Acute Unclassified Leukemia Research Articles

Clinico-Hematological Profile of Acute Leukemia Cases in Bihar: A Multiparameter Study in a Tertiary-Care Hospital.

Objective The actual incidence and demographic profile of hematological malignancies are unknown in Bihar because of lack of population-based cancer registry (PBCR) data and specialized tertiary cancer center facilities. The objective of this study was to estimate the prevalence, clinico-hematological profile and subtyping of acute leukemia cases by retrospective medical records. Materials and Methods A retrospective study was conducted in the Department of Hematology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India, over 2 years from July 2019 to June 2021. A total of 176 cases with relevant clinical features and hematological findings were involved in the study. Medical records were studied and data were retrieved. Statistical Analysis Data were recorded and analyzed using SPSS version 25. Results A total of 176 cases with relevant clinical features and hematological findings were involved in the study. Acute myeloid leukemia (AML) was most prevalent (52.8%), followed by acute lymphoblastic leukemia (ALL) (34.1%) and unclassified acute leukemia cases (13.1%). Flow cytometry correlation was available in 150 cases. The ratio of males (62.5%) to females (37.5%) is 1.6:1. There was statistically significant difference in physical examination findings between AML and ALL patients. Splenomegaly, lymphadenopathy, and sternal tenderness were more often seen in ALL than in AML patients ( p < 0.05). Pallor was more significantly associated with AML than with ALL patients ( p < 0.05). Anemia and leucocytosis were found to be significantly associated with acute leukemia patients ( p < 0.000). Conclusion AML M2 was the most common subtype of AML, and B-ALL was the most common subtype of ALL cases.

Efficacy of liposomal amphotericin against febrile neutropenia in pediatric patients receiving prophylactic voriconazole.

The risk factors for invasive fungal infection have gradually become evident for pediatric patients with hematological diseases. Here we analyze the efficacy of liposomal amphotericin (L-AMB) for pediatric patients with febrile neutropenia using prophylactic voriconazole (VRCZ). We administered L-AMB (2.5mg/kg/day) in patients with febrile neutropenia who were receiving prophylactic VRCZ (10mg/kg/day, orally) and were resistant to second-line antibiotics therapy. Thirteen patients (5 males, 8 females) with 19 febrile neutropenia episodes were targeted in this analysis. The median age of the patients was 14years (range, 1-19years). Eighteen out of 19 episodes occurred in patients with acute myeloid leukemia, with the remaining episode occurring in a patient with acute unclassified leukemia. The median period from start of L-AMB administration to resolution of fever was 4days (1-27days). In 15 out of 19 episodes, fever resolved within 5 days from commencement of L-AMB administration. Using criteria proposed by T. J. Walsh et al., the success rate of L-AMB for febrile neutropenia was 89.5% in this study. Although the sample size of our study was small, the extremely high efficacy of L-AMB warrants its administration in patients with febrile neutropenia who are receiving VRCZ.

Hematological malignancies in relation with ABO blood group at a teaching hospital, Varanasi, India

Background:There has been enormous progress in the diagnosis and treatment modalities of leukemia, but its pattern and prevalence vary throughout India. This inter-regional variation may be due to geographic, cultural or racial variation or maybe due deficiency in case of notification, especially in rural areas.Objective:The aim of our study is to determine the prevalence of different types of hematological malignancies with the ABO blood group at a teaching hospital in Varanasi, India. This cross-sectional study of 77 cases was conducted during 2016–2017 at a tertiary care center. We analyzed the age, sex, subtype of leukemia, blood group, clinical features, and laboratory parameters of patients. Age ranged between 1 year and 81 years with a male to female ratio of 1.9:1. A total of 66.3% of patients were suffering from acute leukemia and 33.7% from chronic leukemia. The most common blood group was B positive (44.8%) but no significant association was found (P = 0.822). Fever (76.6%) and generalized weakness/easy fatigability (46.75%) were common complaints, whereas pallor (68.8%) and splenomegaly (51.9%) were common signs. Cases were from 23 districts of eastern Uttar Pradesh and western Bihar. The majority of the cases (70.12%) were from 8 districts (Jaunpur, Varanasi, Azamgarh, Ballia, Bhadhoi, Gazipur, Kaimur, Rohtas) of Uttar Pradesh, India and Aurangabad district of Bihar. Acute leukemia is more prevalent than chronic leukemia with more male preponderance. Acute lymphoblastic leukemia (ALL) were commonly found in children, acute myeloid leukemia (AML) was found in both children and adults. Chronic Myeloid Leukemia (CML) and chronic lymphocytic leukemia (CLL) mainly noted in adults. Unclassified acute leukemia was seen mainly in children and young adults. Anemia was more severe in acute conditions and thrombocytopenia was also more in acute leukemia. The most common blood group was B positive.

