[Investigation of the effects and mechanisms of berberine on a mouse model of polycystic ovary syndrome: based on intestinal flora analysis].

Objective: To examine the impact of berberine on polycystic ovary syndrome (PCOS) in mice, and to investigate the effects of berberine on the intestinal flora and the intestinal flora on PCOS. Methods: A mouse model of PCOS was established by administering dehydroepiandrosterone in combination with high fat diet, and the mouse model was given a berberine treatment. The study consisted of a blank control group (C group), a PCOS model group (M group) and a berberine treatment group (T group). During the experiment, the mice were closely monitored through timed body weight measurements and estrous cycle monitoring; intraperitoneal glucose tolerance test and insulin tolerance test were done. Upon completion of the pharmacological intervention, the wet weights of liver, ovary and fat deposits of mice were assessed and subjected to HE staining to confirm the success of PCOS modeling and the efficacy of berberine. Additionally, fecal samples were analyzed for intestinal flora through 16S rRNA analysis. Results: The PCOS model was established successfully, berberine alleviated the disturbance of estrous cycle in mice, and significantly alleviated fat accumulation and metabolic abnormalities of glucose in mice. The cross-sectional area of fat pad cells in T group was (2 858±146) µm², which was significantly lower than that in M group [(9 518±347) µm²], and the difference was statistically significant (P<0.001). The blood glucose levels in T group were significantly lower than those in M group (P<0.05). The composition and structure of intestinal flora in mice of M group with PCOS (compared with C group) and in mice of T group after berberine intervention (compared with M group) were significantly altered. However, alpha diversity did not change significantly among three groups (P>0.05). Conclusion: Berberine could alleviate PCOS by intervening in the alterations of gut microbiota.

[The correlation of CD49d expression pattern with molecular genetics and hotspot gene mutants in patients with chronic lymphocytic leukemia].

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.

[Study of cytogenetics and molecular biology in typical and atypical immunophenotypic chronic lymphocytic leukemia].

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.

[Influence of the number of high-risk cytogenetic abnormalities on the clinical characteristics and prognosis in 360 patients with newly diagnosed multiple myeloma].

Objective: To investigate the influence of the number of high-risk cytogenetic abnormalities (HRCA) on the clinical characteristics and prognosis of patients with newly diagnosed multiple myeloma (MM) . Methods: A total of 360 patients with newly diagnosed MM admitted to Jiangsu Province Hospital between November 2013 and September 2020 were included in this study. Cytoplasmic light chain immunofluorescence with fluorescence in situ hybridization (cIg-FISH) was used to detect HRCA. Cytogenetic abnormalities were combined with clinical characteristics and outcomes for further analysis. Results: Among the 360 patients, 120 patients (33.3%) presented with no HRCAs, and 175 (48.6%) , 61 (16.9%) , and four (1.1%) patients had one, two, and three HRCA (s) , respectively. Patients were divided into three groups, including the no-HRCA group, one-HRCA group, and ≥two-HRCA group, according to the number of HRCAs. There were significant differences in the R-ISS stage, hemoglobin level, albumin level, and the proportion of bone marrow plasma cells among the three groups (P<0.05) . The COX proportional-hazards model identified extramedullary disease (P=0.018) , HRCA ≥ 2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P<0.001) as independent risk factors for progression free survival (PFS) and identified lactate dehydrogenase (LDH) level ≥ 220 U/L (P<0.001) , HRCA ≥2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P=0.005) as independent risk factors for overall survival (OS) . The median PFS was 28 months, 22 months, and 14 months (P=0.005) for the three cohorts, and their OS was not reached,60 months, and 30 months (P=0.001) , respectively. Conclusions: HRCA ≥ 2 is an independent risk factor for decreased survival in patients with newly diagnosed MM. More HRCAs result in heavier tumor burden, as well as a higher risk of disease progression and death.

[Clinical analysis of 20 cases of small B lymphocyte proliferative disease with t (14;19) (q32;q13)].

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.

