Cases Of Acute Undifferentiated Leukemia Research Articles

The association of complex genetic background with the prognosis of acute leukemia with ambiguous lineage

Acute leukemia with ambiguous lineage (ALAL) is a rare and highly aggressive malignancy with limited molecular characterization and therapeutic recommendations. In this study, we retrospectively analyzed 1635 acute leukemia cases in our center from January 2012 to June 2018. The diagnose of ALAL was based on either EGIL or 2016 WHO criteria, a total of 39 patients were included. Four patients diagnosed as acute undifferentiated leukemia (AUL) by both classification systems. Among the patients underwent high-throughput sequencing, 89.5% were detected at least one mutation and the median number of gene mutation was 3 (0–8) per sample. The most frequently mutated genes were NRAS (4, 21%), CEBPA (4, 21%), JAK3 (3, 16%), RUNX1 (3, 16%). The mutations detected in mixed-phenotype acute leukemia (MPAL) enriched in genes related to genomic stability and transcriptional regulation; while AUL cases frequently mutated in genes involved in signaling pathway. The survival analysis strongly suggested that mutation burden may play important roles to predict the clinical outcomes of ALAL. In addition, the patients excluded by WHO criteria had even worse clinical outcome than those included. The association of the genetic complexity of blast cells with the clinical outcomes and rationality of the diagnostic criteria of WHO system need to be evaluated by more large-scale prospective clinical studies.

ACUTE UNDIFFERENTIATED LEUKEMIA WITH BCR-ABL TRANSLOCATION: CASE REPORT AND LITERATURE REVIEW

The 2016 WHO classification defines acute undifferentiated leukemia (AUL) as a rare type of acute leukemia (AL) without defining markers for either lymphoid or myeloid lineage commitment. Literature suggests AUL does not harbour the genetic mutations commonly seen in lymphoblastic leukemia or acute myeloid leukemia (AML) including FLT3, WT1, rearrangements of KMT2A or BCR-ABL1 and it highly expresses genes associated with poor prognosis in AML (BAALC, ERG, and MN1); therefore, AUL is often associated with dismal outcome (Arber et al. 2016). This case report depicts an unusual genetic presentation of an AUL with BCR-ABL fusion gene. A 54-year-old female patient presented to our hospital in July 2021 with fatigue and adynamia one month before. She was also found with deep vein thrombosis in the left leg and segmental pulmonary thromboembolism. Laboratory exams showed Hb 9,7 g/dL, WBC 75.77×10 9 /L (77% blasts), platelets 175×10 9 /L. Coagulogram and fibrinogen were normal. Bone marrow (BM) aspiration was hypercellular, showing 86% of agranular blasts. Flow cytometry (FC) from BM blasts exhibited expression of CD34, CD45, HLA-DR, CD33, CD38, and partial expression of CD10 and CD123. The blasts were negative for MPO, CD79a, CD19, CD7, CD3, CD13, C117, CD16, CD11b, CD64, CD15, CD14, CD300e, CD61, NG2, CD71, CD4, CD36, CD22, CD42b. Conventional cytogenetics revealed a Ph+ disease (46,XX,t(9;22)(q34q11.2)[7]/46, idem, add(4)(q35)[7]/46,XX[6]). Real-time quantitative PCR showed positive BCR-ABL with p190 transcripts. FLT3 and NPM1 were found negative. Diagnosis of AUL Ph+ was made. AML chemotherapy was started with 3+7 standard doses regimen plus imatinib 600 mg/day. On day 14th BM evaluation found 89% of blasts with the same immunophenotype as in the diagnosis. The patient presented neutropenic colitis with the need to delay rescue therapy. We searched articles in the Pubmed database using the following strategy: “Bcr-Abl Tyrosine Kinase”AND “undifferentiated leukemia”, “Philadelphia chromosome”AND “undifferentiated leukemia”between 1976 and 2021. We found two case reports that described patients with AUL and positive BCR-ABL gene. In 1983, Smadja, N et al. reported a patient with AL considered at diagnosis to be undifferentiated. Neither the diagnosis cytogenetic nor FC data was provided by the authors, and the AL classification was based only on morphological characteristics. After 17 months, the patient developed Ph+ chronic myelogenous leukemia with a complex translocation (2; 9; 22). Hattori, M et al., in 1995, described a patient that following treatment to non-Hodgkin lymphoma developed myelodysplastic syndrome (MDS) which progressed to AUL, both found with the presence of chimeric BCR-ABL transcripts. The FC showed blasts that expressed CD7 and HLA-DR, and were negative for lineage markers: CD14,CD33,CD2,CD3,CD4,CD5,CD8,CDl0,CD19,CD20 and CD41. Qasrawi, A. et al., in 2019 and 2020 performed two studies analyzing data from 24 and 1444 cases of AUL, respectively. The most common pathogenic mutations were PHF6, SRSF2, RUNX1, ASXL1, and BCOR. No BCR-ABL mutation was described. We believe that with the advent of target therapies, the prognosis of AUL can improve. The patient described has the BCR-ABL gene as a potential target to a tyrosine kinase inhibitor. Considering this is not a usual finding, we hope this report can further help treatment decisions.