Outcome of Hematopoietic Stem Cell Transplantation in the Patients with Pediatric Acute Lymphoblastic Leukemia Enrolled in the Japan Association of Childhood Leukemia Study (JACLS) ALL-02

Background: Though the outcome of patients with acute lymphoblastic leukemia (ALL) has been greatly improved, some high-risk patients still need hematopoietic stem cell transplantation (HSCT). In this study, we evaluated clinical characteristics of children with ALL who were treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL-02 trial and received HSCT to determine prognostic factors for the outcome of HSCT. Methods: Between April 1, 2002 and March 31, 2008, 1,252 patients aged 1-18 years with newly diagnosed ALL were enrolled in JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Of all the 1,252 patients, 211 (16.8%) patients were reported to receive HSCT, of which 206 patients with adequate information of 1st HSCT were subjected to further analysis. In this study, HSCT in 1st complete remission (CR) in JACLS ALL-02 trial was indicated for the patients with extremely high risk (ER: BCP-ALL with PPR and/or evidence of t(4;11) (or KMT2A-AFF1 positive), hypodiploidy (≤ 44) and/or acute mixed lineage leukemia/ acute unclassified leukemia). As a precaution, in ER patients, the timing of HSCT differs depending on whether they have a matched related donor. Results: Of all the 206 patients, 83 patients received HSCT in first CR (CR1), 68 patients in CR2, 54 patients in non-CR and 1 patient in induction failure (IF). 5-year overall survival (5y-OS) for 206 patients was 50% (95% CI 42.7-56.8). In detail, 73.8% (95% CI 62.6-82.1) for CR1 patients, 49.4% (95% CI 35.9-61.5) for CR2 patients, and 14.8% (95% CI 6.9-25.5) for non-CR patients, respectively. In univariate analysis, JACLS risk classification, disease status at HSCT (CR or non-CR or IF), stem cell source were significant prognostic factors. In multivariate analysis, only disease status retained as prognostic factor (p&lt;0.01, HR=1.77). For CR1 patients, stem cell source was significant prognostic factor on multivariate analysis (5y-OS = 41.7%, 66.7%, and 79.3% for peripheral blood, cord blood, bone marrow; p = 0.023). When we focused on immunophenotype, stem cell source retained their effect only for patients with T-ALL. For CR2 patients, central nerves system involvement at diagnosis (p=0.02, HR=7.2), S classification (p=0.03, HR=1.2), and NCI risk (p=0.04, HR=2.5) were significant prognostic factors on multivariate analysis. S classification retained its prognostic impact only for patients with BCP-ALL (p=0.01, HR=1.5). For non-CR patients, NCI or JACLS risk classification at first diagnosis, S classification, frequency of relapse before HSCT, stem cell source, and conditioning for HSCT did not affect treatment outcome. Interestingly, non-CR patients with high-hyperdiploid (HHD) (n=5) had a significantly better 5y-OS (80%) than the other subtypes. In multivariate analysis, age at diagnosis (&lt;10 years, p=0.04, HR=2.0)) and HHD (p=0.006, HR=1.74) contribute to the better outcome of HSCT in non-CR. Conclusion: The current study showed that the prognostic factors for HSCT varied according to the disease status or disease immunophenotype. Also, it is noteworthy that HHD patients may be rescued even in non-CR status by HSCT. Disclosures No relevant conflicts of interest to declare.

Minor population with weak cytoplasmic CD3 expression in acute unclassified leukemia.

Minor population with weak cytoplasmic CD3 expression in acute unclassified leukemia.

Risk-Adjusted Therapy of Acute Lymphoblastic Leukemia Can Optimize the Indication of Stem Cell Transplantation and Cranial Irradiation: Results of Japan Association Childhood Leukemia Study Group (JACLS) Protocol ALL-02

