Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

[Immunophenotyping and molecular genetic analysis of diffuse large B-cell lymphoma].

OBJECTIVE: To perform immunophenotyping and molecular genetic analysis for diffuse large B-cell lymphoma (DLBCL), and to explore their correlation and implication for prognosis. METHODS: Immunohistochemical streptavidin peroxidase (SP) method was used to determine the expression of CD10, BCL6 and MUM1 in 59 cases of DLBCL. A Hans algorithm was used to classify DLBCL into germinal center B-cell (GCB) and non-GCB subtypes. Interphase fluorescence in situ hybridization (FISH) assay was performed on paraffin-embedded lymphoma tissue sections to detect translocations and amplifications of BCL6, BCL2 and MYC genes with dual-color break-apart BCL6 probe, dual-color dual-fusion IgH/ BCL2 probe and dual-color break-apart MYC probe, respectively. RESULTS: In the 59 cases of DLBCL, 28.8% (17/59) belonged to GCB subtype, and 71.2% (42/59) belonged to non-GCB subtype. The incidences of BCL6, BCL2 and MYC gene translocations were 24.1% (14/58), 1.7% (1/59) and 5.3% (3/57), respectively. The incidences of BCL6, BCL2 and MYC gene amplifications were 17.2% (10/58), 22.0% (13/59) and 21.1% (12/57), respectively. BCL6 amplification was not correlated with BCL6 translocation (P=0.424), but was correlated with amplifications of BCL2 and MYC (C=0.405 and 0.403, respectively, P <0.01). The incidence of BCL6 translocation in GCB type was higher than that in non-GCB type, and amplifications of BCL6, BCL2 or MYC were more frequently encountered in non-GCB type, though no statistical significance was detected (P=0.089 and 0.106, respectively). By univariate analysis, immunophenotyping and international prognostic index (IPI) exerted a significant effect on overall survival (OS) (P=0.047 and 0.001, respectively), but to which BCL6 translocation and amplification of the 3 genes were not related (P=0.150 and 0.444, respectively). By multivariate analysis, IPI score was the only independent prognostic factor for OS (RR =3.843, P=0.017). CONCLUSION: The GCB subtype of DLBCL is less common in the patient cohort. Common genetic aberrations have included BCL6 translocation and BCL6, BCL2 and MYC amplifications. Amplification of the 3 genes is strongly correlated with each other, and the incidence of BCL2 translocation is low. Immunophenotyping only has minor significance for the prognosis. Genetic aberrations cannot predict the clinical outcome of DLBCL.

[Clinical and laboratory investigation of pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11) in Ph-positive leukemia].

OBJECTIVE: To investigate clinical and molecule genetics features of four Ph-positive leukemia patients characterized by pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11). METHODS: Cytogenetic analysis was carried out on bone marrow directly or after short-period culture. R banding was used for karyotype analysis. BCR/ABL fusion gene was detected with interphase fluorescence in situ hybridization (FISH), and chromosome painting was carried out using specific probes. RT-PCR was used to detect BCR/ABL chimeric transcripts. RESULTS: One patient with acute myeloid leukemia (AML) presented three clones, which included one with a normal karyotype, one with t(9;22)(q34;q11), and one with inv(9)(p22q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). The inv(9)(p22q34) has always co-occurred with der(9)t(9;22)(q34;q11) accompanied by der(22)t(9;22)(q34;q11) in all metaphases from the three patients with chronic myeloid leukemia (CML). B3a2 transcript was detected in all patients by RT-PCR. Inv(9)(p22q34) was found in both CML and AML, and was associated with poor prognosis. CONCLUSION: Inv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Leukemia with der(9)t(9;22) and inv(9)(p22q34) has unique clinical and laboratory characteristics.

[Deletions and rearrangements of PAX5 gene in B-lineage acute lymphoblastic leukemia].