CD2 and CD7 are sensitive flow cytometry screening markers for T-lineage acute leukemia(s): a study of 465 acute leukemia cases

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a rare acute leukemia that expresses cytoplasmic CD3 (cCD3) and frequently lacks surface CD3. Given that routine flow cytometric testing for cCD3 may not be feasible and cCD3 interpretation may be difficult, we investigate if surface CD2 and/or CD7 expression on blasts can be used by flow cytometry to screen for T-lineage acute leukemia. We retrospectively reviewed flow cytometric data from 233 acute leukemias (36T-ALL/LBL, 8 mixed-phenotype acute leukemia T/myeloid, 80 acute myeloid leukemia, 97B-ALL/LBL, 8 mixed-phenotype acute leukemia B/myeloid, and 4 acute undifferentiated leukemia cases). Uniform expression (≥75% of blasts) of CD2 and/or CD7 was seen in all 44 cCD3-positive cases but in only 11% (20/189) of cCD3-negative acute leukemias, thus demonstrating 100% sensitivity and 89% specificity in the identification of cCD3-positive (T-lineage) acute leukemia. To avoid selection bias, we prospectively studied 232 consecutive acute leukemias for which cCD3, CD2, and CD7 were automatically performed in all cases. Similar to the retrospective study, uniform expression of CD2 and/or CD7 on blasts showed 100% sensitivity and 88% specificity in the screening for cCD3-positive (T-lineage) acute leukemia. Therefore, acute leukemias with uniform expression of CD2 and/or CD7 warrant further testing for cCD3 to evaluate for T-lineage acute leukemia. Blasts that lack both uniform CD2 and CD7 expression do not require additional cCD3 testing. We propose that CD2 and CD7 could be utilized in a limited antibody flow cytometry panel as a sensitive, robust, and cost-effective way to screen for T-lineage acute leukemia.

Acute undifferentiated leukemia: data on incidence and outcomes from a large population-based database

Acute undifferentiated leukemia (AUL) is rare and defined by the absence of bona fide myeloid and lymphoid markers. Little is known about its incidence, survival and optimal management in the recent time period. Based on a case observed in our clinic, we queried the Surveillance, Epidemiology, and End Results database between 2000 and 2016. A total of 1,888 cases of AUL were diagnosed (1.34 per million person-years). The incidence of AUL has significantly decreased over time. Compared to other acute leukemias, patients with AUL have the highest median age (74 years); in contrast to acute myeloid leukemia (AML, 65) and acute lymphoblastic leukemia (ALL, 12). Excluding patients with preexisting malignancies, 1,444 patients with AUL were analyzed for survival. Only 35% of AUL patients had received chemotherapy. Comparatively, 94% of ALL and 71% of AML cases received chemotherapy. Among AUL patients who received chemotherapy, the median survival was 12 months as opposed to 1 month in the group who did not receive chemotherapy (or unknown status). Among adults, AUL patients had the worst prognosis, with a median overall survival (OS) of 9 months, compared to 27 months in ALL and 13 months in AML. Among children, the median OS was superior for all three groups of leukemias, the OS of AUL patients being better than in AML and very similar to ALL. On multivariate analysis, older age and time period were associated with worse outcome. We describe here the largest series of cases with AUL published to date.

