Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western adults, although the incidence of CLL is relatively low in Asian populations. However, with the aging population, the incidence of CLL is increasing in China. The interaction between CLL cells and the microenvironment plays a crucial role in the recognition of antigens by the B-cell receptor immunoglobulin (BCR IG). The mutational status of the immunoglobulin heavy variable region (IGHV) is a classical prognostic marker for CLL. Over 40% of CLL patients exhibit biased usage of IGHV and highly similar amino acid sequences in the heavy complementarity-determining region 3 (HCDR3), known as the BCR stereotypy. Different subgroups of stereotyped BCR exhibit distinct biological and clinical features. Among them, subset #2 with mutated IGHV and poor prognosis, as well as the subset #8 with a high risk of Richter transformation, have been recommended by the European Research Initiative on CLL to be included in clinical reports on IGHV mutational status. This review summarizes the definition, distribution, biological characteristics, and clinical significance of clonality patterns of the BCR in CLL.

Objective:To analyze the sensitivity of cytoplasmic light-chain immunofluorescence with fluorescence in situ hybridization in bone marrow smears (new FISH) for detecting cytogenetic abnormalities in multiple myeloma (MM) .Methods:42 MM patients admitted to the First Affiliated Hospital of Nanjing Medical University from April 2022 to October 2023 were enrolled. The patients with MM were detected by new FISH and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) or cytoplasmic immunoglobulin FISH (cIg-FISH) to analyze cytogenetic detection results using combination probes which included 1q21/1p32, p53, IgH, IgH/FGFR3 [t (4;14) ], and IgH/MAF [t (14;16) ].Results:In 23 patients with MM, the abnormality detection rates of cIg-FISH and new FISH were 95.7% and 100.0%, respectively ( P>0.05). The detection rates of 1q21+, 1p32-, p53 deletion, and IgH abnormalities by cIg-FISH and new FISH were consistent, which were 52.2%, 8.7%, 17.4%, and 65.2%, respectively. The results of the two methods further performed with t (4;14) and t (14;16) in patients with IgH abnormalities were identical. The positive rate of t (4;14) was 26.7%, whereas t (14;16) was not detected. In 19 patients with MM, the abnormality detection rates of MACS-FISH and new FISH were 73.7% and 63.2%, respectively ( P>0.05). The positivity rate of 1q21+, 1p32- and IgH abnormalities detected by MACS-FISH were slightly higher than those detected by new FISH; however, the differences were not statistically significant (all P values >0.05) . Conclusion:The new FISH method has a higher detection rate of cytogenetic abnormalities in patients with MM and has good consistency with MACS-FISH and cIg-FISH.

Objective:To investigate the vaccination status, characteristics and prognosis of patients suffering from a combination of COVID-19 and chronic lymphocytic anemia (CLL) in China.Methods:Clinical data of 328 patients with chronic lymphocytic leukemia (CLL) who were first diagnosed with COVID-19 and treated in the Department of Hematology of Jiangsu Provincial People’s Hospital between November 2022 and February 2023 were retrospectively analyzed. Univariate and multivariate analysis of data of patients with severe/critical COVID-19 were conducted by applying the binary logistic regression model.Results:The median age of the CLL patients was 60 (24-87) years. 23.5% (77/328) of these patients suffered from severe/critical COVID-19 infection. Univariate analysis of the data demonstrated that a combination of factors including age >67 years ( OR=2.15, 95% CI 1.24- 3.73, P=0.006), diabetes ( OR=2.09, 95% CI 1.05-4.20, P=0.037), chronic hepatitis B ( OR=2.91, 95% CI 1.30-6.51, P=0.010), CLL progressive ( OR=3.79, 95% CI 1.57-9.15, P=0.003) and CD20 antibody-based treatments within three months prior to the COVID-19 infection ( OR=2.79, 95% CI 1.35-5.77, P=0.006) were the risk factors for severe/critical COVID-19. According to the multivariate analysis, CLL progressive ( OR=2.98, 95% CI 1.10-8.10, P=0.033) was an independent risk factor for severe/critical COVID-19 and administration of the BTK (Bruton tyrosine kinase) inhibitor monotherapy might exert a protective effect and influence a positive outcome of the COVID-19 infection ( OR=0.38, 95% CI 0.16-0.90, P=0.028). Among the 242 patients who were followed up until October 2023, 9.1% (22/242) had multiple subsequent COVID-19 infections (≥3), and 2.1% (5/242) had persistent COVID-19 infections (patients with persistent positive test for the SARS-CoV-2 antigen testing until missing follow-up for any reason). The peak value of the anti-SARS-CoV-2-IgG titres was observed between three and four months post symptom onset (median: 3.511 S/CO vs 1.047 S/CO, P<0.05). The levels of immunoglobulin A gradually decreased following infection with COVID-19, and its trough levels were attained between two to four weeks post infection (median: 0.30 g/L vs 0.74 g/L, P<0.05). According to this study the mortality of patients suffering from a combination of COVID-19 infection and CLL was 2.7% (9/328), and the main reason for their death was respiratory failure and heart failure. Conclusions:A low rate of COVID-19 vaccination and a high rate of severe/critical COVID-19 infection was observed in the CLL patients. CLL progressive was associated with severe/critical COVID-19. Anti-CD20-based treatments received within the past three months might be a risk factor for exacerbation of COVID-19 infection, whereas a monotherapy with BTK inhibitors exert a protective effect and improve outcome of COVID-19 infection.