OBJECTIVE: To determine the frequency paired-box domain 5 (PAX5) gene alterations in B-lineage acute lymphoblastic leukemia (B-ALL) harboring 9p abnormalities and its implication for clinical prognosis. METHODS: Bacterial artificial chromosomes RP11-344B23 and RP11-652D9 encompassing the PAX5 gene were selected. DNA was extracted with conventional method and labeled with fluorescein by nicking transition. Fluorescence in situ hybridization (FISH) was used to determine the rearrangement or deletion of the PAX5 gene in B-ALL harboring chromosome 9p abnormalities. Clinical and laboratory features of patients were analyzed. RESULTS: Fifty cases were analyzed with FISH. Complete deletion was observed in 23 patients (46%), partial deletion was observed in 2 patients (4%), and rearrangement was detected only in 1 case. The total frequency of abnormalities was 52% (26/50). No significant difference was found in clinical features of patients with or without PAX5 rearrangement or deletion. CONCLUSION: The frequency of PAX5 gene alterations in B-ALL harboring 9p abnormalities was 52%. However, no significant difference was found between patients with and without PAX5 alterations.

[Clinical and cytogenetic study of 6 cases of hematological disorders associated with 20q- and t (20;21) (q11;q11) abnormalities].

OBJECTIVE: To analyze clinical and cytogenetic features of hematological disorders associated with 20q- and t (20;21) (q11;q11) abnormalities. METHODS: Following short-term culture of bone marrow cells, karyotypic analysis was carried out with R-banding. 20q- and t(20;21) (q11;q11) was detected by fluorescence in situ hybridization (FISH) using dual-color 20q11/12 probe, ST 20qter /ST 21qter probes, SE20(D20Z1)/SE 13/21 probes, and WC20/WC21 probes. RESULTS: Six (2.3%) of the 257 patients with 20q- detected by conventional karyotypic analysis were found to have t(20;21) (q11;q11) abnormality. Five cases had myelodysplastic syndrome, 1 had acute lymphoblastic leukemia. Above results were all confirmed by FISH. CONCLUSION: i (20q-), t(20;21) (q11;q11) seems to be a rare but recurrent chromosomal abnormality which is specifically associated with myeloid disease, late occurrence and poor prognosis. The translocation between chromosome 20q11 and 21q11 may form a novel fusion gene which has an important role in the pathogenesis of the disease.

[Clinical and experimental study of a multiple myeloma case with low hypodiploidy].

OBJECTIVE: To report the clinical and laboratory characterization of a case of multiple myeloma with low hypodiploid complex karyotyptic abnormalities. METHODS: Cytogenetic examination of bone marrow performed by 24 h culture method. R-banding technique was used to analyze the karyotype. Interphase fluorescence in situ hybridization (FISH) was performed using chromosome probes such as 13q14, p53, Rb1, 1q21 and IgH/CCND1. The DNA content was detected by flow cytometry. RESULTS: Chromosome analysis revealed complex chromosomal rearrangement. Five cells had a low hypodiploid karyotype with 35 chromosomes. Three cells had the duplication of the low hypodiploid karyotype. Four cells had a normal karyotype. Monosomy 1, 13, 14, 17 and a mark chromosome 1 derived from chromosome 11 resulting in the amplication of CCND1 gene were confirmed by interphase FISH. Flow cytometric analysis displayed a low hypodiploid peak with the DNA index of 0.8426. CONCLUSION: These results indicated that the low hypodiploidy is a rare abnormality in multiple myeloma. Interphase FISH is a reliable method for detecting molecular abnormalities in multiple myeloma.

[Clinical and experimental studies of childhood acute myeloid leukemia with 11q23/MLL rearrangements].