Clinical, immunophenotypic, and genomic findings of acute undifferentiated leukemia and comparison to acute myeloid leukemia with minimal differentiation: a study from the bone marrow pathology group

Acute undifferentiated leukemia is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited and it is uncertain if acute undifferentiated leukemia is biologically distinct from acute myeloid leukemia with minimal differentiation, which also shows limited myeloid marker expression and has been reported to have a poor prognosis. We identified 92 cases initially diagnosed as acute undifferentiated leukemia or acute myeloid leukemia with minimal differentiation from pathology databases of nine academic institutions with available diagnostic flow cytometric data, cytogenetic findings, mutational and clinical data. Outcome analysis was performed using Kaplan Meier test for the 53 patients who received induction chemotherapy. Based on cytogenetic abnormalities (N = 30) or history of myelodysplastic syndrome (N = 2), 32 cases were re-classified as acute myeloid leukemia with myelodysplasia related changes. The remaining 24 acute undifferentiated leukemia patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the remaining 30 acute myeloid leukemia with minimal differentiation patients. Compared to acute myeloid leukemia with minimal differentiation, acute undifferentiated leukemia cases were characterized by more frequent mutations in PHF6 (5/15 vs 0/19, p = 0.016) and more frequent expression of TdT on blasts (p = 0.003) while acute myeloid leukemia with minimal differentiation cases had more frequent CD123 expression (p = 0.042). Outcome data showed no difference in overall survival, relapse free survival, or rates of complete remission between acute undifferentiated leukemia and acute myeloid leukemia with minimal differentiation groups (p > 0.05). Acute myeloid leukemia with myelodysplasia-related changes patients showed shorter survival when censoring for bone marrow transplant as compared to acute undifferentiated leukemia (p = 0.03) and acute myeloid leukemia with minimal differentiation (p = 0.002). In this largest series to date, the acute undifferentiated leukemia group shows distinct characteristics from acute myeloid leukemia with minimal differentiation, including more frequent PHF6 mutations and expression of TdT.

Clinical, Immunophenotypic and Genomic Findings of Acute Undifferentiated Leukemia and Comparison to AML with Minimal Differentiation: A Study from the Bone Marrow Pathology Group