BACKGROUNDThe trial JACLS ALL-02 for treatment of childhood acute lymphoblastic leukemia (ALL) was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in unfavorable-risk groups by treatment intensification. These aims were pursued through a refined stratification strategy using white blood cell count, age, immunophenotype, unfavorable genetic aberrations, and treatment response providing an excellent discrimination of risk groups. PATIENTS AND METHODSBetween April 2002 and March 2008, 1252 children with newly diagnosed de novo ALL with 1-18 years of age were enrolled to JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Patients with BCP-ALL were stratified into 3 groups: standard-risk (SR), high-risk (HR), extremely high-risk (ER), based on initial prednisolone (PSL) response and the modified National Cancer Institute(NCI) workshop criteria. Prednisolone poor response (PPR) was determined after 7 days of monotherapy with prednisone and one intrathecal dose of methotrexate. PSL good responders (PGR; < 1,000 blasts/microL) were divided into SR and HR according to the modified NCI workshop criteria by WBC 10K and age 10, and received conventional therapy. BCP-ALL with PPR (≥ 1,000 blasts/microL) or t(4;11), and acute mixed lineage leukemia/ acute unclassified leukemia were assigned to ER and received intensified post-induction therapy . Patients with T-ALL were treated by a specific protocol that was different from the protocol used for BCP-ALL. Bone marrow response was also evaluated in aspiration smears on day 15 and 33 of induction treatment, and those who had slow early bone marrow response, defined as an M3 marrow on day 15 or M2/3 marrow on day33, shifted to the higher risk and received augmented post-induction therapy. Cranial irradiation was restricted to patients with initial central nerve system involvement or T-ALL with high WBC (≥10K) at diagnosis. Alternatively, protracted TIT was given during induction, intensification and maintenance depending on the risk group (12 doses in SR/T and 15 in HR/ER). Slow early responders who had an M3 marrow on day 15 in ER/T or M2/3 marrow on day33 in any risk, underwent to stem cell transplantation (SCT). PPRs without slow early bone marrow response underwent to SCT only if a matched sibling donor was available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTSEstimated 4-year EFS and OS for all 1252 patients was 83.7% (SE=1.1) and 90.3% (SE=0.89), slightly better in EFS than the former study (ALL-97; 79.3%, SE=1.7, p=0.054). The SR group (N=457, 37%) achieved excellent 4-year EFS of 90.4% (95%CI: 87.2, 92.7) and 4-year OS 97.3% (95.3, 98.5); in SR without slow early bone marrow response, patients with hyperdiploid and triple trisomy showed excellent 4-year EFS/OS of 100%. In the HR group (N=542, 43%), the estimated 4-year EFS and 4-year OS were 84.9% (81.5, 87.7) and 89.3% (86.3, 91.6), respectively; older age (≥ 6 years) was predictive for inferior OS (hazard ratio=1.14, p<0.0001), and ETV6-RUNX1 was predictive for favorable OS (HR=0.182, p<0.03). In the ER group (N=139, 11%), intensification of consolidation treatment using high-dose dexamethasone and AML-oriented agents led to considerable improvement yielding a 4-year EFS of 74.1% (66.0, 80.6) and a 4-year OS of 87.0 (80.2, 91.6). In T group (N=107, 8%), the dose reduction of L-asparaginase and anthracyclines compared with the ALL-97 resulted in inferior outcome in 4-year EFS 65.1% (54.9, 73.6), but not in 4-year OS 83.2% (74.6, 89.1), although the incidence of pancreatitis decreased. In non-T-ALL, The 5-year cumulative risk of an isolated and total CNS relapse was lower in ALL-02 than in ALL-97 (isolated CNS relapse: 0.8% vs 2.7%, p=0.017, total CNS relapse: 1.4% vs 4.5%, p=0.01). The proportion of CRT and SCT were 2%, 8% in ALL-02 respectively, whereas 31%, 11% in ALL-97 respectively. CONCLUSIONSRefined stratification and risk-adjusted therapy in the JACLS ALL-02 trial can bring about the abolition of prophylactic CRT in non-T ALL and the optimized indication of SCT without compromising the treatment results. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3984: Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of anchored multiplex PCR-based next-generation sequencing

Abstract In this study, we demonstrate real-time clinical application of novel anchored multiplex PCR (AMP) technology in hematologic disease. A 78-year-old man with a history of hairy cell leukemia treated with Cladribine (2013) and bladder cancer (2014) developed dyspnea in August 2015. Work up revealed bicytopenia and leukocytosis. Outside bone marrow biopsy was read as B lymphoblastic leukemia/lymphoma. Rebiopsy was performed at our NCI-designated cancer center with extensive work up resulted in reclassification as acute unclassifiable leukemia (rare) with deferral to molecular diagnostics for guidance regarding classification. MLL, BCR-ABL, and MDS FISH panel were negative. Next generation sequencing (NGS) interrogating more than 400 genes showed FLT3-ITD, truncation of RUNX1 exon 6, ASXL1 p.G646Wfs*12, MLL p.V297L, and EZH2 p.V674M and p.C443Y mutations. The molecular profile favored therapy-related AML. Interestingly, cytogenetics revealed 46,Y,t(X;21)(q26;q22)[20]. FISH confirmed RUNX1 rearrangement but the partner was unknown. The t(X;21)(q26;q22) appears to be novel. However, the rare t(3;21)(q26.2;q22) has been cited to segregate with therapy-related MDS/AML. Subsequent aberrant activation of RUNX1 and MECOM is postulated to be pivotal in pathogenesis. Therefore, it is of great interest to determine the translocation partner associated with RUNX1 in our case. This type of analysis is not easily amenable to either Sanger or conventional NGS approaches. Fortuitously, with the recent advent of AMP technology (Archer FusionPlex) novel fusion detection has become possible. AMP was key in the characterization of this sample and promotes discovery of novel fusions by targeting one (known) of the two genes involved in a translocation event. Application of this cutting edge technology lead to the discovery of the novel fusion RUNX1-G6PD in this case. The identity of the novel fusion partners was supported by the breakpoints originally observed by the karyotype analysis. G6PD has been previously reported to be upregulated in acute leukemia. This discovery raises the possibility convergent pathogenic pathways and carries potential biological, diagnostic, and therapeutic implications. Citation Format: Laura Johnson, Helen Wang, Katelyn Trifilo, Brian Kudlow, Peter R. Pappenhausen, Mohammad Hussaini. Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of anchored multiplex PCR-based next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3984.