OBJECTIVE: To explore clinical and experimental features of 28 cases of childhood acute myeloid leukemia (AML) with 11q23/MLL gene rearrangements. METHODS: Karyotypes of 234 cases of de novo childhood AML were analyzed using short-term culture of bone marrow cells and R-banding. The fusion transcripts involving MLL gene and partial tandem duplication of MLL (MLL-PTD) were detected by multiple reverse transcription polymerase chain reaction (RT-PCR) assay. Two cases with 11q23 translocation by karyotypic analysis but with negative result of multiple RT-PCR were studied with MLL-dual-color fluorescence in situ hybridization (D-FISH). RESULTS: R-banding karyotypic analysis has revealed 20 cases with 11q23 translocation (14 cases with M5, 4 cases with M4, 2 cases with M2), including 12 cases with t(9;11)(p22;q23), 3 cases with t(1;11)(q21;q23), 2 cases with t(6;11)(q27;q23), 1 case with t(11;19)(q23;p13), 1 with t(5;11)(q31;q23), and 1 with t(X;11)(q24;q23). Eighteen cases with 11q23 translocation having fusion transcripts involving MLL genes were confirmed with multiple RT-PCR; 2 cases showed negative results, but they were confirmed to have MLL rearrangements by D-FISH. MLL-PTD was also detected in 8 cases (4 cases M5, 2 cases M4, M2 and M6, one case each) from the other childhood AML cases. The total incidence of 11q23/MLL gene rearrangements was 11.97% (28/234), and most of patients(85.7%, 24/28) were M4/M5. The complete remission (CR) rate after treatment for the 28 cases with MLL rearrangements was 53.8%, the difference was significant by statistics (P< 0.05) compared with 90.5% for the control group (M4/M5 childhood AML with other karyotypic abnormalities or normal karyotype). Of them, 2 cases receiving intensive chemotherapy survived for 81 and 66 months, respectively, 4 cases receiving allogeneic stem cell transplantation survived for 21, 20, 16 and 11 months, respectively, and are still alive with CR. The medium survival (MS) time for 28 cases with 11q23/MLL rearrangements was 11 months, whereas the MS for control group was 15 months. The difference was not statistically significant(P> 0.05). CONCLUSION: The 11q23/MLL rearrangements is highly correlated with the occurrence of monocytic leukemia (M4 and M5). The 11q23 translocation and MLL-PTD are mutually exclusive, though both are indicative of poor prognosis. Intensive chemotherapy and allogeneic stem cell transplantation may ameliorate the clinical outcome. Multiple RT-PCR combined with karyotypic analysis and D-FISH are useful for screening the 11q23/MLL rearrangements in childhood AML.

[Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with inv(16)/t(16;16)(p13.1;q22) and/or CBFß-MYH11].

OBJECTIVE: To summarize the clinical and laboratory characteristics of patients with acute myeloid leukemia (AML) with inv(16)/t(16;16) (p13.1;q22), and to analyze the risk factors affecting the prognosis of the patients. METHODS: AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFß-MYH11+ admitted to the Department of Hematology, The First Affiliated Hospital of Soochow University from January 1, 2008 to October 30, 2019 were retrospective analyzed, the clinical and laboratory indicators, as well as treatment plans and efficacy evaluations of the patients were all recorded. Furthermore, related factors affecting the overall survival (OS) and event-free survival (EFS) of the patients were analyzed. RESULTS: Among 151 AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFß-MYH11+, the percentage of additional chromosomal abnormalities was about 27.8%, and the most common additional chromosomal abnormality was +22 (33/151, 21.8%), followed by +8 (11/151, 7.3%). There were 112 patients with perfect NGS examination, and the result showed the most common accompanying gene mutations were KIT mutation (34/112, 30.4%) and FLT3 mutation (23/112, 20.5%). Univariate analysis showed that factors affecting EFS included: NE≤0.5×109/L (P=0.006) and combined K-RAS mutation (P=0.002); Factors affecting OS included: Age≥50 years old (P<0.001) and NE≤0.5×109/L (P=0.016). Multivariate analysis showed that NE≤0.5×109/L (P=0.019) was the risk factors affecting OS. The proportion of bone marrow eosinophilia (BME)≥10.00% (P=0.029) was the risk factors affecting EFS. CONCLUSION: The prognosis for those newly diagnosed AML patients who were of advanced age, the high proportion of bone marrow eosinophils, K-RAS mutations, and agranulocytosis is poor. The treatment plans can be adjusted in the early stage to improve the prognosis of such patients.

[Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with inv(16)/t(16;16)(p13.1;q22) and/or CBFß-MYH11].