Introduction: Acute undifferentiated leukemia (AUL) is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited: the largest study to date reported 16 AUL cases but did not use the current WHO classification and included limited genetic data on 5 cases (Ann Hematol 2013 92:747-758). Moreover, it is uncertain if AUL is biologically distinct from acute myeloid leukemia with minimal differentiation (AML MD), which also shows limited myeloid marker expression and has been reported to have a poor prognosis.Methods: A total of 95 cases (36 AUL cases 59 AML MD) were identified from pathology databases of eight academic institutions with available diagnostic flow cytometric data, cytogenetic findings, and clinical data by searching for diagnoses of “AUL” or “AML MD”. Diagnosis of AUL required absence of any lineage-defining markers including MPO, CD19 and CD3. Using the WHO classification, diagnosis of AML with MD required expression of at least 2 myeloid markers (CD117, CD13 or CD33), absence of myeloid maturation (CD15) or monocytic markers (CD64, CD11b, lysozyme or non-specific esterase). Next generation sequencing with extensive mutational panel data was available in 78 cases. Outcome analysis for overall survival (OS) and relapse-free survival (RFS) and were performed using Kaplan Meier and log rank test for patients who received induction chemotherapy.Results: Based on cytogenetic abnormalities (N=27) or history of MDS (N=2), according to the 2016 WHO Classification, 28 cases (6 AUL and 22 AML MD) were re-classified as AML with myelodysplasia related changes (AML MRC). The remaining 30 AUL patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the 37 AML MD patients (all p > 0.05). Comparison of immunophenotype in the two groups showed that AUL blasts had more frequent expression of TdT (p=0.0003) and lacked myeloid markers (CD117, CD13 or CD33 p<0.0001), while AML MD cases had more frequent CD123 expression (p=0.0421). No differences in B (CD19, CD20, CD10, CD22, CD79a) or T lymphoid antigen (CD2, CD3, CD4, CD5, CD7, CD8) or monocyte marker (CD11b, CD64) co-expression on blasts (p>0.05) were seen between these two groups. The frequency of abnormal karyotype was similar between AUL and AML MD (16/30 [53%] vs 15/37 [41%], respectively). The most common mutations identified in AUL were PHF6 (7/18), SRSF2 (7/18), RUNX1 (7/23), ASXL1 (6/23) and BCOR (5/16). Compared to AML MD, AUL cases were characterized by frequent mutations in PHF6 (7/18 vs 1/23, p=0.013) and SRSF2 (7/18 vs 2/22 p=0.028). Limiting AUL cases to only those with 1 myeloid marker or less also showed similar findings with more frequent mutations in PHF6 (7/16 vs 1/25, p=0.0031), SRSF2 (6/15 vs 3/26 p= 0.018) and trend towards higher BCOR frequency (5/15 vs 2/26, p=0.078) in AUL patients as compared to AML MD. RUNX1 mutation was seen in 7/23 AUL and 8/29 AML MD (p>0.05). 19/30 AUL patients received induction chemotherapy (AML-type regimen in 18 cases and an ALL-type regimen in 1 case) and 15/30 achieved complete remission. In 10 AUL patients who relapsed, 9 showed identical immunophenotype and one case showed expression of CD13 and CD33. Outcome data in the subset of patients who received induction showed no difference in OS, RFS, or rates of complete remission between AUL and AML MD groups (p>0.05). The 28 AML MRC cases that were originally classified as AUL or AML MD presented with lower WBC (p=0.026), more frequent abnormal karyotype (27/28) specifically complex karyotype (20/28 p=0.002), frequent TP53 mutations (p=0.0002) when compared to the AUL group. AML MRC patients showed worse overall OS (p=0.029) as compared with AUL patients and a trend toward worse outcome as compared with AML MD (p=0.068).Conclusions: In this largest series to date, AUL group shows distinct characteristics from AML MD, including more frequent PHF6 and SRSF2 mutations and expression of TdT. However, clinical outcome is similar between the two groups in patients treated with induction chemotherapy. Cases reclassified as AML-MRC had shorter survival compared to de novo AUL and trend towards worse outcome when compared to AML MD patients. These results suggest a genetic rationale for the separation of AUL as a distinct entity from AML MD and also support the WHO classification of cases with history of prior MDS and/or MDS-type karyotype findings as AML-MRC. DisclosuresGarcia:Celgene: Consultancy.

Molecular and Immunophenotypic Characteristics of Adult Acute Leukemias of Ambiguous Lineage