Multiplex fluorescence in situ hybridization in identifying chromosome involvement of complex karyotypes in de novo myelodysplastic syndromes and acute myeloid leukemia.

Complex chromosomal aberrations (CCA) can be detected in a substantial proportion of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), which are associated with very poor prognosis. Conventional cytogenetics (CC) cannot accurately define the specific alterations in CCA. Multiplex fluorescence in situ hybridization (M-FISH) allows the comprehensive identification of CCA. In this study, M-FISH was used in 16 patients with de novo MDS and 22 with AML with CCA detected by R-banding CC, and revealed 206 aberrations involved all 24 chromosomes, including 73 numerical chromosomal abnormalities and 133 structural abnormalities. The chromosomes most often involved were, by decreasing incidence, 5, 17, 8, 11, 7 and 21 in 57.9%, 55.3%, 44.7%, 36.8%, 34.2% and 34.2% of the cases, respectively. There were 98 unbalanced translocations, which were the most frequently observed aberrations in our study. Derivative chromosome 5 and 8 were implicated most often. The other derivatives were der(11), der(12), der(7), der(14), der(15) and der(17). Fourteen balanced translocations were detected in our series, and the most frequent reciprocal translocations was t(8;21). Fifty-five monosomies, 15 partial deletions, and 18 trisomies were found in all patients. The most frequently observed were -5/5q-, -17/17q-, -7, -18, -21, -19, and trisomy of chromosome 8 and 6. There were some abnormalities that have not been previously described, including two complex t(8;21) and seven unbalanced translocations. M-FISH could refine CCA, find or correct the missed or misidentified aberrations by CC analysis. Our findings confirmed that M-FISH was a powerful molecular cytogenetic tool to characterize complex karyotypes in MDS and AML.

Molecular cytogenetic aberrations in 21 Chinese patients with plasma cell leukemia.

Plasma cell leukemia (PCL) is a rare malignant plasma cell disorder. Cytogenetic studies performed on plasma cell disorders are scarce and difficult because of the low proliferation rate of plasma cells (PCs). Fluorescence in situ hybridization (FISH) analysis is an attractive alternative for evaluation of chromosomal changes in PCL. To explore the molecular cytogenetic abnormalities in Chinese patients with PCL, interphase FISH studies with three probes for the regions containing 13q14.3 (D13S319), 14q32 (IGHC/IGHV) and 1q12(CEP1) were retrospectively performed in 21 PCL patients. FISH with LSI IGH/CCND1 and LSI IGH/FGFR3 probes were used to detect t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with 14q32 rearrangement. Among 21 PCL patients, molecular cytogenetic aberrations were found in 18 (81.8%) patients, four (19.0%) patients simultaneously had 13q14 deletion, illegitimate IgH translocation and 1q abnormality. 13q14 deletion was detected in 13 (61.9%) cases and illegitimate 14q32 rearrangement in 16 (76.2%) including six with t(11;14) and three with t(4;14). Chromosome 1 abnormality was found in seven (33.3%) patients, one with deletion of 1q, six with at least three copies amplifications of 1q12 (Amp1q12). 14q32 rearrangement and 13q14 deletion were found concurrently in 11 (52.4%) cases. It was showed that most PCL had chromosomal abnormalities, 14q32 rearrangement, 13q14 deletion and chromosome 1 abnormality are the frequent abnormalities, and over half of the 14q32 rearrangement were t(11;14) or t(4;14). t(4;14) and 13q14 deletion were correlated in PCL. FISH is a highly sensitive technique at detecting molecular cytogenetic aberrations in PCL and should be used in the routine evaluation of PCL.