OBJECTIVE: To summarize the clinical and laboratory characteristics of patients with acute myeloid leukemia (AML) with inv(16)/t(16;16) (p13.1;q22), and to analyze the risk factors affecting the prognosis of the patients. METHODS: AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFß-MYH11+ admitted to the Department of Hematology, The First Affiliated Hospital of Soochow University from January 1, 2008 to October 30, 2019 were retrospective analyzed, the clinical and laboratory indicators, as well as treatment plans and efficacy evaluations of the patients were all recorded. Furthermore, related factors affecting the overall survival (OS) and event-free survival (EFS) of the patients were analyzed. RESULTS: Among 151 AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFß-MYH11+, the percentage of additional chromosomal abnormalities was about 27.8%, and the most common additional chromosomal abnormality was +22 (33/151, 21.8%), followed by +8 (11/151, 7.3%). There were 112 patients with perfect NGS examination, and the result showed the most common accompanying gene mutations were KIT mutation (34/112, 30.4%) and FLT3 mutation (23/112, 20.5%). Univariate analysis showed that factors affecting EFS included: NE≤0.5×109/L (P=0.006) and combined K-RAS mutation (P=0.002); Factors affecting OS included: Age≥50 years old (P<0.001) and NE≤0.5×109/L (P=0.016). Multivariate analysis showed that NE≤0.5×109/L (P=0.019) was the risk factors affecting OS. The proportion of bone marrow eosinophilia (BME)≥10.00% (P=0.029) was the risk factors affecting EFS. CONCLUSION: The prognosis for those newly diagnosed AML patients who were of advanced age, the high proportion of bone marrow eosinophils, K-RAS mutations, and agranulocytosis is poor. The treatment plans can be adjusted in the early stage to improve the prognosis of such patients.

[Fluorescence in situ hybridization study of acute myeloid leukemia with cryptic chromosome rearrangements].

OBJECTIVE: To detect specific chromosome rearrangements in acute myeloid leukemia (AML) using interphase-fluorescence in situ hybridization (FISH). METHODS: All cases were studied by R-band karyotypic analysis using direct method and/or short-term culture for chromosomes preparation. Interphase-FISH was performed in 108 cases of AML with M5, M4, M2, M3 subtypes including 103 cases with normal karyotypes, 4 cases with chromosomal abnormalities other than specific chromosomal rearrangements using chromosome translocation probe such as AML1/ETO, PML/RARα, CBFß/MYH11 and MLL. RESULTS: Of 38 cases of M2-AML without t(8;21) on conventional cytogenetics(CC) analysis, 4 cases showed positivity for AML1/ETO fusion transcript, which included 2 cases with typical signal model and 2 with insertion. Of 9 cases of M3-AML without t(15;17) on CC analysis, 6 showed positivity for PML/RARα fusion transcript including 2 with typical signal model, 3 with insertion, one without PML/RARα rearrangement on reverse transcription-PCR and FISH assay using PML/RARα probe. FISH assay using the RARα dual color, break-apart rearrangement probe indicated a partial deletion of RARα. Of 23 cases with M4 or M4EO-AML without inv(16) on CC analysis, 3 showed positivity for CBFß/MYH11 fusion transcript. Of 38 cases without 11q23 translocation on CC analysis, all cases were negative for MLL rearrangement. CONCLUSION: Interphase-FISH can detect specific chromosome rearrangements such as AML1/ETO, PML/RARα or CBFß/MYH11 in some AML cases with normal karyotype, though it seemed less useful for the detection of MLL rearrangement.

[Clinical Features and Prognostic Factors of Patients with Multiple Myeloma].