Acute undifferentiated leukemia (AUL) and mixed phenotype acute leukemia (MPAL) are acute leukemias of ambiguous lineage representing less than 5% of all adult leukemias. Ontogenetically, they are thought to be caused by precursor lesions in early pluripotent hematopoietic progenitor cells having the ability to differentiate into both lymphoid and myeloid lineages. In contrast, common lymphoid and myeloid leukemias are likely derived from precursor lesions in committed progenitor cells. The molecular characteristics of the cell of origin of these leukemias have not been fully characterized.Given the ambiguous classification of these disorders, we used NGS to evaluate the molecular profiles of this unique subset of leukemias. Our panel tested for the 60 most commonly mutated genes in myeloid malignancies (MM), which can be used to further elucidate driver mutations that play a role in the pathogenesis of this unique entity. Molecular data was available for 18 of 35 patients (pts) (51%), of whom 8 were T/myeloid, 7 were B/myeloid, and 3 were AUL. 14 pts out of the 18 had samples at the time of initial diagnosis, 3 AUL cases harbored mutations commonly seen in MM, including one patient with DNMT3A and a germ line JAK3 mutation, another with U2AF1 and PHF6 mutations, and one pt with SF3B1 and MECOM mutations. Of the 5 B/myeloid cases, 4 had only one mutation detected by the sequencing panel, including TET2, PTPN11, KDM6A, and PHF6, and one had no mutations, but did have complex cytogenetics. Of interest, we identified four patients with T/myeloid MPAL that harbored a FLT3-ITD mutation. Notably, one of these cases had additional DNMT3A and TET2 mutations while another case had a WT1 mutation. We assert that the mutational data for MPAL supports that T-myeloid MPAL encompasses a borderland between early T precursor (ETP)-ALL/AML in addition to more typical cases of T-ALL/AML given its similar immunophenotype to ETP ALL (CD117, CD13 and CD33 positive). The overlap between these two entities is illustrated in the figure below. With this immunophenotyping and molecular profiling we propose a new provisional entity, ETP-myeloid MPAL in addition to already existing ETP ALL.Of 1250 pts with acute leukemia diagnosed from 1997-2016 at Cleveland Clinic, 35 had immunophenotypic characteristics of ambiguous leukemia based on WHO criteria. The median age at diagnosis was 50 years, and 22 (63%) were male. There were 31 MPAL (16 T/myeloid, 15 B/myeloid) and 4 AUL cases. Among the 31 MPAL patients, 2 were mixed lineage chimera (subclones), and 29 had true biphenotypic features. 48% of the cohort had normal cytogenetics at diagnosis, 23% had other cytogenetic abnormalities, 14% had complex karyotype, 9% had MLL gene rearrangement, and 6% had t(9;22). 62% of pts were treated with acute lymphoblastic leukemia (ALL) directed induction chemotherapy (IC), while 38% were treated with acute myelogenous leukemia (AML) directed IC. Rates of complete remission (CR) were significantly higher in pats treated with ALL-IC regimen (82%) versus AML-IC regimen (23%) (p=.003). In our study, two cases with the recurrent lymphoid mutations WT1 and KDM6A, that achieved CR with upfront ALL-IC, while one case with FLT3-ITD, DNMT3A, and TET2 mutations achieved CR with AML-IC treatment. Pts <40 yrs of age had significantly better median OS (41 months) than patients over 40 yrs old (16 months) (p=0.03). MPAL pts with normal cytogenetics had worse median OS (16 months) compared to any other cytogenetic group (25 months) (p=.05).In conclusion, clinical evaluations of ambiguous leukemias demonstrate that those with normal karyotype have the worst outcome of all pts diagnosed with MPAL. This can be partly explained by mutations that are detected at the time of diagnosis such as FLT3-ITD and DNMT3A, which have been associated with poor survival in AML with normal karyotype. Identification of actionable targets like FLT3-ITD can improve clinical outcomes for this subset of patients prior to BMT or for those ineligible for BMT. The existence of a unique subset of pts with immunophenotypic overlap between ETP-ALL/AML, that were classified in routine practice as T/myeloid leukemia, are important to recognize due to their unique actionable phenotype. Molecular profiling of these cases identified gene mutations more akin to MM. We propose that sequencing may play an important role in optimizing the therapeutic approach for patients with ambiguous lineage leukemia. [Display omitted] DisclosuresCarraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Celgene: Research Funding, Speakers Bureau.

Immunophenotypic Profile of Acute Leukemia: Critical Analysis and Insights Gained at a Tertiary Care Center in India

