[Mutational features of immunoglobulin heavy chain variable region gene in patients with chronic lymphocytic leukemia].

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.

Variation in ovarian cancer care in Australia: An analysis of patterns of care in diagnosis and initial treatment in New South Wales.

OBJECTIVE: Ovarian cancer has the highest mortality of all gynaecological cancers. This study aimed to identify the extent to which women across New South Wales experienced variation in their care in diagnosis and initial treatment for ovarian cancer against the national optimal care pathway for ovarian cancer. METHOD: Clinical audit methodology was utilised to explore variations for women with primary ovarian cancer; 171 eligible cases were identified through by the NSW Cancer Registry for the period of 1 March 2017 to 28 February 2018. RESULTS: Limited variation was detected with 86% of women being reviewed by a specialist gynaecological oncology multidisciplinary team; 54% of women received their first treatment within 28 days of their first specialist appointment, 66% of women having their first surgery completed by a gynaecological oncologist and 45% of women received their first treatment in a specialist gynaecological oncology hospital. CONCLUSION: Deviation from effective ovarian cancer care is apparent particularly in the location and timeliness of first treatment, with implications for the quality of care received and care outcomes. Understanding factors that contribute to variation is critical to ensure optimal and appropriate ovarian cancer care and to tackle systemic barriers to the provision of effective care.

Investigating the system effect of reporting multidisciplinary care measures for cancer services in New South Wales, Australia.

BACKGROUND: Multidisciplinary cancer care to facilitate the provision of patient centred and evidence-based care is considered best practice internationally. In 2016 multidisciplinary care measures were developed for all local health districts across NSW. The aim of this study was to identify system-level changes and quality improvement activities across the NSW cancer system linked to reporting on these measures. METHODS: Focus group discussions were used to generate a synergy of ideas from key stakeholders. An exploratory descriptive approach was used within the ontological position of Framework Analysis, the analysis method chosen for this research study, sitting most closely within pragmatism. The use of Framework Analysis in the analytic strategy is because it is well-suited to addressing policy issues and maintaining specific focus within a wider dataset. RESULTS: Two focus groups were held with a total of 18 purposively selected participants. Four primary themes emerged: value of electronic documentation; role clarity; relationships; and future development of measures. Key findings included that the reporting of performance measures has expedited the development of electronic documentation and data extraction from the multidisciplinary team meeting (MDT), identified barriers and facilitators to MDT data collection and supported MDT improvement activities across NSW. CONCLUSIONS: The findings of this study have highlighted that MDTs and their meetings across NSW are harnessing technological advancements to support and further develop their MDTs, as well as the challenges of implementing new processes within the MDTM. This study adds a unique contribution to knowledge of how the reporting of measures can assist in understanding variation in the development and implementation of multidisciplinary teams, as well as highlighting future programs of work to decrease variation in multidisciplinary team meetings and quality improvement activities.

[Analysis of fusion gene expression in acute myeloid leukemia].

Objective: To analyze the genetic landscape of multiple fusion genes in patients with de novo acute myeloid leukemia (AML) and investigate the characteristics of immunophenotypes and mutations. Methods: The results of multiple fusion genes from 4192 patients with de novo AML were retrospectively analyzed from 2016 to 2020. In addition, the immunophenotypical data and the mutational results from high-through put method were statistically investigated and correlated as well. Results: ①Among the 52 targets, 29 different types of fusion genes were detected in 1948 patients (46.47%) with AML, which demonstrated an "exponential distribution" . ② As the age increased, the number of patients with fusion gene increased first and then decreased gradually. The total incidence rate of fusion genes and MLL rearrangment in children were significantly higher than those in adults (69.18% vs 44.76%, 15.35% vs 8.36%) . ③The mutations involving FLT3 and RAS signaling pathway contributed most in patients with MLL rearrangment. ④No specific immunophenotypic characteristics were found in AML patients with MLL or NUP98 rearrangements. Conclusion: Nearly half of AML patients were accompanied by specific fusion gene expression, the proportions of different fusion genes in pediatric and adults patients were different by multiple PCR. The gene mutations and immunophenotype of these AML patients have certain rules.

[Clinical and laboratory characteristics in patients with myeloid neoplasms complicated with clonal T large granular lymphocyte proliferation].