The Characteristics of Blastic Plasmacytoid Dendritic Cell Neoplasms Presenting Leukemia Dissemination, Diagnosed By Nation-Wide Immunophenotyping for Pediatric Hematological Malignancy in Japan

[Background] According to the nation-wide retrospective study for pediatric leukemia phenotyping, immunological diagnostic committee of Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) established diagnostic criteria and panel to pediatric hematological malignancies in 2008. Then Nation-wide standardized central immnophenotyping in Japan was started from 2011, and done in more than 2,500 cases by the end of 2014. Most cases were determined immunological diagnosis, but it was of note that we found 11 acute unclassified leukemia cases, including typical and atypical blastic plasmacytoid dendritic cell neoplasms (BPDCN). BPDCN is a rare subtype of leukemia/lymphoma, which has been categorized in the 2008 World Health Organization (WHO) classification of hematological diseases under acute myeloid leukemia and related precursor neoplasms. It was well known for high incidence of extra medullary regions. Most cases of BPDCN were found in elder people, so pediatric cases were extremely rare. We report here the characteristics of pediatric BPDCN cases, including clinical manifestations and peculiar immunophenotype.[Materials and Methods] Immunophenotyping was done on bone marrow (BM) aspiration samples according to diagnostic panel and criteria established by JPLSG immunological diagnostic committee. Immunophenotyping data from July 2011 to June 2015 were collected from JPLSG immunophenotyping centers and analyzed. BPDCN cases were screened by typically CD4+ 56+ HLA-DRhi profile. Other lineage markers such as CD7, CD34, CD13, CD33, CD36, CD64, CD123, and NG2 were also analyzed. As it is well known that existence of atypical phenotype in a substantial proportion, including absence of CD56 and/or CD4, immunophenotype data were prudently investigated and compared with 21 acute myelogenous leukemia (AML) presenting M5 characteristics in FAB criteria.[Result] Out of more than 2,500 cases, 4 cases were diagnosed as typical BPDCN (CD4+ 56+ HLA-DRhi), and 2 atypical BPDCN (1 CD56-, 1 CD4-) by immunophenotyping. All 6 cases presented leukemic dissemination, but only 4 typical cases indicated extra medullary regions. Gender ratio (M: F) was 3:1 and 0:2, and median age were 6.9 years (3.3 to 11.3) and 13.8 (12.8, 14.7) respectively. All 6 cases were negative for CD34 and CD13, and positive for NG2. CD123 was also positive in all (5/5) cases. CD33 and CD36 were positive in 5 cases respectively. It was of note that CD64, typical monocytic marker, was negative in all 6 cases although it was positive in all AML-M5 cases. All BPDCN cases could be distinguished with expression patterns of NG2, CD7, CD33, CD36 (positive) or CD13, CD64 (negative). Additionally, CD2 was positive in 3/6 cases, TdT was positive in 2/6 cases. CD117 was positive in 2 cases (1 typical, 1 atypical).[Discussion] BPDCN is a rare, clinically aggressive hematologic malignancy that most commonly manifests as cutaneous lesions with or without bone marrow involvement and leukemic dissemination. Diagnosis of BPDCN has been usually made by histopathologic examination with cutaneous lesions, which is typically positive for interleukin-3 receptor (CD123), blood dendritic cell antigen 2 (BDCA2)/CD303, and TCL-1. Morphologic and immunophenotypic studies were usually performed on BM and/or peripheral blood prior to histopathologic examination, however diagnosis with these examinations as BPDCN is not so easy because of the lack of morphological peculiarity or traditional lineage-specific markers. Treatments for BPDCN patients have been also inconsistent, because there are no prospective clinical trial data to define the optimal frontline treatment. AML-like or acute lymphoblastic leukemia (ALL)-like regimens have been used for induction therapy, as well as lymphoma-like regimens. The difficulty of BPDCN diagnosis might affect to choose inconsistent frontline therapy. Recently some reports documented ALL oriented therapy indicated more effectiveness to BPDCN, so the diagnosis should be made precisely to evaluate the treatment effectiveness. Here we indicated new diagnostic strategy by immunophenotyping with NG2, CD7, CD33, CD36, CD13, and CD64 as well as CD4, CD56 and HLA-DR. This approach contributes not only to select treatment regimen but also to investigate molecular pathogenesis such as whole exome sequence. DisclosuresNo relevant conflicts of interest to declare.

Absolute Lymphocyte Counts at the End of Induction Is a Prognostic Indicator in Childhood Acute Lymphoblastic Leukemia