The purpose of this retrospective study, the largest series in our country, was to illustrate the spectrum of subtypes and the immunophenotypic features of cases referred as acute leukemia (AL). Morphological diagnosis was correlated with cytochemical stains, immunophenotyping, and molecular genetic studies including fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Two thousand five hundred and eleven consecutive new referral cases of Acute Leukemia (AL) between January 1, 2003 and December 31, 2006 were evaluated based on WHO classification. It included 1464 cases (58.3%) of Acute Lymphoblastic Leukemia (ALL), 962 cases (38.3%) of Acute Myeloid Leukemia (AML), 45 cases (1.8%) of Chronic Myelogenous Leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL and 2 cases of Acute Undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%) which comprised of intermediate stage/CALLA positive (73%), early precursor/Pro BALL (3%). T-cell ALL constituted 24% (348 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), AML t(8;21) (9%). CMLBC included myeloid blast crisis (40 cases), lymphoid blast crisis (2 cases), and biphenotypic leukemia (3 cases). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. An immunoprofile of CD34+, HLADR+, CD14+ and CD33− is unlikely to be APML. CD117 is not a sensitive marker for APML and shows negative to weak continuous expression on promyelocytes. CD19 was expressed in 45% of FAB AMLM2 with t(8;21)(q22;q22). At least five cases of hepatosplenic gamma delta T-cell lymphomas were misdiagnosed as T-ALL based on a minimal selective panel where blasts expressed surface CD3. These cases were correctly diagnosed at relapse. Overall incidence of AML in adults is low (53% only). T-ALL is common in adolescent males. We recommend at least ten antibodies mainly CD13, CD33, CD117, CD10, CD19, HLADR, CD7, CD5 (or CD2), CD45 and CD34 as a primary minimal panel for a case of AL. Tdt is invaluable in work-up of pediatric T-cell neoplasm, to differentiate T-ALL from hepatosplenic gamma delta T cell lymphoma. Only 8% cases required additional markers (including intracytoplasmic markers) in the secondary panel for a lineage assignment. cCD22 is more sensitive than cCD79a for B-cell lineage assignment. Drawbacks of this study included referral bias, no clinical correlation, and three color immunophenotyping with FSC/SSC gating. Our approach for immunophenotyping was semi directed, a primary minimal panel followed by additional antibodies, as and when required. Cytoplasmic lineage specific markers (used in additional panel) for a lineage assignment were required only in 8% cases (n=199). We used only 14–20 antibodies per case (mean = 16), a number much less then many other laboratories. WHO classification has made it mandatory to do FISH/PCR to diagnose various leukemias which otherwise could be diagnosed with reasonable accuracy based on morphology, cytochemistry and flow cytometry like CML, APML, AMLM4Eo etc. Significant number of our FAB AMLM2 and AMLM4E0 cases did not show associated translocations. Few patients were partially treated by referring physician (steroids, blood transfusion, heavy metals etc), a category not addressed in WHO classification. Cytogenetics made a major difference in therapeutic decision in 0.8% cases only. Though flow cytometry is extremely popular amongst hematopathology laboratories, cytogenetics is available at few selected centers in countries with limited resources.

Immunophenotypic profile of acute leukemia: Critical analysis and insights gained at a tertiary care center in India

To analyze the spectrum of various types and subtypes of acute leukemia. Two thousand five hundred and eleven consecutive new referral cases of acute leukemia (AL) were evaluated based on WHO classification. It included 1,471 cases (58%) of acute lymphoblastic leukemia (ALL), 964 cases (38%) of acute myeloid leukemia (AML), 45 cases (1.8%) of chronic myelogenous leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL, and 2 cases of acute undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%), which comprised of intermediate stage/CALLA positive (73%), early precursor/proBALL (3%). T-cell ALL constituted 24% (351 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), and AML t(8;21) (9%). CMLBC was commonly of myeloid blast crisis subtype (40 cases). B-cell ALL was the commonest subtype in children and AML in adults. Overall incidence of AML in adults was low (53% only). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. A minimal primary panel of nine antibodies consisting of three myeloid markers (CD13, CD33, and CD117), B-cell lymphoid marker (CD19), T-cell marker (CD7), with CD45, CD10, CD34, and HLADR could assign lineage to 92% of AL. Cytogenetics findings lead to a change in the diagnostic subtype of myeloid malignancy in 38 (1.5%) cases.