Despite exciting achievements with some malignancies, immunotherapy for hypoimmunogenic cancers, especially glioblastoma (GBM), remains a formidable clinical challenge. Poor immunogenicity and deficient immune infiltrates are two major limitations to an effective cancer-specific immune response. Herein, we propose that an injectable signal-amplifying nanocomposite/hydrogel system consisting of granulocyte-macrophage colony-stimulating factor and imiquimod-loaded antigen-capturing nanoparticles can simultaneously amplify the chemotactic signal of antigen-presenting cells and the "danger" signal of GBM. We demonstrated the feasibility of this strategy in two scenarios of GBM. In the first scenario, we showed that this simultaneous amplification system, in conjunction with local chemotherapy, enhanced both the immunogenicity and immune infiltrates in a recurrent GBM model; thus, ultimately making a cold GBM hot and suppressing postoperative relapse. Encouraged by excellent efficacy, we further exploited this signal-amplifying system to improve the efficiency of vaccine lysate in the treatment of refractory multiple GBM, a disease with limited clinical treatment options. In general, this biomaterial-based immune signal amplification system represents a unique approach to restore GBM-specific immunity and may provide a beneficial preliminary treatment for other clinically refractory malignancies.

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 investigate the clinical significance of cc-chemokine receptor 7 (CCR7) as a potential diagnostic or differential marker for chronic lymphocytic leukemia (CLL).Methods:A total number of 643 patients with B-cell chronic lymphoproliferative diseases (B-CLPD) admitted to the First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2018 were enrolled. The patients included 327 cases of CLL, 58 cases of mantle cell lymphoma (MCL), 34 cases of follicular lymphoma (FL), 36 cases of marginal zone lymphoma (MZL), 10 cases of hair-cell leukemia or its variants (HCL/HCLV-v), 40 cases of Waldorf′s macroglobulinemia (WM), 48 cases of CD5 +B-cell chronic lymphoproliferative disease unclassified (B-CLPD-U) and 90 cases of CD5 -B-CLPD-U. At the same time, 20 samples from healthy people from the medical examination center of our hospital were used as normal controls. Flow cytometry was used to detect the immune-phenotype and CCR7 expression level in B-CLPD patients, and Fluorescence in situ hybridization (FISH) was used to analyze the genomic alterations: the ataxia telangiectasia mutant gene (ATM) deletion, the 13q14 deletion, the P53 deletion and trisomy 12. Sanger sequencing was used to analyze gene mutations of splicing factor 3B subunit 1 (SF3B1), NOTCH1, tumor protein 53 (TP53) and immunoglobulin heavy chain variable region (IGHV). Measurement data were compared by Mann-Whitney test, and the positive rates were compared by chi-square test. The diagnostic value and optimal positive cutoff value of CCR7 were calculated using receiver operating characteristic (ROC) curve. Results:The positive rates of CCR7 expression in typical CLL and atypical CLL were 90.8% (257/283) and 84.1% (37/44), respectively, and there was no significant difference of the positive rates (χ 2=1.228, P=0.268) between groups. The positive expression rates of CCR7 in CLL, MCL, CD5 +B-CLPD-U, CD5 -B-CLPD-U, FL, WM, HCL/HCL-v and MZL were 89.9% (294/327), 10.3% (6/58), 6.3% (3/48), 8.9% (8/90), 0, 0, 0 and 13.9% (5/36) respectively, and the median mean fluorescence intensity (MFI) was 278 (246, 307), 114 (106, 128), 112 (106, 117), 110 (104, 121), 108 (105, 119), 111 (105, 124), 112 (108, 115) and 109 (105, 120) respectively. Compared with CLL, the positive expression rates of CCR7 in other types of B-CLPDs were lower significantly (χ 2=181.3, 177.8, 232, 164.7, 180.8, 62.6, 129, P<0.01). In addition, the sensitivity, specificity and accuracy of CCR7 for distinguishing CLL from other types of B-CLPD were 89.9%, 93.0% and 92.3%, respectively. The positive expression rate of CD49d in CCR7 +CLL patients was 13.9%, which was significantly lower than that in CCR7 -CLL patients (42.1%) (χ 2=7.6, P=0.01). The coincidence rate of 13q14 deletion was 50.3% in CCR7 +CLL patients, which was significantly higher than that in CCR7 -CLL patients (20%) (χ 2=6.56, P=0.01). Conclusions:The CC-chemokine receptor 7 (CCR7) antigen is an effective marker for the diagnosis and identification of chronic lymphocytic leukemia (CLL). The expression level of CCR7 in clinical specimens can distinguish CLL from other pathological subtypes of B-CLPDs.

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: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.