Molecular cytogenetic aberrations in patients with multiple myeloma studied by interphase fluorescence in situ hybridization.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological disorder characterized by the accumulation of malignant plasma cells within the bone marrow (BM). The clinical heterogeneity of MM is dictated by the cytogenetic aberrations present in the clonal plasma cells (PCs). Cytogenetic studies in MM are hampered by the hypoproliferative nature of plasma cells in MM. Therefore, fluorescence in situ hybridization (FISH) analysis combined with magnetic-activated cell sorting (MACS) is an attractive alternative for evaluation of numerical and structural chromosomal changes in MM. METHODS: Interphase FISH studies with three different specific probes for the regions containing 13q14.3(D13S319), 14q32(IGHC/IGHV) and 1q12(CEP1) were performed in 48 MM patients. Interphase FISH studies with LSI IGH/CCND1, LSI IGH/FGFR3, and LSI IGH/MAF probes were used to detect t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23) in patients with 14q32 rearrangement. RESULTS: Molecular cytogenetic aberrations were found in 40 (83.3%) of the 48 MM patients. 13 patients (27.1%) simultaneously had 13q deletion/monosomy 13[del(13q14)], illegitimate IGH rearrangement and chromosome 1 abnormality. Del (13q14) was detected in 21 cases (43.7%), and illegitimate IGH rearrangements in 29 (60.4%) including 6 with t (11;14) and 5 with t(4;14). None of 9 patients with illegitimate IGH rearrangements and without t(11;14) or t(4;14) we detected had t(14;16)(q32;q23). 24 of the 48 MM patients (50%) had chromosome 1 abnormalities. Among 21 patients with del (13q14), 15 patients had Amp1q12;16 had IgH rearrangements. Whereas, among 27 cases without del (13q14), 8 had Amp1q12; 13 had IgH rearrangements. There was a strong association between del(13q14) and Amp1q12 ( = 8.26, p < 0.01), and between del (13q14) and IgH rearrangement ( = 3.88, p < 0.05). CONCLUSION: 13q deletion/monosomy 13, IGH rearrangement and chromosome 1 abnormality are frequent in MM. They are not randomly distributed, but strongly interconnected. Interphase FISH technique combined with MACS using CD138-specific antibody is a highly sensitive technique at detecting molecular cytogenetic aberrations in MM.

Conventional and molecular cytogenetic features of myelodysplastic syndrome in China.

BACKGROUND: Myelodysplastic syndrome (MDS) constitutes a heterogeneous group of hematopoietic stem cell disorder characterized by peripheral blood cytopenia(s), in the presence of hypercellular bone marrow with features of ineffective hematopoiesis, and susceptibility to acute leukemia (AL). Although the precise pathogenesis of MDS remains to be clarified, cytogenetic abnormalities seem to be involved in its pathogenesis and are considered as an important factor in diagnosis and predicting clinical outcome. OBJECTIVE: To explore the cytogenetic features of Chinese patients with myelodysplastic syndrome (MDS). METHODS: Conventional cytogenetic analysis was performed in 88 MDS patients and among them, 34 cases were studied by interphase fluorescence in situ hybridization (I-FISH) with precisely chromosome 8 centromere specific DNA probe and DNA specific probes for 7q32 , 5q31. RESULTS: Of the 88 patients, 45 (51.1%) showed clonal karyotypic abnormalities by CC at diagnosis, including numerical changes (18 cases, 20.5%), structural changes (12 cases, 13.6%), and numerical and structural changes simultaneously(15 cases, 17.0%). Trisomy 8, -5/5q-, and -7/ 7q- account for 20.5%, 15.9%, and 5.7% respectively. Complex karyotypes were observed in 17 patients, the incidence being 19.3% in the whole series of cases. Among 34 MDS patients studied by I-FISH, -5/5q-, -7/7q- and trisomy 8 occurring in 4, 2 and 10 cases respectively for CC were confirmed by I-FISH. 5 cases in 30 cases who did not show -5/5q- by CC displayed this abnormality by I-FISH. 3 cases without -7/7q- by CC presented this aberration by I-FISH. 5 cases with trisomy 8 for I-FISH was not identified this change by CC. CONCLUSIONS: The frequent abnormalities are trisomy 8, -5/5q- and -7/ 7q-. FISH is very useful in detecting these alterations in MDS and it is an important complement to CC.