Cytogenetic and clinicobiological features of acute leukemia with stem cell phenotype: Study of nine cases

Morphologic, immunologic, cytogenetic, and clinical features were studied in 9 cases of acute undifferentiated leukemia (AUL). These patients were unclassifiable by FAB criteria, they were CD34+ and did not express myeloid- or lymphoid-associated antigens (CD13, CD33, CD14, CD15, CD61, CD19, CD10, CD22, CD7, CD2, CD5, CD3). Clonal abnormalities were seen in 8 of 9 cases. Del(5q) as the sole anomaly was observed in 3 cases; +13 was the primary change in 3 cases, and isolated trisomy 12 was found in 1 patient. A complex karyotype with trisomy 12q, in association with del 17p and trisomy 21q was detected in 1 case. One patient with 5q- relapsed with refractory anemia with excess of blasts; the presence of dysgranulopoiesis and a few blasts with possible monocytoid morphology in the remaining 2 patients point to a “myeloid nature” of these leukemias. Analysis of cytologic features in our 3 patients with +13, in combination with previously reported cases, suggests the occurrence of immature stem cell involvement with limited differentiation potential, possibly more along the myeloid than the lymphoid lineage. The significance of trisomy 12q in this subset of leukemia remains elusive; some clues of minimal differentiation towards the myeloid lineage in our cases are provided by positivity for the CD117 (c-kit) antigen and by relapse with acute myeloid leukemia without maturation (M1) in one patient. We conclude that, with presently available diagnostic techniques, AUL is a rare subset of leukemia, in which cytogenetic changes are confined to a few chromosomes, with prevalent involvement of 5q and of chromosomes 13 and 12. Chromosome findings may be of value in clinical practice, especially in those cases with “myeloid-oriented” karyotype.

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Heterogeneity of Acute “Undifferentiated” Leukemia of Childhood

The present study was undertaken in an attempt to reclassify the 19 cases of childhood acute undifferentiated leukemia (AUL) diagnosed at our institution during the past 12 years. Based on ultrastructural and immunophenotypic data, seven of the cases were reclassified as lymphoid, nine as myeloid, and three remain unclassifiable. Clinical features, clonal karyotypes, and responses to treatment were also examined. Abnormal clonal karyotypes were found in 16 of 17 cases, including eight cases with translocations, three with monosomy 7 or 7q, and one with numerous complex structural rearrangements. Fourteen patients had greater than 10% French-American-British L2 blasts in bone marrow. Although nine of 15 patients who initially received induction therapy for acute lymphoblastic leukemia (ALL) achieved remission, only one patient is a long-term survivor. Only one of 10 patients who received therapy for acute nonlymphoblastic leukemia during the course of their disease remains a long-term survivor. These data suggest that the majority of cases of AUL can be reclassified as either myeloid or lymphoid leukemias, that AUL is associated with a high frequency of chromosomal abnormalities, and that AUL carries a very poor prognosis.

A Monoclonal Antibody Reacting With Human Basophils

AbstractBsp-1 is an IgM murine monoclonal antibody raised against the human erythroblastic leukemia cell line (HEL) that reacts with basophils but not neutrophils or eosinophils. Western blotting techniques showed that Bsp-1 reacts with a 45-kilodalton surface antigen on HEL cells. The distribution of Bsp-1 antigen on leukemic cells is confined to a basophilic leukemia cell line, KU812, chronic myeloid leukemia with basophilia, and some cases of acute undifferentiated leukemia. Bsp-1 might therefore be a useful reagent for the study of basophil function and differentiation.

A monoclonal antibody reacting with human basophils

Bsp-1 is an IgM murine monoclonal antibody raised against the human erythroblastic leukemia cell line (HEL) that reacts with basophils but not neutrophils or eosinophils. Western blotting techniques showed that Bsp-1 reacts with a 45-kilodalton surface antigen on HEL cells. The distribution of Bsp-1 antigen on leukemic cells is confined to a basophilic leukemia cell line, KU812, chronic myeloid leukemia with basophilia, and some cases of acute undifferentiated leukemia. Bsp-1 might therefore be a useful reagent for the study of basophil function and differentiation.

Heterogeneity of acute undifferentiated leukemia at the immunoglobulin and T-cell receptor genes level.