Objective: To analyze the clinical manifestations and laboratory features in patients with myeloid neoplasms complicated with clonal T large granular lymphocyte (T-LGL) proliferation. Methods: The clinical data of 5 patients with myeloid neoplasms complicated with clonal T-LGL proliferation from November 2017 to November 2018 in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College were analyzed retrospectively. Results: The median age was 60 years old. All patients had a history of abnormal peripheral blood cell counts for over 6 months. The absolute lymphocyte count in peripheral blood was less than 1.0×10(9)/L. In addition to the typical T-LGL phenotype, the immunophenotype was heterogenous including CD4(+)CD8(-) in 2 patients, the other 3 CD4(-)CD8(+). Four patients were αß type T cells, the other one was γδ type. STAT3 mutation was detected in 1 patient by next-generation sequencing, the other 4 cases were negative. Conclusions: Clonal T-LGL proliferation with myeloid neoplasm develops in an indolent manner, mainly in elderly patients. Hemocytopenia is the most common manifestation. The diagnosis of T-LGL proliferation does not have specific criteria, that it should be differentiated from other T cell proliferative disorders, such as T-cell clones of undetermined significance. STAT3 or STAT5b mutation may help distinguish.

[Prognostic factors of cyclosporine A combined with androgen in the treatment of transfusion dependent non-severe aplastic anemia].

Objective: To analyze the prognostic factors of transfusion-dependent non-severe aplastic anemia (TD-NSAA) patients treated with cyclosporine A (CsA) and androgen. Methods: Clinical data of 77 consecutive TD-NSAA patients treated with CsA and androgen were retrospectively analyzed between 2010 and 2013. We obtained clinical manifestations and baseline parameters of routine blood test from responders, and compared those with non-responders. All data were analyzed by univariate analysis and multivariate analysis. Results: In 77 patients, there were 43 (55.8%) patients achieved hematological response after 6 months'treatment, and 53 (68.8%) patients got response after 12 months. Univariate analysis showed that platelets baseline was the only factor related to hematological response [19 (6-61) ×10(9)/L vs 13.5 (5-45) ×10(9)/L, P=0.001] after 6 months therapy. After 12 months, the statistical differences were maintained, which were platelets baseline [18 (6-61) ×10(9)/L vs 10.5 (5-45) ×10(9)/L, P<0.001], absolute reticulocytes [0.03 (0.01-0.06) ×10(12)/L vs 0.029 (0.02-0.06) ×10(12)/L, P=0.043], transfusion-dependent of platelet (P=0.007) , transfusion-dependent of platelet and erythrocyte (P=0.012) . Multivariate analysis showed that platelets baseline could be an independent prognostic factor of hematological response (P=0.010 or 0.009) . Cutoff value of platelets by receiver operating characteristic curve was 15.5×10(9)/L. Conclusion: Baseline of higher platelets, higher reticulocyte, and no transfusion dependence of platelet are favorable prognostic factors. When platelets baseline is higher than 15.5×10(9)/L, CsA and androgen regimen is rational.

[Application of immunophenotypic analysis and molecular genetics in the diagnosis of acute promyelocytic leukemia].

Objective: To investigate the application values of immunophenotypic analysis and molecular genetics in the diagnosis of acute promyelocytic leukemia (APL) . Methods: The retrospective analyses of flow cytometric (FCM) immunophenotypic anyalysis, chromosome karyotype and chromosome fluorescence in situ hybridization (FISH) of 798 outpatient or hospitalization APL patients referred to our hospital between May 2012 and December 2017 were performed to further study the application values of FCM and molecular genetics in the diagnosis of APL. Results: The sensitivity and specificity of FCM were 91.9% and 98.7% respectively. The typical characteristic immunophenotype for APL was as of follows: a high SSC, absence of expression of cluster differntiation (CD) CD34 and HLA-DR, and expression or stronger expression of CD33, consistent expression of CD13, CD9, CD123, expression of CD56, CD7, CD2 (sometimes) . The rest 10% of the cases harbored atypical APL phenotypes, generally accompanied by CD34 and/or HLA-DR expression, decreased SSC and often accompanied by CD2 expression, it was difficult to definitively diagnose APL by this FCM phenotype, and their diagnoses depended on the results of genetics or molecular biology tests. Compared with normal individuals, complex karyotypes APL with t (15;17) translocation, other variant translocations and variant t (11;17) , t (5;17) had no significant differences in terms of their FCM phenotypes. Conclusions: FCM could rapidly and effectively diagnose APL. Despite the fact that complex karyotypes with various additional chromosomal abnormalities were detected in approximately one third of APL cases in addition to the pathognomonic t (15;17) (q22;q21) , they had no observable impact on the overall immunophenotype. Molecular and genetic criteria were the golden criteria for the diagnosis of APL. About 10% of immunophenotyping cases relied on molecular genetics for diagnosis.

[Targeted sequencing analysis of hyper-eosinophilic syndrome and chronic eosinophilic leukemia].

