cases of TCL, 40 cases of normal controls and 50 cases of patients without T lymphocyte proliferative diseases (non-TCL) admitted to the Department of Hematology, The First Affiliated Hospital of Nanjing Medical University. The diagnostic value of TCRVβ repertoire analysis, TCR gene rearrangement and TRBC1 restricted expression detection in TCL was evaluated.Results:The positive rates of CD4 +T and CD8 +T cell subsets TRBC1 in normal control group were (39.6±6.5) % and (39.3±4.4) %. The positive rates of CD4 +T and CD8 +T cell subsets TRBC1 in non-TCL were (39.1±3.8) % and (36.0±8.4) %. All 30 cases of TCL were CD3 +TCRγδ -, and the positive rate of TRBC1 was >92.3% or <12.7%. All cases showed restrictive expression pattern (monoclonal expression) , which was significantly different from those of the normal control and the non-TCL cases ( P<0.001) . In terms of the diagnostic performance of T cell clonality, the sensitivity of TRBC1 was 100%, the positive detection rate of TCR gene rearrangement was 92.8%, and the sensitivity of TCRVβ detection was 94.1%. Kappa test showed high consistency among the three detective methods. Conclusion:Multi-parameter flow cytometry detection of TRBC1 expression level can quickly and efficiently diagnose mature T-cell lymphoma, which has good clinical application value.

Objective:The clinical characteristics and prognosis of 20 patients with small B-lymphocyte proliferative disease with t (14;19) (q32; q13) were analyzed to improve the understanding of such rare cases.Methods:The clinical data of 20 patients with t (14; 19) (q32; q13) small B lymphocyte proliferative disease treated in the First Affiliated Hospital of Nanjing Medical University from April 2013 to December 2020 were retrospectively collected and analyzed. Among them, 10 cases were chronic lymphocytic leukemia (CLL) and 10 cases were other small B-cell malignancies.Results:Among the 20 cases, 10 were male and 10 were female, and the median age at diagnosis was 53.5 (35-88) years old. All patients had absolute lymphocytosis, 19 patients had lymphadenopathy, and 10 patients had splenomegaly. With a median follow-up of 36 (4-163) months, three patients died, and 11 patients had a time to treatment (TTT) ≤12 months. Ten patients (50%) were accompanied by +12, two patients (2/17, 12%) were accompanied by 13q-. Moreover, we found that t (14;19) was associated with unmutated immunoglobulin heavy-chain variable (IGHV) somatic mutation (17/19, 89%) and a biased use of IGHV4-39 (7/17, 41%) was observed. Next-generation sequencing detected one or more gene mutations in 14 (14/17, 82%) cases and a total of 25 gene mutations had been revealed, of which the most frequent were NOTCH1 (35%) , followed by SF3B1 (24%) and KMT2D (18%) . For 10 CLL patients, five (50%) were defined as Rai Ⅲ/Binet C. It is noteworthy that among the 20 cases, two cases actually involved Richter transformation.Conclusions:Small B-cell malignant tumors with abnormal t (14; 19) show unique clinical biological characteristics, often accompanied by a variety of adverse prognostic factors, and tend to have an aggressive clinical course.

Objective:To investigate the clinical, genetic, and clonality related aspects of individuals with Richter transformation (RT) .Methods:From January 2019 to December 2021, 18 RT patients with diagnoses at the First Affiliated Hospital of Nanjing Medical University (Pukou CLL center) were retrospectively examined. The immunoglobin heavy variable (IGHV) gene usage and IGHV-D-J rearrangement pattern of diagnosed CLL/SLL and transformed diffuse large B-cell lymphoma (DLBCL) were compared to determine the clonality relatedness. To investigate the risk factors of RT, Clinical and laboratory data from patients with newly diagnosed CLL/SLL and transformed DLBCL were gathered.Results:The median age of RT was 56.5 (41-75) years old. 17 patients transformed to DLBCL and 1 transformed to Hodgkin lymphoma (HL) . Of 17 individuals who had DLBCL transformation, 15 had CLL/SLL-related clonality and 2 had unrelated clonality. Next-generation sequencing (NGS) analysis of 11 paired initially diagnosed treatment-naive CLL/SLL and RT DLBCL found that EGR2、TP53 and NOTCH1 were among the most frequently mutated genes both in treatment-naive CLL/SLL and in RT DLBCL. In several cases, specific mutations were gained or lost throughout RT, indicating clonal evolution. Among 10 patients before exposure to BTK inhibitors before RT, four patients acquired BTK mutation. The aforementioned mutations should be considered high-risk variables for transformation; in addition, TP53 and EGR2 mutations could be linked to a poor prognosis following RT in patients receiving a cocktail of new medicines.Conclusion:Most RT DLBCL patients in our center are clonality related (15/17, 88.2% ) and we recommend all qualified centers to evaluate clonality relatedness of RT DLBCL patients. There was some variability in the mutational landscape between DLBCL that had undergone a transformation and initially diagnosed, treatment-naive CLL/SLL. The underlying molecular mechanism of RT needs more research.