Phenotypic markers of leukemic cells from 76 children with acute leukemia were examined. Of these cases, 7 were diagnosed as acute undifferentiated leukemia (AUL) whose leukemic cells were negative for myeloperoxidase and did not react with lineage-specific or lineage-associated monoclonal antibodies. Then, we analyzed the configuration of both immunoglobulin (Ig) and T-cell receptor beta-chain (T beta) gene in these 7 AUL cases. Three cases had no rearrangement of Ig or T beta genes which was suggestive of non-lymphoid origin of these cases. In contrast, 4 cases showed rearrangements of Ig and/or T beta genes. One of these 4 cases demonstrated T beta gene rearrangement with retention of the germline configuration of Ig genes. Two cases with both Ig and T beta gene rearrangements also showed kappa-chain gene rearrangements. These findings indicate the heterogeneity of AUL at the DNA level, and may cast new light on the early differentiation of hematopoietic progenitor cells.

Characterization of Acute Undifferentiated Leukemia by Combined Analysis of Plasma Membrane-Associated ?-Glutamyltranspeptidase and Soluble Terminal Transferase

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma-GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma-GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT-blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma-GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma- GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma- GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT- blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma- GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

Monoclonal Antibody to Human Granulocytes: Cellular Specificity and Functional Studies

Antigenic specificity and functional studies of G2, a monoclonal antibody to human granulocytes, prepared by fusing spleen cells from immunized Balb/c mice to the nonimmunoglobulin (Ig) secretor line SP2/0, are described. The antibody was reactive with the HL60 and K562 cell lines and with human peripheral blood granulocytes; and unreactive toward human lymphocytes, erythrocytes, a variety of T and B cell lines, as well as toward leukemic cells obtained from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and acute myelocytic leukemia (AML). The G2 monoclonal antibody identified cell surface antigens on cells from cases of acute myelomonocytic leukemia (AMMoL) and on cells from 2 of 12 cases of acute undifferentiated leukemia (AUL). G2 was capable of inhibiting oxygen consumption by human polymorphonuclear leukocytes (PMNL) stimulated with aggregated human immunoglobulin (IgG), opsonized zymosan, f-met-leu-phe (FMLP), and the calcium ionophore A23187. Inhibition of the PMNL response to phorbol myristate acetate (PMA) and digitonin was dependent upon the dose of the stimulant. G2 should facilitate elucidation of the mechanisms of granulocyte membrane perturbation and subsequent activation of various functions via a selective interaction with key cell surface antigens.

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl- β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl- β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.

Antisera to acute lymphoblastic leukemia cells

Antisera to acute lymphoblastic leukemia cells have been raised in rabbits by injecting ALL cells coated in vitro with rabbit antibodies directed against normal lymphocyte antigens. The resultant antisera were absorbed with various normal tissues and tested by immunofluorescence for their ability to discriminate between ALL and other leukemias, and between ALL and normal cells. Fourteen out of 19 ALL patients at presentation had circulating cells reactive with the antisera. Three of these had a bone marrow diagnosis of ALL with no apparent abnormal cells in the peripheral blood. Of the five unreactive cases, three were the only T cell-like leukemias in the ALL group. Several patients considered to be in complete hematological remission had a small number of anti-ALL binding cells in their blood. Of several other types of leukemia and lymphoreticular cancers studied, the only other anti-ALL reactive cells were from two cases of acute undifferentiated leukemia, four out of five cases of chronic myeloid leukemia (CML) in blast crisis (CMLs at presentation were unreactive), and very weak reactivity in a proportion of cases of acute myeloid leukemia (AML). The reactivity of myeloid leukemic cells could be abolished by absorption with leukemic myeloblasts, leaving ALL reactivity intact. The only cells in normal fetal or adult tissues to show reactivity were a very small proportion of cells in bone marrow and spleen and a variable proportion of cells in fetal liver. Various control studies suggest that the antigen detected by anti-ALL sera is not a cell cycle, fetal, or cryptic “normal” antigen of lymphocytes. We conclude that antisera to ALL may define an antigen which may be restricted in expression to a large subgroup of ALL cases, and which offers considerable diagnostic and prognostic potential.