OBJECTIVE: To summarize the clinical and Laboratory characteristics of patients with multiple myeloma (MM) and analyze the prognostic factors. METHODS: Two hundred MM patients were retrospectively analyzed for the following parameters, including peripheral blood, bone marrow morphology, cytogenetics, clinical staging, and response to the chemotherapy in order to summarize related factors affecting overall survival (OS). The prognostic factors were also analyzed. RESULTS: 200 patients with MM were divided into 3 groups according to bone marrow plasma cell percentage (BMPC%) in bone marrow smears: ＜10% group (74 cases, 37.0%), 10%-50% group (75 cases, 37.5%), ＞50% group (51 cases, 25.5%). Compared with the other two groups, patients in BMPC%<10% group were characterized by lower clinical staging levels, lower rates of 13q14 deletion and t(11;14) positive, better response to chemotherapy and favorable three-year OS rate. The univariate analysis showed that prognostic factors indicating favorable outcome as evaluated by OS included age≤55 years old, BMPC%＜10%, WBC＜7.5×109/L, Hb≥68 g/L, PLT≥150×109/L, ß2-MG＜5.5 mg/L, LDH≤230 U/L, Durie-Salmon staging A, achievement of VGPR or better outcome after the first chemotherapy, achievement VGPR or better outcome after the fourth chemotherapy, and presence of autologous hematopoietic stem cell transplantation(auto-HSCT)(P<0.05). The multivariate analysis showed that prognostic factors indicating favorable outcome as evaluated by OS included age≤55 years old, BMPC%≤50%, WBC＜7.5×109/L, Hb≥68 g/L, achievement of VGPR or better outcome after the fourth chemotherapy (P<0.05). CONCLUSION: The clinical characteristics are different among MM patients with different BMPC% in bone marrow smears at initial diagnosis, and prognostic analysis shows that the BMPC% in bone marrow smears has an effect on OS rate. BMPC% in bone marrow smears at initial diagnosis, age, WBC, Hb, response to the fourth chemotherapy are also the main factors impacting the prognosis of patients.

A Recurrent Cryptic MED14-HOXA9 Rearrangement in an Adult Patient With Mixed-Phenotype Acute Leukemia, T/myeloid, NOS.

To define the fusion genes in T/myeloid mixed-phenotype acute leukemia (T/M MPAL), we performed transcriptome sequencing of diagnostic bone marrow samples from 20 adult patients. Our analysis identified a second instance of a recurrent MED14-HOXA9 chimeric gene resulting from the in-frame fusion of exon 23 of MED14 and exon 1 of HOXA9, the first in an adult patient. The MED14-HOXA9 fusion gene was detected in both the diagnostic and relapsed blasts with reverse transcription-polymerase chain reaction and Sanger sequencing. The patient received combined conventional chemotherapy but suffered relapse at 11 months and died of disease progression one year after the initial diagnosis. Our data suggest that MED14-HOXA9 is a cryptic recurrent aberration in T/M MPAL, which might indicate an aggressive clinical course and inferior outcome after conventional chemotherapy. Further studies will be carried out to reveal the effects of the MED14-HOXA9 fusion on the differentiation and proliferation of leukemia stem cells, as well as suitable treatment strategies for this emerging entity.

LPXN, a member of the paxillin superfamily, is fused to RUNX1 in an acute myeloid leukemia patient with a t(11;21)(q12;q22) translocation.

RUNX1 (previously AML1) is involved in multiple recurrent chromosomal rearrangements in hematological malignances. Recently, we identified a novel fusion between RUNX1 and LPXN from an acute myeloid leukemia (AML) patient with t(11;21)(q12;q22). This translocation generated four RUNX1/LPXN and one LPXN/RUNX1 chimeric transcripts. Two representative RUNX1/LPXN fusion proteins, RL and RLs, were both found to localize in the nucleus and could bring the CBFB protein into the nucleus like the wild-type RUNX1. Both fusion proteins inhibit the ability of RUNX1 to transactivate the CSF1R promoter, probably through competition for its target sequences. Unlike RL and RLs, the LPXN/RUNX1 fusion protein LR was found to localize in the cytoplasm. Thus, we believe it has little impact on the transcriptional activity of RUNX1. We also found that fusion proteins RL, RLs, LR, and wild-type LPXN could confer NIH3T3 cells with malignant transformation characteristics such as more rapid growth, the ability to form colonies in soft agar, and the ability to form solid tumors in the subcutaneous tissue of the BALB/c nude mice. Taken together, our data indicated that the RUNX1/LPXN and LPXN/RUNX1 fusion proteins may play important roles in leukemogenesis and that deregulation of cell adhesion pathways may be pathogenetically important in AML. Our study also suggests that LPXN may play an important role in carcinogenesis.