Objective: Analysis of the molecular characteristics of eosinophilia. Methods: Targeting sequence to 24 patients with chronic eosinophilic leukemia (CEL) with rearrangement of PDGFRA, PDGFRB, or FGFR1 and 62 patients with hyper-eosinophilic syndrome (HES). Mutation annotation and analysis of amino acid mutation using authoritative databases to speculate on possible pathogenic mutation. Results: Thirty-seven kinds of clonal variant were detected from 17 patients with CEL, no recurrent mutation site and hot spot region were found. No pathogenic mutation was detected in 19 patients with PDGFRA rearrangement, but pathogenic mutations of ASXL1, RUNX1 and NRAS were detected from 2 patients with FGFR1 rearrangement who progressed to acute myeloid leukemia and 1 patient with PDGFRB rearrangement who progressed to T lymphoblastic lymphoma, respectively. One hundred and two kinds of clonal abnormalities were detected in 49 patients with HES. The main hot spot mutation regions included: CEBPA Exon1, TET2 Exon3, ASXL1 Exon12, IDH1 Y208C, and FGFR3 L164V. CRRLF2 P224L and PDGFRB R370C point mutations were detected separately in 2 patients with HES who treated with imatinib monotherapy and achieved hematologic remission. Conclusion: The pathogenesis of CEL with PDGFRA, PDGFRB or FGFR1 rearrangement is usually single, and the progression of the disease may involve other driver mutation. A variety of genes with hot mutation regions may be involved in the pathogenesis of HES, and some mutation sites are sensitive to tyrosine kinase inhibitors.

[Spectrum of somatic mutations and their prognostic significance in adult patients with B cell acute lymphoblastic leukemia].

Objective: To investigate the spectrum of gene mutations in adult patients with B-acute lymphoblastic leukemia (B-ALL), and to analyze the influences of different gene mutations on prognosis. Methods: DNA samples from 113 adult B-ALL patients who administered from June 2009 to September 2015 were collected. Target-specific next generation sequencing (NGS) approach was used to analyze the mutations of 112 genes (focused on the specific mutational hotspots) and all putative mutations were compared against multiple databases to calculate the frequency spectrum. The impact of gene mutation on the patients' overall survival (OS) and recurrence free survival (RFS) was analyzed by the putative mutations through Kaplan-Meier, and Cox regression methods. Results: Of the 113 patients, 103 (92.0%) harbored at least one mutation and 29 (25.6%) harbored more than 3 genes mutation. The five most frequently mutated genes in B-ALL are SF1, FAT1, MPL, PTPN11 and NRAS. Gene mutations are different between Ph+ B-ALL and Ph- B-ALL patients. Ph- B-ALL patients with JAK-STAT signal pathway related gene mutation, such as JAK1/JAK2 mutation showed a poor prognosis compared to the patients without mutation (OS: P=0.011, 0.001; RFS: P=0.014,<0.001). Patients with PTPN11 mutation showed better survival than those without mutation, but the difference was not statistically significant (P value > 0.05). Besides, in Ph+ B-ALL patients whose epigenetic modifications related signaling pathway genes were affected, they had a worse prognosis (OS: P=0.038; RFS: P=0.047). Conclusion: Gene mutations are common in adult ALL patients, a variety of signaling pathways are involved. The frequency and spectrum are varied in different types of B-ALL. JAK family gene mutation usually indicates poor prognosis. The co-occurrence of somatic mutations in adult B-ALL patients indicate the genetic complex and instability of adult B-ALL patients.

[Clinical characteristics of high-grade B-cell lymphomas with rearrangement of MYC, bcl-6 and bcl-2].

Objective: To investigate the clinicopathologic features of patients with high-grade B-cell lymphomas (HGBL) that have rearrangements of MYC, bcl-6 and bcl-2. Methods: One hundred and fifty-eight B-cell lymphomas patients from Institute of Hematology and Blood Diseases Hospital from January 2016 to April 2017 were detected by fluorescence in situ hybridization (FISH) with double color split-apart probes. Results: Among 158 B-cell lymphomas, 3 cases with MYC, bcl-2 and bcl-6 rearrangements were identified, 1 of which also had CCND1/IgH translocation. All three patients were of older age, with poor prognostic parameters, multiple organs involvements, elevated LDH and advanced-tumor stage. Two of the three patients were treated with high-intensity chemotherapy and had no remission with an overall survival of 9 months and 11 months respectively. One patient had follow-up with no treatment. Histologically, all three cases showed a spectrum of morphologic features. Although initially categorized as lymphoblastic lymphoma, diffuse large lymphoma and mantle cell lymphoma respectively, two cases were associated with germinal center B-cell (GCB) immunophenotype and 1 case with non-GCB immunophenotype. They had a high proliferation index as assessed by immunostaining for Ki-67 (60%-90%). Conclusions: MYC(+) bcl-2(+) bcl-6(+) HGBL is an aggressive disease with multiple organ involvement, high serum LDH levels, advanced stage disease, poor prognosis and shorter patient survival. The diagnosis should be made by histopathology combined with FISH analysis. Its separation from other types of B cell large cell lymphoma is of clinical importance.

Sequence-based typing identification of the novel HLA-C*07:565 variant in a Chinese family.