BACKGROUND: Recently, several studies have demonstrated that absolute lymphocyte counts (ALC) after induction therapy predicted treatment outcome. To address this issue, we here assessed the impact of the ALC at the end of induction therapy on outcomes in childhood ALL.METHODS: We reviewed 141 cases of pediatric ALL with 1-21 years of age treated on the Japan Association Childhood Leukemia Study group ALL-02 series of treatment trials between 2002 and 2013. Patients with Philadelphia chromosome-positive ALL were excluded. Variables retrospectively analyzed included ALC at several time points during remission induction, age at diagnosis, gender, initial white blood cell count (WBC), cytogenetics, immunological phenotype, stratified risk, treatment response for bone marrow (the percentage of blasts at day 15), and outcome. Events in the analysis of event-free survival (EFS) included induction failure, death, relapse and secondary malignant neoplasm. The comparison of categorical variables between groups was performed by chi-square test. The probability of EFS and overall survival (OS) were analyzed with the use of the Kaplan–Meier method and a stratified log-rank test. A multivariate analysis of survival was performed with the use of a Cox proportional-hazard model to evaluate the treatment effect with adjustment for stratification factors.RESULTS: The subjects included 121 of B-precursor ALL, 10 of T cell ALL, and 4 of acute mixed lineage leukemia/ acute unclassified leukemia. We found high WBC count at diagnosis (>100K/microL) and slow early responder for bone marrow at day 15 to be an unfavorable prognostic indicator, and also the ALC at the end of induction (day29) to be a statistically significant predictor of improved OS and EFS in our cohort. Patients with ALC ≥ 800/microL had a superior 5-year overall survival (100 ± 1.7% vs 88.1 ± 4.3 %, p=0.0001) and EFS (98.3 ± 1.7% vs 81.8 ± 5.0 %, p=0.0001). Multivariate analysis demonstrated that ALC at day29 was an independent, clinically significant predictor of improved EFS and OS after controlling WBC at diagnosis, gender, age at diagnosis, and cytogenetics. Multiple regression analysis adjusting for initial WBC count, peripheral blast counts at day8, and cytogenetics, also revealed an independent relationship (p=0.005) between treatment response (the percentage of blasts at day 15) and ALC at day29.CONCLUSIONS: ALC is a simple, statistically significant prognostic factor in childhood ALL that may refine current risk stratification algorithms. DisclosuresNo relevant conflicts of interest to declare.

The Expression of I-Mfa in Adult Patients with De Novo Acute Myeloid Leukemia and Its Clinical Significance

Background: I-mfa has been identified as an inhibitor of MyoD and other related myogenic basic helix-loop-helix proteins. I-mfa contains a cysteine-rich C-terminal domain, and has been reported to function as transcriptional regulator of different pathways including Wnt signaling, c-jun N-terminal kinase signaling, and the regulatory properties of I-mfa depend on the C-terminal domain. Furthermore, recent studies have found that the I-mfa domain may have a close correlation with the development of myeloid neoplasms, however the role of I-mfa in adult patients with de novo acute myeloid leukemia still remain unclear.Aims: The aim of this study was to determine I-mfa expression in adult patients with de novo acute myeloid leukemia and its clinical significance.Methods: BM samples form 110 adult patients with de novo AML were analyzed. Of the 110 AML patients, 66 were males and 44 were females, with a mean age of 32 years( range from 12 to 77 years). Among them, 1 out of 110 patients was M1, 49 were M2, 14 were M4, 28 were M5, 1was M6 and 17 were acute unclassified leukemia. All patients received 1 to 2 cycles of induction of standard-dose cytarabine continuous infusion×7 days with idarubicin or daunorubicin×3days, fellowed by consolidation therapy with HiDAC and then stem cell transplantation according to patient’s condition. Real-time reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the expression of I-mfa gene in 110 de novo adult AML patients, and the patients were divided into high and low I-mfa expression groups accordint to the median expression of I-mfa mRNA. Comparisons were performed using Mann-Whitney U test, Chi-square test and Kaplan-Meier method.Results:Distribution of I-mfa gene expression in different FAB subtypes was with no significant differences (P=0.169). The median age of AML pateints in low and high I-mfa gene epxression groups were 35 and 40 years old(P=0.162), and the median expression of I-mfa in 44 female patients and 66 male patients was 0.018 and 0.013 separately(P=0.728). What’s more, there was no significant difference of WBC, Hb level, PLT, bone marrow blast counts between the two groups (P＞0.05), and the I-mfa expression level was also not correlated with chromosome risk stratification and the expression of CD34 (P＞0.05). High I-mfa expression group had a lower complete remission rate than that in the low expression group (81.8% vs 63.6%, P=0.032), However, the overall survival rate was with no significant difference in the low and hign I-mfa gene expression groups(76.4% vs 76.4%, P=0.471).Conclusions: Our results showed high I-mfa expression correlates with a poor treatment response, the OS rate was with no significant difference in the two groups. There is somewhat correlation between the expression level of I-mfa gene and prognosis and the expression of I-mfa may be a prognostic factor for adult patients with de novo acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

The Expression of Nrf2 in Adult Patients with De Novo Acute Myeloid Leukemia and Its Clinical Significance