[The different signal patterns of two FISH probes in the FISH detection of Ph-positive leukemia and their clinical significance].

OBJECTIVE: To compare the signal patterns of dual color extra-signal BCR/ABL probe (ES-FISH) and dual color dual fusion BCR/ABL probe (D-FISH) in the fluorescence in situ hybridization (FISH) detection of Ph-positive leukemia, and to explore their diagnostic value. METHODS: ES-FISH probe and D-FISH probe were used, respectively, to detect the BCR/ABL fusion gene in 74 cases with typical t(9;22)(q34;q11) and 37 cases with variant t(9;22)(q34;q11) translocation or complex karyotypic abnormalities containing Ph translocation. RESULTS: The BCR/ABL fusion gene in all cases with typical t(9;22)(q34;q11) could be detected by both FISH probes. D-FISH had a signal pattern of 1O1G2F, while ES-FISH showed a signal pattern of 2O1G1F. ES-FISH enables the minor breakpoint cluster region to be identified in 9 cases (12.2% ) of Ph-positive leukemia, whereas D-FISH could not differentiate the minor breakpoint cluster region from major breakpoint cluster region. D-FISH could distinguish simple ABL gene deletion from simultaneous deletion of the ABL and BCR genes in 8 cases (10.8%) of Ph-positive leukemia patients, but ES-FISH could not. For variant Ph translocation or complex karyotypic abnormalities containing Ph translocation, each FISH probe showed four or six types of signal pattern, most of which were atypical. The exact interpretation was dependent on conventional karyotypic analysis and FISH on metaphases. CONCLUSION: ES-FISH and D-FISH probes displayed different signal patterns in Ph-positive leukemia due to their differences in size and covered regions. ES-FISH and D-FISH probes may be selected as better probe for Ph-positive acute lymphocytic leukemia and Ph-positive chronic myeloid leukemia, respectively. When imatinib was used for treatment, there was no preference between ES-FISH and D-FISH probe, because major breakpoint cluster region, minor breakpoint cluster region and partial sequence deletion of derivative chromosome 9, would not affect the prognosis of Ph-positive leukemia. However, considering that ES-FISH probe has a better cost-performance than D-FISH probe does, it is recommended as first choice.

[Clinical Significance of Common Gene Mutations in 53 Patients with Acute Myeloid Leukemia Harboring 11q23/MLL Rearrangements].

OBJECTIVE: To investigate the clinical significance of AML patients with 11q23/MLL rearrangement, and to evaluate the effect of those mutations on the AML patients. METHODS: 53 cases involving translocations of chromosome 11q23 were identified by chromosome banding analysis. MLL rearrangements were detected by fluorescence in situ hybridization and/or multiplex nested PCR. The samples were screened for mutations in the candidate genes FLT3-ITD, FLT3-TKD, TET2, N-RAS, ASXLI, EZH2, DNMT3, C-Kit, NPM1, WT1, CEBPA by using genomic DNA-PCR and deep-sequencing. RESULTS: 21/53 MLL-rearranged AML cases showed at least one additional chromosomal aberrations. The most common additional aberration was +8. Gene mutations were observed in 23 cases (43.4%) and most cases showed singal mutation. N-RAS mutation was more frequent (8 cases, 15.1%), followed by WT1 mutation in 4 cases (7.5%), FLT3-ITD mutation in 3 cases, ASXL1 mutation in 2 cases, DNMT3A mutation in 2 cases, EZH2 mutation in 1 case, c-Kit17 mutation in 1 case, FLT3-TKD mutation in 1 case, and FLT3-ITD and TKD mutation coexistent in 1 case. No mutation was detected in CEBPA, NPM1, C-KIT8, TET2. Median OS for gene mutated patients was 8.5 months and 13 months for no mutated patients. Median OS for patients who received hematopoietic stem cell transplantation (HSCT) was 22.5 months and 7.5 months for patients who olny received chemotherapy. CONCLUSION: A relatively high mutation frequency is observed in AML patients with 11q23/MLL rearrangements and most cases shows single mutation. The RAS signaling pathway alterations are most common. Gene mutation does not affect the OS of these patients, who show poor prognosis. A significantly higher Hb at initial diagnosis in FLT3 mutated patients is significantly higher than that in FLT3 wild-type cases. Patients who underwent HSCT show a better prognosis than those only received chemotherapy.