HLA-C*07:565 differs from HLA-C*07:02:01:01 (400C->T, exon 3, L110F).

[Screening of adult Ph-like acute lymphoblastic leukemia by multiplex real-time quantitative PCR].

Objective: To investigate the feasibility of multiplex real-time RT-PCR with fluorescent probes in early screening of Ph-like acute lymphoblastic leukemia (ALL) and analyze the clinical feature and prognos. Method: A total of 118 adult B-ALL patients diagnosed between October 2010 and March 2016 were enrolled in this study. Multiplex RT-PCR was used to detect the Ph-like ALL related fusion gene and CRLF2 expression in 58 BCR-ABL and MLL rearrangement negative patients. The clinical features, treatment response and prognosis were analyzed in Ph-like fusion gene positive and/or CRLF2 over-expression patients. Result: Among 58 patients, 9 patients (9/58, 15.5%) showed Ph-like ALL related fusion genes positive and 10 patients (10/58, 17.2%) showed CRLF2 over-expression. There were statistical differences in age, WBC count, immunophenotypes, cytogenetics and risk stratification among Ph-like fusion gene positive or CRLF2 over-expression patients, Ph(+) patients, MLL(+) patients and B-other patients. The 2-year overall survival rates were 65%, 47%, 64% and 74% respectively among these four groups (P=0.043) . The 2-year relapse free survival rates were 51%, 39%, 62% and 70% respectively among these four groups (P=0.010) . Conclusion: Routine screening of Ph-like ALL by multiplex RTPCR is feasible.

A novel allele HLA-B*27:149 identified by sequence-based typing in a Chinese individual.

HLA-B*27:149 differs from HLA-B*27:04:01 (488C->T, exon 3, A139V).

[Clinicopathologic characteristics of plasma cell myeloma with marrow fibrosis].

Objective: To study the clinicopathologic features of plasma cell myeloma(PCM) with bone marrow fibrosis (MF). Methods: The clinicopathologic data of 175 cases of newly diagnosed PCM patients were retrospectively analyzed. Based on reticular fiber staining, these cases were divided into PCM-MF and non-PCM-MF groups. Results: Sixty-three cases were PCM-MF(36%), 112 were non-PCM-MF (64%). No statistical difference in gender, age, hemoglobin level, platelet counts, the classification of immunoglobulin, ISS staging, immunohistochemical phenotypes and genetic features was found between PCM-MF and non-PCM-MF groups (P>0.05). Compared to non-PCM-MF group, lactate dehydrogenase (LDH)level and renal impairmentrate were higher in PCM-MF group (P<0.05). The degree of bone marrow hyperplasia, the percentage of myeloma cells and cells with plasmablastic morphology were significantly higher in PCM-MF group(P<0.05). Conclusion: The higher LDH level, renal impairment rate, and more significant bone marrow hyperplasia, proliferation of plasma cells and plasmablastic myeloma cells infiltration indicate poor prognosis of PCM-MF patients.

Identification of a novel HLA-C*06 variant allele, HLA-C*06:166, by sequence-based typing in a Chinese individual.

HLA-C*06:166 differs from HLA-C*06:02:01:01 (146T → C, exon 2, V25A).

[Characterization of mutational pattern in patients with Ph negative myeloproliferative neoplasms].

OBJECTIVE: To characterize the molecular profile in patients with Ph negative myeloproliferative neoplasms (MPN) by exploring 49 gene mutations. METHODS: Targeted gene sequencing were performed to analyze 49 MPN-associated genes in 51 patients with Ph negative MPN, of which CARL (exon 9), NPM1 (exon 12) and CEBPA (TAD, BZIP domains) were investigated by using Sanger sequencing simultaneously, while FLT3-ITD was assessed by PCR method. RESULTS: Mutations were detected in 73.5% (36/49) of genes, and the mutational rates of JAK2-V617F, CALR (exon 9) and MPL were 60.8%(31/51), 7.8%(4/51) and 7.8%(4/51) respectively, whereas the mutational rates of ASXL1, SETBP1, and SF3B1 were around 10%. In addition, 96.1% (49/51) of patients harbored at least one mutation, and more than half of the patients (52.9%, 27/51) possessed 3 or 4 gene mutations. The amount of gene mutations was significantly higher in patients with JAK2-V617F mutation than those without JAK2-V617F or CALR (exon 9) mutation (P<0.05). The last finding was that there was no statistically significant difference in the amount of mutations among four MPN subtypes (PV, ET, PMF, and MPN-U). CONCLUSION: Most patients with Ph negative MPN possesses three or more gene mutations, with various mutational profiles.

A new HLA-B allele, B*52:44, sequenced in a Chinese individual.

One nucleotide replacement in codon 154 (GAG>GAT) of HLA-B*52:01:01:01 results in a novel allele formation, HLA-B*52:44.