Background: Nuclear factor erythroid-2 related factor-2(Nrf2), a helix-loop-helix basic leucine zipper transcription factor, is a key regulator in the cellular defense system against oxidative stress. Nrf2 is binding with Keap1 in a physiological manner, activation of Nrf2 by ROS results in the induction of a series of anti-oxidative stress/detoxifying enzymes and proteins, such as HO-1, NQO-1, UGT and GST. Previous studies suggest that, Nrf2 is aberrant activated in many cancer cells, which is playing an important role in the drug-resistance of the patients. What’s more, it was reported that there’s also an aberrant activation of Nrf2 in the AML cells, which leading to the chemotherapy resistance of AML patients. But the value of Nrf2 in prognosis of adult acute myeloid leukemia(AML) still remain unknown.Aims: The aim of this study was to determine Nrf2 expression in adult patients with de novo acute myeloid leukemia and its clinical significance.Methods: BM samples form 110 adult patients with de novo AML were analyzed. Of the 110 AML patients, 66 were males and 44 were females, with a mean age of 32 years (range from 12 to 77 years). Among them, 1 out of 110 patients was M1, 49 were M2, 14 were M4, 28 were M5, 1was M6 and 17 were acute unclassified leukemia. All patients received 1 to 2 cycles of induction of standard-dose cytarabine continuous infusion×7 days with idarubicin or daunorubicin×3days, fellowed by consolidation therapy with HiDAC and then stem cell transplantation according to patient’s condition. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of Nrf2 gene in 110 de novo adult AML patients, and the patients were divided into high and low Nrf2 expression groups accordint to the median expression of Nrf2 mRNA. Comparisons were performed using Mann-Whitney U test, Chi-square test and Kaplan-Meier method.Results:Distribution of Nrf2 gene expression in different FAB subtypes was with no significant differences (P=0.0.385). The median age of AML pateints in low and high Nrf2 gene epxression groups were 35 and 39 years old (P=0.385), and the median expression of Nrf2 in 44 female patients and 66 male patients was 0.034 and 0.030 separately (P=0.696). What’s more, there was no significant difference of WBC, Hb level, PLT, bone marrow blast counts between the two groups (P＞0.05), and the Nrf2 expression level was also not correlated with chromosome risk stratification and the expression of CD34 (P＞0.05). The patients in high Nrf2 expression group got the same complete remission (72.7%) with the low Nrf2 epxression group. The survival curve gave the clues that the median survival period of hig and low Nrf2 expression groups were 218d and 361d, the overall survival rate was also with no significant difference in the two groups(72.7% vs 80.0%，P=0.667).Conclusions: Our results showed that no matter the expression level of Nrf2 gene, there are no significant difference of CR rate and OS rate in the patients with de novo acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

The Utility of the Multiplex Reverse Transcriptase- Polymerase Chain Reaction Assay in the Detection of Hematologic Malignancies

The Utility of the Multiplex Reverse Transcriptase- Polymerase Chain Reaction Assay in the Detection of Hematologic Malignancies

Plasmacytoid Dendritic Cell Leukemia in Children

CD4/CD56 malignancies are rare hematologic neoplasms, which have recently been shown to represent the malignant counterpart of plasmacytoid dendritic cells (pDC). A 5-year-old boy initially presented with multiple subcutaneous lesions on his upper and lower extremities. Skin biopsy results showed large atypical lymphoid cells in the dermis. The blast cells were stained with CD4 and CD56. In the bone marrow aspirate, 20% of the blast cells were found. The patient was diagnosed as acute unclassified leukemia and received chemotherapy designed for the treatment of acute myeloid leukemia. He achieved a complete remission that lasted for 8 months. However, multiple subcutaneous lesions recurred 1 month after the end of the therapy, with increasing blast cells in his blood. Immunophenotypically, the blast cells were positive for CD2, CD4, CD7, and CD56, and negative for CD3, CD13, CD19, CD33, and CD34 antigens. The blast cells were positive for CD123 (interleukin-3 receptor alpha chain) and blood dendritic cell antigen-2, which are expressed on pDC. The patient was diagnosed as acute leukemia derived from pDC. The CD4, CD56, CD3, CD13, CD19, CD33 profile is highly suggestive of this disease, and the CD123 and blood dendritic cell antigen-2 markers are useful in helping to diagnose pDC leukemia.

Risk factors for leukemia in Thailand

A case-control study of adult-onset leukemia was conducted in Bangkok, Thailand to explore the contribution of cellular telephone use and other factors to the etiology of the disease; 180 cases (87 acute myeloblastic leukemia, 40 acute lymphoblastic leukemia, 44 chronic myelogenous leukemia, eight chronic lymphocytic leukemia, one unclassified acute leukemia) were compared with 756 age- and sex-matched hospital controls. Data were obtained by interview; odds ratios (ORs) were estimated by unconditional logistic regression. There was no clear association with cellular telephone phone use, but durations were relatively short (median 24-26 months), and there was a suggestion that risk may be increased for those with certain usage practices (ORs, 1.8-3.0 with lower confidence intervals >1.0) and those who used GSM service (OR, 2.1; 95% confidence interval, 1.1-4.0). Myeloid leukemia (acute and chronic combined) was associated with benzene (OR, 3.9; 95% confidence interval, 1.3-11), a nonspecific group of other solvents (2.3; 1.1-4.9), occupational pesticides that were mostly unspecified (3.8; 2.1-7.1), and working with or near powerlines (4.3; 1.3-15). No associations were found for diagnostic X-rays, cigarette smoking, or other occupational exposures.