[Fluorescence in situ hybridization studies on a myeloid leukemia patient with ins(8;21)(q22;q22.1q22.3)].

OBJECTIVE: To report a case of acute myeloid leukemia (AML) with the insertion (8;21)(q22;q22.1q22.3). A 33-year-old Chinese woman was referred to our hospital. Hematologic data showed WBC 42.7 x 10(9)/L with monocytosis (monocyte counts 7.296 x 10(9)/L). Bone marrow aspirate was hypercellular with 4.5% monoblasts and 7.5% promonocytes. At first she was diagnosed with chronic myelomonocytic leukemia (CMML) according to the FAB criteria. Initially the patient received supportive care only, but her general condition rapidly became worse three months later. The monoblasts and promonocytes in the bone marrow rose to 20.5%. After two cycles of combined chemotherapy she obtained complete remission. METHODS: Chromosome specimens were prepared by short-term culture of bone marrow cells. Karyotype analysis was carried out by R-banding technique. Three fluorescence in situ hybridization (FISH) analyses were performed using AML1-ETO dual color, dual fusion probe, whole chromosome painting 8 and 21 probes, and cen-8 and Tel 21qter probes, respectively. Reverse transcription polymerase chain reaction (RT-PCR) assay for detecting the AML1-ETO fusion transcript was also performed. RESULTS: Conventional cytogenetic analysis showed a karyotype of 46,XX,ins(8;21) (q22;q22.1q22.3)[7]/46,XX[3]. FISH tests confirmed the insertion. RT-PCR analysis detected the AML1-ETO fusion transcript. CONCLUSION: We consider that this patient should be rediagnosed as acute myeloid leukemia according to the criteria proposed by World Health Organization (WHO) and that FISH and RT-PCR play an important role in verification of the ins(8;21).

[Clinical Features and Prognosis of 188 Patients with Acute Myeloid Leukemia-M2].

OBJECTIVE: To summarize the clinical characteristics of patients with acute myeloid leukemia-type M2 (AML-M2) and analyze the factors affecting the prognosis. METHODS: One hundred eighty-eight AML-M2 patients were retrospectively analyzed for the following parameters including peripheral blood, immune phenotypes, fusion genes and cytogenetics to explore their significance for the overall survival (OS) and progression-free survival (PFS). The prognostic factors were also analyzed. RESULTS: Among 188 patients with AML-M2, the chromosomal abnormality with t (8;21), normal chromosome and other abnormalities accounted for 37% (70/188), 41% (77/188) and 22% (41/188), respectively. For the immunopheno typing of M2 patients, the hematopoietic progenitor cell differentiation antigen CD117 (96.1%) were mainly expressed, CD34 (81.6%) and HLA-DR (55.9%), and myeloid-associated antigen of CD13 (90.5%) and CD33 (89.4%) were also highly expressed. There were lymphoid-associated antigens expressed in some patients, among which the positive expression rate of CD19 was highest (29.6%), and the next was CD7 (28.5%). The most common accompanied mutations was FLT3 mutation (30.2%). The univariate analysis showed that the patients at age＜50 years old, without extramedullary infiltration, with positive expression of CD19, NPM-1 (-), CEBPA double mutation(+), and HSCT were significant superior in OS and PFS (P＜0.05); the multivariate analysis showed that the patient at age＜50 years old, without extramedullary infiltration, with positive expression of CD19 and CEBPA double mutation (+) were significant superior in OS and PFS (P＜0.05). The analysis indicated that the Karytypes affected only OS (P＜0.05), while the NPM-1 gene mutation positive affected only PFS (P＜0.05). The univarate analysis of factors affecting the survival in 70 AML-M2 patients with t (8;21) abnormatity showed that the C-KIT gene mutation was a poor factor for OS and PFS. CONCLUSION: The clinical characteristics are different between M2 patients with different karyotype, and prognostic analysis shows that the karytypes have an impact on overall survival; age, extramedullary infiltration, CD19 expression and CEBPA double mutation are also the main factors impacting the prognosis of patients.