Effective Risk-Adapted Therapy for Childhood B-Precursor Acute Lymphoblastic Leukemia (ALL) with the Japan Association of Childhood Leukemia Study (JACLS) ALL-97 Protocol.

BACKGROUND: With current intensified chemotherapy, more than 70% of children with acute lymphoblastic leukemia (ALL) are curable. However, prognosis of the patients at higher risk has been unsatisfied. More appropriate risk assignment and innovative treatment is expected to be developed. We conducted a clinical trial of JACLS ALL-97 including multi-agent block therapy followed by hematopoietic stem cell transplantation (SCT) for the selected higher risk patients.PATIENTS AND METHODS: Between April 1997 and March 2002, 674 patients aged 1 to 15 years with newly diagnosed ALL (excluding mature B-cell ALL) were enrolled on the JACLS ALL-97 protocol. Excluding 75 T-ALL, 26 mixed lineage leukemia other than B-precursor ALL with myeloid markers, 9 acute unclassified leukemia and 1 B-precursor ALL with pretreatment, 563 patients with B-precursor ALL were eligible for this analysis. Treatment group was divided 5 groups according to the modified National Cancer Institute (NCI) workshop criteria. Standard risk (SR) and IR (intermediate risk) divided by WBC10 × 103 account for NCI-SR. High risk (HR) and ER (extremely high risk) divided by WBC 100 × 103 account for NCI-HR. The patients with ALL positive for Philadelphia chromosome and for translocation with chromosome11q23 were assigned to the highest risk group F. The patients in complete remission (CR) at day 35 with M2/M3 at the day 14 marrow were assigned to shift higher risk after induction therapy. Treatment of ALL-97 consists of early phase and maintenance phase. Early phase includes induction therapy, consolidation therapy, sanctuary therapy, and re-induction therapy for 19 to 26 weeks dependent on risk group. Early phase in IR/HR protocol is identical to the SR protocol except the addition of two doses of DNR and three doses of CPM. Maintenance phase contains standard MTX and MP with monthly VCR and PSL intensification for SR, and rotational therapy of a set of MTX and MP with a set of VCR, PSL, ASP, and (DNR or CPM) for IR/HR/ER/F. The IR protocol is identical to the HR protocol without cranial irradiation. Treatment duration is 24 months for any risk. The patients assigned ER and F were candidate for allogeneic stem cell transplantation by the end of early phase. Transplant procedures depended on the institute.RESULTS: Number of patients at each risk was 204 for SR, 158 for IR, 128 for HR, 36 for ER, 27 for Ph+ and 10 for 11q23. Six of them were treated with incorrect risk protocol. Thirty-four patients received SCT in first CR. Following the induction therapy, 550 of 564 patients (97.5%) achieved CR. 23 patients(8 in SR, 9 in IR, 4 in HR and 2 in ER, 4.4% of all) were shift to higher risk due to the findings of day 14 marrow. Five-year overall survival rate (OS) and event-free survival rate (EFS) for all patients was 90.6% and 77.0%, respectively. Five-year EFS for NCI-SR and HR was 81.6% and 67.6%, respectively. According to risk group, 5-year EFS for SR, IR, HR, ER, Ph+ ALL, and ALL with 11q23 were 86.6%, 77.0%, 71.9%, 68.7%, 40.7%, and 70.0%, respectively. 5-year OS for them were 94.0%, 93.9%, 92.1%, 83.8%, 55.6%, and 68.6%, respectively.CONCLUSIONS: By the risk-adapted therapy in the JACLS ALL-97 trial, high cure rate could be achieved for children with B-precursor ALL.

Acute Unclassified Leukemia with CD7 and CD13 Positivity

Acute Unclassified Leukemia with CD7 and CD13 Positivity

Detection of myeloperoxidase by flow cytometry in acute leukemia.

The value of flow cytometric detection of myeloperoxidase (MPO) in the differential diagnosis of acute leukemia was evaluated in 57 cases of acute leukemia and in 9 leukemia cell lines. Cells were fixed and permeabilized with Fix & Perm cell permeabilization kit at room temperature for 15 min each, and stained with anti-MPO monoclonal antibody (MPO-7) by direct immunofluorescence. One myeloid cell line, HL-60, was MPO-positive, while the other myeloid cell lines (KG-1, K-562, and MEG-01) as well as lymphoid cell lines (KM-3, NALM-6, Raji, REH, and T-ALL-1) were MPO-negative as previously described. Among acute leukemias, MPO was detected in 23 of 26 cases of acute myeloid leukemia (AML), 7 of 23 cases of B-lineage acute lymphoblastic leukemia (ALL), 1 of 6 cases of T-lineage ALL (T-ALL), and 1 of 2 cases of acute unclassified leukemia (AUL). The intensity of MPO expression in 6 of 7 B-lineage ALL cases was weak compared with AML labeling. There was no detectable cytochemical MPO in the cells of ALL, AUL, or AML that stained negative for anti-MPO. No relationship between the expression of MPO and myeloid lineage surface antigens was observed in ALL. Three cases of MPO-positive ALL and AUL could be reclassified as biphenotypic leukemia according to the revised Catovsky scoring system. These results indicate that anti-MPO is an excellent marker for the diagnosis and classification of acute leukemia and can be reliably detected by flow cytometry. This rapid technique should be a valuable addition to routine immunophenotyping of acute leukemia.

Sensitive detection technique of myeloperoxidase precursor protein by flow cytometry with monoclonal antibodies.

This report describes the analysis of culture cells and blast cells separated from the heparinized bone marrow and whole blood of patients with acute leukemias by means of a density-gradient technique (Ficoll-sodium metrizoate d = 1.077 g/cm3). Cell-surface antigens were analyzed by a fluorescence-activated cell sorter using a panel of monoclonal antibodies (MAbs). The blast cells and culture cells were fixed by 3% paraformaldehyde in phosphate-buffered saline. A low level of expression of MPO precursor protein was found in THP-1. K-562 and HEL, MEG-01, erythro-megakaryocytic leukemia cell lines, Jurkat, MOLT-3, MOLT-4, RPM18402, ATL-5, T-cell leukemia cell lines, Raji, Daudi. BALL-1, B-cell leukemia cell lines, and AGNK1 showed negative reaction. The de novo MPO-negative acute leukemias, middle level of expression of MPO precursor protein, was found in the blasts of MPO-negative AML (AML, M0), which coexpressed CD13, CD33, CD34, and CD38. A high level of expression of MPO protein was found in all cases of AML, M1, and M2. The MPO expression was not found in all cases of acute lymphoblastic leukemia. The highest level of MPO expression was found in cases of AML, M3, and AML, M3v, suggesting the diagnostic value for this type of leukemia. The detection of MPO precursor protein by flow cytometric analysis with monoclonal antibodies is essential for the determination of lineage and precise diagnosis of acute unclassifiable leukemia, and should contribute substantially to the development of an effective form of therapy and cure.

Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines.

In this study, we examined a large number of patients to clarify the distribution and frequency of a recently described FLT3 tandem duplication among hematopoietic malignancies, including 112 acute myelocytic leukemia (AML), 55 acute lymphoblastic leukemia (ALL), 37 myelodysplastic syndrome (MDS), 20 chronic myelogenous leukemia (CML), 30 non-Hodgkin's lymphoma (NHL), 14 adult T cell leukemia, 15 chronic lymphocytic leukemia (CLL) and 38 multiple myeloma (MM). We also evaluated 71 cell lines derived from 11 AML, 31 ALL, two hairy cell leukemia, three acute unclassified leukemia, 10 CML, 12 NHL including six Burkitt's lymphoma, and two MM. Using genomic PCR of exon 11 coding for the juxtamembrane (JM) domain and first amino acids of the 5'-tyrosine kinase (TK) domain, this length mutation was found only in AML (22/112, 20%) and MDS (1/37). According to the FAB subclassification, they were 5/18 (28%) of M1, 4/29 (14%) of M2, 3/17 (18%) of M3, 6/24 (25%) of M4, 4/20 (20%) of M5 and 1/9 of refractory anemia with excess of blast in transformation. In the various cell lines examined, this abnormality was determined in only one derived from AML and never found in other hematological malignancies. The sequence analysis of the abnormal PCR products revealed that 23 of 24 showed internal tandem duplication with or without insertion of nucleotides. In one AML, insertion and deletion without duplication was determined. All 24 lengthened sequences were in-frame. Duplication takes place in the sequence coding for the JM domain and leaves the TK domain intact. In conclusion, we emphasize that the length mutation of FLT3 at JM/TK-I domains were restricted to AML and MDS. Since all these mutations resulted in in-frame, this abnormality might function for the proliferation of leukemic cells.

Characterization of Acute Leukemia with t(4;12)

Acute leukemia with t(4;12)(q11–13;p12–13) is rare but has unique characteristics. The incidence of t(4;12) in acute leukemias was about 0.6% in our laboratory. Twelve patients with acute leukemia with t(4;12) have been reported until now. They included eight acute myeloid (AML: M0 2, M1 3, M2 1, M4 1, and M7 1), three acute lymphoblastic (ALL: L1) and one acute unclassified leukemia (AUL). There were some differences between adults and children with t(4;12). The eight adult patients included seven with AML and one with AUL, two of whom had a history of exposure to mutagenic agents and/or genotoxic therapy. Three patients had the CD7+HLA-DR+CD13+CD34+c-kit+ phenotype, suggesting that the leukemic cells were of stem cell origin. Four children expressed the B lymphoid phenotype (HLA-DR+CD10+CD19+) although one had myeloperoxidase positivity. It was difficult for adult patients to achieve complete remission with the usual therapy regimen, whereas children with t(4; 12) seemed to be easier to treat. Rearrangement of the TEL gene located on the short arm of chromosome 12 (12p13), was investigated in two adult patients. FISH analysis using the YAC probe that covers the TEL gene region, revealed split signals in these patients, suggesting a break inside or near the TEL gene. The t(4;12) abnormality is associated with unique characteristics of acute leukemia namely stem cell or secondary AML in adults, and B lymphoid leukemia in children.