Translocation t(2;8)(p12;q24) in two patients with B Cell chronic lymphocytic leukemia.

It is well known that translocation between chromosomes 2 and 8, t(2;8)(p12;q24), has a strong association with Burkitt's lymphoma. It has rarely been seen in indolent lymphoproliferative disorders. In this study, we report for the first time on 2 cases of chronic lymphocytic leukemia (CLL) with t(2;8). They were diagnosed as having typical CLL (case 1) and CLL/prolymphocytic leukemia (case 2), respectively, based on morphology and immunophenotyping. Karyotypic analysis of the bone marrow cells using the R-banding technique revealed a karyotype of 47,XY, t(2;8)(p12;q24), +4,[17]/46, XY[9] in case 1 and a karyotype of 45,X, t(Y;7)(q12;q21),t(2;8)(p12; q24),del(12)(p12),-17[5]/46,XY[9] in case 2. T(2;8) translocation was confirmed by whole chromosome painting. Rearrangement of the MYC gene and loss of one p53 allele were detected by FISH only in case 2. Both cases had negative ZAP70 and CD38 expressions; however, only case 1 showed good response to therapy. We consider that MYC rearrangement, loss of one p53 allele as well as other factors, such as more prolymphocytes in the blood and bone marrow and complex chromosome abnormalities, also had adverse effects on the poorer prognosis of case 2.

[Effect of Additional Chromosomal Abnormalities on the Outcome of CML-CP Patients Receiving TKI Therapy].

OBJECTIVES: To explore the effect of additional chromosomal abnormalities on the prognosis and outcome of CML-CP patients receiving imatinib therapy. METHODS: The clinical and genetic data of 589 CML-CP patients receiving imatinib treatment between May 2009 and October 2014 in the 1st Affiliated Hospital of Soochow University were analyzed, the 589 patients were divided into 5 groups according to the karyotypes at the initial diagnosis. The OS(overall survival), PFS (progression-free survival), EFS (event-free survival), Cumulative MMR (major molecular remission) and Cumulative CCyR (complete cytogenetic remission) were calculated by using the Kaplan-Meier method and compared by using the log-rank text by Graphpad 6.0. The χ2 test was used to compare the frequency of optimal molecular response at 3, 6, 12 months among the 5 groups. RESULTS: There was significant difference about the frequency of optimal molecular response at 3 and 6 months between CML-CP patients with additional chromosomal abnormalities and those with classic t(9;22) ï¼»50%（12/24） vs. 73.94%(261 /353), P<0.05; 50%(10 /20) vs. 72.05%(232 /322) (P<0.05)ï¼½, and the same significant difference was found at 6 months between the group with variant translocations and that with classic t(9;22) ï¼»53.3% (16 /30) vs. 72.05%(232 /322) (P<0.05)ï¼½. The P values of cumulative CCyR (P<0.05) and EFS (P<0.01) for 4 years were statistically significant between CML-CP patients with additional chromosomal abnormalities and the other 4 groups. Compared one to another, there was the significant difference in cumulative CCyR and EFS for 4 years between CML-CP patients with additional chromosomal.abnormalities and those with classic t(9;22) (47.25% vs. 84.01%)(P<0.05); (75.03% vs. 90.01%)(P<0.01). CONCLUSION: The additional chromosomal abnormalities influence the outcome of CML-CP patients receiving imatinib treatment, which make poor prognosis.